BIOPROSPECTING IN THE DELTAIC SUNDARBANS FOR MARINE MICROORGANISMS PRODUCING COMMERCIALLY IMPORTANT BIOACTIVE COMPOUNDS

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) OF JADAVPUR UNIVERSITY KOLKATA 700032 INDIA

BY

DEBASHISH GHOSH
B.Pharm., M.Tech.

M.PHIL. PROGRAMME IN ENVIRONMENTAL SCIENCE AND DEPARTMENT OF LIFE SCIENCE & BIOTECHNOLOGY JADAVPUR UNIVERSITY KOLKATA 700032 INDIA 2004

BIOPROSPECTING IN THE DELTAIC SUNDARBANS FOR MARINE MICROORGANISMS PRODUCING COMMERCIALLY IMPORTANT BIOACTIVE COMPOUNDS

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) OF JADAVPUR UNIVERSITY KOLKATA 700032 INDIA

BY

DEBASHISH GHOSH
B.Pharm., M.Tech.

M.PHIL. PROGRAMME IN ENVIRONMENTAL SCIENCE AND DEPARTMENT OF LIFE SCIENCE & BIOTECHNOLOGY JADAVPUR UNIVERSITY KOLKATA 700032 INDIA 2004

CERTIFICATE FROM THE SUPERVISOR

This is to certify that the thesis entitled “Bioprospecting In The Deltaic Sundarbans For Marine Microorganisms Producing Commercially Important Bioactive Compounds” submitted by Sri Debashish Ghosh, who got his name registered on January 24, 2003 for the award of Ph.D. (Science) degree of Jadavpur University, is absolutely based upon his own work under the supervision of Dr. Joydeep Mukherjee and that neither this thesis nor any part of its has been submitted for any degree / diploma or any other academic award anywhere before.

_______________________________________ Dr. Joydeep Mukherjee Thesis Supervisor

To The gallant people who sacrificed their lives in “Sundarban Tiger Reserve Forest” .

Satadal Das.De and Prof. Asok Nath Bose and Mr. Krishnakalidi.G. with which he guided me all throughout this work. I am indebted to them. they became more than my . K. I have had the opportunity of coming in contact with many a person but for whose help and cooperation I could not have completed my works. Prof. Here I pay homage to all other teachers who guided me all through my life. Jadavpur University and Dr. The reminiscence of my association with my present colleagues Mr. Miss Manjira Biswas and Mr. Department of Life Science and Biotechnology. Roy Research Centre. Priyadarshinidi. for their active encouragements and inspiring suggestions for orienting the line of my thoughts. Reader. Jadavpur University. Dean of Science. The pathogenic strains for performing antimicrobial study of the antibiotic lead compounds were also supplied by him. Debobrata Garai. I feel duty bound to convey my heartiest gratitude to all those who extended their hands for the completion of my work. Prof. Subrata Kr. Rajat Bondyopadhya. with satisfaction to my mentor Dr. Jack Nicklaus said. A. Joydeep Mukherjee.ACKNOWLEDGEMENTS During the year of my study in Jadavpur University. for his discreet advice. Parimal Karmakar. Kolkata. I would also like to thank Dr. Malay Saha and Mr. hence with deep sense of gratitude.K. “Achievement is largely the product of steadily raising one’s level of aspiration and expectation”. Rousseau said “Gratitude is a duty which ought to be paid and it’s the heart’s memory. Head of the Department of Life Science and Biotechnology. U. Jadavpur University. which helped me to a great extent. for his valuable guidance. Chief Pathologist. Ratan Gachhui. Pal. I also express my respectable and heartfelt thanks to our Vice Chancellor. But as the time went on. I express my deep heartfelt thanks to Prof. It is he who introduced me into the field of research.” So my thanks is also due to Prof. In the time of course of my doctoral work I had the opportunity to share my emotions to some of my seniors Dhritimanda.Datta for their immense appreciation and also for encouraging me to have perseverance. constant encouragement and extraordinary forbearance. Barindra Sana as well as my juniors Mr. Peerless Hospital & B. I wish to express my sincere gratitude to Dr. Amalesh Chowdhury. Registrar of Jadavpur University. Gopinath Ghosh will always be a source of joy and delight for me. Shubhoda and Sabyasachida. I acknowledge and owe my thanks to the affectionate encouragement for finishing this project.

Rajib. which helped me a lot. Apurba. Mridulda. Pobitra and Mananda. Tanmay. They were my fellow daily passengers of train who accompanied me everyday on my way to the University. Santanu. I remember with love and affection the cooperation that I received from my friends Anasuya. I warmly thank every person in the Department of Life Science and Biotechnology and Department of Environmental Science who in one way or the other helped me throughout this work. They always treated me as their younger brother. Sougatada. DST and ICMR for the necessary fundings and equipments provided by them. Narottom. Poulami. I had with them many fruitful discussions. I am thankful to “Dhar Brothers” and “A-House” for their excellent job.seniors. and above all. Brotoda. “Bioprospecting In The Deltaic Sundarbans . I am unable to mention a few more persons by name without whose help and support I would hardly realize my objective. For want of space. Last of all I must mention that it would have been an uphill task for me to accomplish what a little work I have done. the optimism they bore with me in regard to my work drove me to translate my dreams to reality. Kuntal. DEBASHISH GHOSH FOREWORD The work embodied in this thesis entitled. I love them. I would like to convey my deep gratitude to some people with who I spent some wonderful times. We enjoyed every moment of the journey. Jyoti. I am indebted to AICTE. if I had not the blessings as well as the mental and financial supports from my dad and mom and endless love and care from all the members of my family. I also extend my gratitude towards my Kaku and Kakima who helped me a lot during the tenure of this work.

....................... Marine enzymes.............1 1... The investigation was carried out by the author in the Environmental Science Programme and the Department of Life Science and Biotechnology............... This thesis is divided into two parts. International status of research in marine enzymes... India...Tech. The next part is the scheme of work..... This doctoral work is a continuation of that dissertation....... .... in his M..................1.. Kolkata July 2004 DEBASHISH GHOSH CONTENTS SUMMARY.. Kolkata............. The entire experimental work has been included in this part....... Joydeep Mukherjee from August 2001 to July 2004........... Microorganisms from sediment and sea water......1 1. India under the guidance of Dr.. It represents the first ever biotechnological exploration in that area..................... The literature survey was done on the basis of the research works of international and national levels published in various journals and articles within last 10 to 15 years......1.................. The first part gives a comprehensive review on the bioprospecting of marine enzymes and marine antibiotics........ ABBREVIATIONS... world’s largest tidal mangrove forest..D....... 1 1............ The discussion and references are presented after the scheme of work.................. Kolkata..................1 Marine biodiversity of bioactive compounds....... The author did a preliminary microbiological survey in the deltaic Sundarbans of Bay of Bengal.................................1...................................................................... (Science) degree of Jadavpur University........ (Biotechnology) dissertation.For Marine Microorganisms Producing Commercially Important Bioactive Compounds” submitted for the award of Ph... INTRODUCTION..... Jadavpur University.....

......... International status of research in marine antibiotics. Tetracyclines...........2............... Sampling data.......1 1...................................... β -lactam antibiotics.................. International status of research in marine antibiotics bioprocessing.....................1............1.............2..2 1...........1....................................2 2 2.................1 1..2...... Sampling techniques.3 Bioprospecting for enzyme........1.......... Marine enzyme bioprocessing..1............. Marine symbiotic microorganisms...........1............3 1.........................2............2 1..............1..............................................1......2.............1...................1................... Marine antibiotics bioprocessing.............................2 1........................................................ Marine symbiotic microorganisms...1 1.......... .............................1.....2.................... Bioprocessing......................1 2... Other antibiotics..2............................ National status of research in marine enzyme bioprocessing........2...1........ Field survey.............1.....1..............2...................................1 1......2 1..................... National status of research in marine enzymes....2 1.............2.......................1..2 1.....................1..................1 1....................................1........................ Lyases......... Medium preparation.. Macrolides................. Oxidoreductases.2........1..6 1... Other enzymes ....1....................1... Marine symbiotic microorganisms..............................1 2.....1..................... National status of research in marine antibiotics bioprocessing..............1........2 1........... Isolation of “true” marine microorganisms................1....................... Polypeptides...........2 1......................................4 1......... Primary stock cultures preparation............1......2.............. Sampling..................1......... Ligases. Microorganisms from sediment and sea water..................... Extremophiles......5 1............. Aminoglycosides.......1..........1........................1................... International status of research in marine enzyme bioprocessing................1..............1 1....1.................................. Sample preparation and spreading.. National status of research in marine antibiotics......................1.............................................2................................ Microorganisms from sediment and sea water......1.............2......3 1..............2 2.....................................1 1.................1....1..............................1 2.................................1....................................................1.1.........3 1..................2..................2.................1............................4 1...................1. Marine antibiotics....... 1 1.................1 1.1..............................3 1..2 1................................2 1...5 1............2.......2 Hydrolases.........................1....2 2............... SCHEME OF WORK.......................................1...

......5 1............................3....3......2..................3 1............3.............. Prot-104 (protease producer)..4 1.......1 1....2.....................4..... Cellulase............................8 1...........................1 1........4................... Microorganism 2/01 (DNase producer)....3.. Assay techniques.....3 1......1......4.4..1.2 1...2 1...........4..............................2........ Reaction conditions.........2 1.........3.......................1...1.. Bioreactor specifications............................ Prot-103 (protease producer).3.....5...............1 1......4......... DNase.4................3...4..........................................2.........3................................................. DNase (Deoxyribonuclease).........1 1.2. Nitrilase..........4........1 1.1 Identifications of true marine microorganisms..2.....2....4....................5 1..............4 1...........4....1....3 1...... Asp-03 (L-asparaginase producer)....................................4...... Prot-105 (protease producer).............................. Asp-04 (L-asparaginase producer)................................ Glut-02 (L-glutaminase producer)...............6 1......................3........3........3 1.....................11 1................2...5.... Reaction conditions...............................3 1. .................. Reaction conditions......................................................... Peroxidase..........................2..........4.........7 1........3....4.. Cellulase....................2 1........... Result........ RNase (Ribonuclease).... Results.....................3.....3..........2.........................3 1...............................1.....5.................3.....9 1..........4 1.4..................5 1....... L-asparaginase...................4...........2 1.4.............................2....2.................................4 1.....3........3 1.........1.......1........2............1 1..........2 1............1 1.... Results........................................................4......4..............1....1...4...2 1...............................3.... Protease...............2 1.........2.. Cel-2/03 (Cellulase producer).......... Amylase..... Results...... L-asparaginase. Results.............. L-glutaminase.... Reaction conditions.............6........1.1..1 1...4.6....................... Result................................4....1.............4 1.......4......... Reaction conditions.............3................................2 1.4.4..................4...4................................... Protease..1 1.... Microorganism 2/47 (xylanase producer)....4.......3........2.... Reaction conditions.4...................4........3...... Asp-05 (L-asparaginase producer). Xylanase..............................1 1.....................4..........................3......6......1.......1 1....1 1..10 1.....1........ Results.....5....... Enzyme screening............5 1.............................................6 1...............2................ Results.........................1..............4..............1.........2......................1...4 1.........3.....5 1............... L-glutaminase..............2..... Asp-01 (L-asparaginase producer)............1............ Prot-101 (protease producer).....2 1......2.....4.........2 1. Asp-02 (L-asparaginase producer)..2 1........ Glut-01 (L-glutaminase producer)......... Bioreactor specifications..................1 1........ Screening for special enzyme properties........................1 1.4........4.3....4........... Prot-102 (protease producer)............................................. Esterase........................4....... Xylanase....4.4.....................

..2....................4...3 1.... Operating conditions.8.............1............4..7.....................4.......... Offline analysis..............7.. R-08 (RNase producer)..3 1....2..................2................................................. Test culture preparation.2 2.......8 1.............2...2 1..............1...................... Results...2...........6 1.. Reaction conditions..........1 3.......3 2....................2....... N-01 ( nitrilase producer).....4...... 2.....................3......7...1 1.................4.. R-09 (RNase producer)............. Media standardization and day wise study.......5 3 3.........................2.......................................2.4 2....... N-05 ( nitrilase producer)............8.....................1........ Es-05 (esterase producer)..........8 1........4............. Bioprocessing...... Medium design for optimum antibiotic activity.. Nitrilase.............................5 1...................................................... Screening.......2 1.9........................1 3.......2 3.........1 1...2..........5 1.......................................1 3.......2...2........... Extraction procedure.................8.................................. Operating conditions..4.2...........3.....9................2 3...........1.....4........2............ Reaction conditions......... Bioreactor Run No.................... Cup-plate method...2.........7.........3............................. 1.................................4...... Culture preparation.................1.1 2................1...............2....................1..........4.............................................................1 1.............2....................4...................................3 2...........................2 3.....2.......................2 1...........4.... N-03 ( nitrilase producer).2 1.........7....................................... Results.............................6....4.....3... Bioprocessing of enzymes......... Es-03 (esterase producer).............................................3 Microorganism 2/22 (DNase producer)....9......2.4.........4....................................................2 2...........4....... Results for antifoam activity......................................1 1.............8..............4...........4..................8......................................2................................7.................................................3 2...........................1..........4.......... R-05 (RNase producer)................................... N-04 ( nitrilase producer).......... Results.................7............ Reaction conditions........7 1.. R-04 (RNase producer)......................5 2 2....................1 1......... Es-02 (esterase producer)..........................4........ Bioreactor Run No........ R-03 (RNase producer)..........................9 1. RNase.......................................9...1 2........ Extraction of antibiotic into organic solvent.4...2... Esterase. R-01 (RNase producer).....4 1...............2...2....7.....9.....2 1..............2......1 3..................4 1........1.2 2........2......1.......1.......................... Antifoam activity............. Results..9..................1 1.......4...................7........... Es-04 (esterase producer)..........2........9...........................................4 2.......4.............................. .....3 1...............3 3..4.....4.... R-07 (RNase producer).............1 2.....................2......... N-02 ( nitrilase producer)..........4.............7.........7 1...................2 1..............7................ Es-01 (esterase producer).......................... Offline analysis..............4........2 1.................................8..2.....9 1...............1....................... Result. Results for media standardization........................................4... R-06 (RNase producer)..4 1.... Bioprospecting for antibiotics.... Results................................4..............3................................ R-02 (RNase producer)...............8.......

....................1.....................1 3........................................4........ Results................... ........1............ 3.....................................2................. Bioreactor Run No......1. Off line analysis...........2..........1................. 2....3 3...........................1.........4.. 5..................1.............. Operating conditions...........................................4 3...............................2. L-asparaginase.... Special enzyme properties......3 3.....................................4........ Result................. Offline analysis...........2 1...........3 1............. 1...........1.......2................3 3.........................................................................2........1 1...... Operating conditions............... Screening of marine enzymes.......4 1............................................. Operating conditions..3...2 3... DISCUSSION...........................................................2...............................4..................... Offline analysis..........4.....................2.............................5 Bioprospecting in deltaic Sundarbans...........................6.............................1.........2........ Results.................................2 3.......................2...... Bioreactor Run No...1.2............................... Bioreactor Run No.............. Protease.............................. Result...............3 3.....................................6 3...............2 1.1......... Operating conditions..........................................................1 3.......................................................1.3............................2..........................4 3. 4............. Bioreactor Run No......5.........3 3......... Bioreactor Run No..1 1..............1..........3 3.......................2..........................................2 3...................1 1.......................1 3..5....2...................3..................... L-glutaminase. Cellulase............................... Off line analysis..2 3.........1.. Off line analysis.........2.........2..............2 3.................3 3......................1.............. Bioprocessing of antibiotics........3....3.1.......1 3.. Bioreactor Run No........... Bioreactor Run No........ Operating conditions................... 4........2............3 3.. Result...........6...............................2 3..1......................1...1......... 1 1................................5 3......1.........................................4............1 3................3 3...2 3................6...................................2 3... Marine enzymes..1..........2..........................1..........................3... Operating conditions............ Offline analysis.........................................2..............1............. Xylanase.......1............................1..1 3...... Results................................. Results...2 3.....................1............................2..1 3....................................5.......2............................1 3................................ 3........2..............................2 3.. Off line analysis............................2..........1...... Offline analysis.............................. 6.............. Operating conditions.......................... Result.......2..............1 3.........2....................... Operating conditions.3 Bioreactor Run No..................................3....

RNase.2 1..........................2 1..................... Nitrilase....3 1.........................2.... Overall assessment on marine antibiotic lead compound....................................................2..................9 1.......................................1 1.........6 1.................................2.............. Extraction of antibiotic lead compound in organic solvent....................3 1........2... Overall assessment of marine enzymes...................... 01 02 03 04 05 06 07 Classifications of enzymes.....................1. Esterase....8 1.........................1. REFERENCES....................1 1......2..1................. Antibiotic profile of screened antibiotic lead compounds........................................... Field data of second field............................2.......... Classification of antibiotics......................................................... Marine antibiotics..................2.............................1...... Field data of third field...................................................1........1............7 1............................................................2 DNase....................................1........ Field data of fourth field............. Standardization of optimum antibiotic production by microorganism DG I................ LIST OF TABLES Table No.... Bioreactor study of the antibiotic producer DG I...................................................................1..... Title Page No...... Field data of first field.......1................ Isolated numbers of true marine microorganisms in four .......2..........................2..............................................

......................... Different assay conditions for testing special enzyme properties of xylanase............................................ Different sample sets for the DNase producing microorganisms.................................................................................... Composition of different flasks during cellulase assay......................... Asp-01 in 100% SDW Marine Difco medium. India............................... Survey area 3rd and 4th field...................................................................................................................................................... ............................ Composition of different flasks during xylanase assay..... LIST OF FIGURES Figure Title Page No...................... Composition of various blanks for testing special properties of DNase.................................. Different assay conditions for testing special enzyme properties of nitrilase................................................................................... Different assay conditions for testing special enzyme properties of esterase.................... Map of Lothian and Sagar Island.......... 01 02 03 04 05 06 07 Location of Sundarbans in West Bengal......... Sajnakhali................................ Production of antibiotic in different production media in different time intervals..................................................... Results of primary and secondary screening of different enzymes........................ Different assay conditions for testing special enzyme properties for L-asparaginase.............................................08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 field surveys...................... Result of primary screening of antibiotic producing microorganism against various test organisms............................................... Specifications of the controls used in case of DNase assay.............................................................................................................................................................. Asp-01 in 100% NSW Marine Difco medium................... Effect of antifoam in the production of antibiotic from DG I in PM1...................................................... Sundarban Tiger Reserve Forest......................................................................................................... Different assay conditions for testing special enzyme properties of RNase............................................................................................. Different assay conditions for testing special enzyme properties of cellulase...................................................................................................................................................................................... Different assay conditions for testing special enzyme properties of DNase..................... Asp-02 in 100% SDW Marine Difco medium........... Different assay conditions for testing special enzyme properties for L-glutaminase. Sajnakhali............................................................... Different assay conditions for testing special enzyme properties for protease.................... Diameters of zones of inhibition of antibiotics produced by DG I before and after extraction..........................

........ Es-01 in 100% NSW Marine Difco medium................ R-05 in 100% SDW and 100% NSW marine Difco medium..... Prot-105 in 100% NSW Marine Difco medium................................................................................................................................................. Prot-103 in 100% NSW Marine Difco medium.. Asp-04 in 100% NSW Marine Difco medium........................... R-09 in 100% SDW and 100% NSW marine Difco medium.......................................... N-03 in 100% SDW Marine Difco medium........ Asp-03 in 100% SDW Marine Difco medium............................... Microorganism 2/01 in 100% NSW Marine Difco medium............................................. Es-03 in 100% SDW Marine Difco medium....................................................................... N-01 in 100% NSW Marine Difco medium................................................................... Prot-102 in 100% NSW Marine Difco medium............. Es-02 in 100% NSW Marine Difco medium.............................................................................................. N-02 in 100% SDW Marine Difco medium........................................................... Microorganism 2/01 in 100% SDW Marine Difco medium...08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Asp-02 in 100% NSW Marine Difco medium......................................... Microorganism 2/22 in 100% NSW Marine Difco medium............................ R-01 in 100% SDW and 100% NSW marine Difco medium......................... Prot-102 in 100% SDW Marine Difco medium........ Prot-103 in 100% SDW Marine Difco medium............................................ Asp-03 in 100% NSW Marine Difco medium................................ N-02 in 100% NSW Marine Difco medium............. N-01 in 100% SDW Marine Difco medium..... Glut-01 in 100% NSW Marine Difco medium.... ........................................................................................................ Prot-105 in 100% SDW Marine Difco medium............................................ R-04 in 100% SDW and 100% NSW marine Difco medium.................................................... Es-01 in 100% SDW Marine Difco medium.................................................................................................. Es-05 in 100% SDW Marine Difco medium.........................1% xylan....................... Es-02 in 100% SDW Marine Difco medium................................................. R-07 in 100% SDW and 100% NSW marine Difco medium................................................ Microorganism 2/22 in 100% SDW Marine Difco medium........... Es-04 in 100% SDW Marine Difco medium...... Glut-01 in 100% SDW Marine Difco medium............................ Asp-05 in 100% SDW Marine Difco medium.......... Prot-101 in 100% NSW Marine Difco medium........................................... Cel-2/03 in 100% SDW based nutrient broth with 0................................................... Asp-05 in 100% NSW Marine Difco medium................ Es-05 in 100% NSW Marine Difco medium................................. Prot-104 in 100% NSW Marine Difco medium................................................... Glut-02 in 100% SDW Marine Difco medium. Microorganism 2/47 in 100% SDW based nutrient broth with 0........................................................................ Prot-104 in 100% SDW Marine Difco medium.. Asp-04 in 100% SDW Marine Difco medium............ Es-04 in 100% NSW Marine Difco medium............................................................................................... R-08 in 100% SDW and 100% NSW marine Difco medium....................................1% CMC................. R-06 in 100% SDW and 100% NSW marine Difco medium.... Es-03 in 100% NSW Marine Difco medium................................................... R-02 in 100% SDW and 100% NSW marine Difco medium........................... Prot-101 in 100% SDW Marine Difco medium...................................................................... Glut-02 in 100% NSW Marine Difco medium................................................. R-03 in 100% SDW and 100% NSW marine Difco medium....

.. Production of protease in Bioreactor by microorganism Prot-105................. Bioreactor Run No...... Production of xylanase in Bioreactor by microorganism 2/47................................................................................................ especially Sundarbans region........... Bioreactor Run No........................................................................................Tech................................ N-04 in 100% SDW Marine Difco medium.................. SUMMARY Bioprospecting for marine microorganisms producing commercially important bioactive compounds is still in its infancy in India..59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 N-03 in 100% NSW Marine Difco medium .............. but Bay of Bengal.............. Bioreactor Run No................................................................................................ 2 for DG I......... remained untapped until this project work was initiated................. N-05 in 100% NSW Marine Difco medium................................................... Production of cellulose in Bioreactor by microorganism 2/03..................... Zone of inhibition of DG I against different test organisms before extraction................ Eyela MBF Bioreactor. Though a few successful steps have been taken in the western sea-coast of the country................................................................................ Production of DNase in Bioreactor by microorganism 2/01....... 3 for DG I........................................................ N-04 in 100% NSW Marine Difco medium............. Production of cellulose in Bioreactor by microorganism 2/47................... Zone of inhibition of DG I against different test organisms after extraction.................... 4 for DG I....... Production of RNase in Bioreactor by microorganism R-01...... Bioreactor Run No....(Biotechnology) dissertation and the promising results led to a thorough ... N-05 in 100% SDW Marine Difco medium.................................... The author has done a preliminary microbiological survey of this area during his M..... 1 for DG I........................... world’s largest tidal mangrove forest................

ABBREVIATIONS 3'. xylanase. They were tested in wide range of pH. during his M. 5 L-glutaminase. Sediment and sea water samples from various locations of the Sundarbans such as Lothian Island. Sagar Island and Sajnakhali regions were collected. Escherichia coli and Klebsiella pneumoniae. After the primary screening the enzyme producers were screened for special enzymatic properties. 5'-cyclic adenosine monophosphate carboxy fluorescein diacetate . About 35 different marine enzyme producing microorganisms were treated for special enzymatic properties and 5 protease. Since the idea was to produce commercially important enzymes from marine source. high temperature and salinity. 5'-cyclic nucleotide phosphodiesterase Acquired Immune Deficiency Syndrome acute lymphoblastic leukemia air membrane surface American Public Health Association American Water Works Association β -hydroxypropionate 3'. The enzymes targeted in this project were protease. DNase. RNase. 2 1 cellulase. some of the microorganisms’ enzyme production was scaled up in bioreactor and increase of production was successfully achieved. The results of the first ever biotechnological exploration of the highly biodiverse Sundarbans can be taken up for further exploitation of these microbial resources. cellulase.Tech. 2 DNase. The antibiotic lead compound was also extracted in organic solvent (ethylacetate) completely. L- asparaginase. Out of 1050 bacteria collected. 5'-CNP AIDS ALL AMS APHA AWWA β -HP c-AMP cFDA 3'. L-glutaminase. This thesis work describes the entire screening work of marine enzymes and antibiotic lead compounds. L-asparaginase. Various parameters like optimum antibiotic production in various production media. dissertation and one of those microorganisms was found to have both antibacterial and antifungal activity along with prominent antimicrobial activity against recalcitrant microorganisms like MRSA. day wise study and effect of antifoam were studied before successful scale up in bioreactor. 9 RNase. 5 esterase and 5 nitrilase producers were found to be thermostable and active in high saline conditions as well as in wide range of pH. 378 were truly marine.microbiological investigation as described in this doctoral thesis. 1 xylanase. esterase and nitrilase. MSSA. Four antibiotic lead compounds producing marine bacteria were screened by the author from sea-shore sediment.

rpm RT s carboxymethylcellulose dextro dimensional 2. IUCN km λ LL LB m M MarBEC MeOH mg min ml mM MPa MRSA mS MSSA NAD(P) nir NMR NSW OO.U.4-diacetylphloroglucinol double distilled water dimethyl sulfide dimethylsulfoniopropionate Deoxyribonucleic acid 3.c.D.CMC DD DAPG ddH2O DMS DMSP DNA DNS DTT FDA g GDH h I.s. r.f.B. ppt PAGE PCR q. 5-dinitrosalicylic acid dithiothreitol fluorescein diacetate gram glucose dehydrogenase hour(s) International Union of Biochemists International Union for Conservation of Nature and Natural Resources kilometre wave length levo litre Lauria-Bertani metre Molar Marine Bioproducts Engineering Centre methanol miligramme minute(s) mililitre miliMolar Mega Pascal methicillin-resistant Staphylococcus aureus mili Siemens methicillin-sensitive Staphylococcus aureus Nicotinamide adenine dinucleotide (phosphate) nitrite reductase gene Nuclear Magnetic Resonance natural sea water orthro Optical Density parts per trillion polyacrylamide gel electrophoresis polymerase chain reaction quantum suficit relative centrifugal force revolution per minute room temperature seconds .

S sq. km SDW SSF TCA TMB µ g µ L µ M U VRE VRSA v/v vvm w/v WPCF Svedberg unit square kilometre single distilled water solid state fermentation tri-chloro acetic acid 3.min-1 weight/volume Water Pollution Control Facilities INTRODUCTION . 3'. 5.volume of medium-1. 5'-tetramethylbenzidine micro gramme microLitre microMolar Unit vancomycin-resistant Enterococcus vancomycin-resistant Staphylococcus aureus volume/volume volume of air.

1.1.1.1.1.1.1.1 1.3 Marine biodiversity of bioactive compounds Marine enzymes International status of research in marine enzymes Microorganisms from sediment and sea water Hydrolases Oxidoreductases Lyases .1 1.1.2 1.1.1.1 1.1 1.1.1.1 1.

1.1.2.2 1.1.1 2.2.2.1.1.1.1.1.1.2.2.2 1.1.1 1.1.2 2.3 1.2.2 2.2 1.1 1.1.2 1.1.4 1.1.2.1.1.1 1.1.2 1.1.1.1.1.1.3 1.1 1.1.4 1.2.1 2.2.1.1.1.2.1.2 Ligases Other enzymes Extremophiles Marine symbiotic microorganisms National status of research in marine enzymes Microorganisms from sediment and sea water Marine symbiotic microorganisms Marine antibiotics International status of research in Marine antibiotics Microorganisms from sediment and sea water β -lactam antibiotics Aminoglycosides Tetracyclines Macrolides Polypeptides Other antibiotics Marine symbiotic microorganisms National status of research in marine antibiotics Bioprocessing Marine enzyme bioprocessing International status of research in marine enzyme bioprocessing National status of research in marine enzyme bioprocessing Marine antibiotics bioprocessing International status of research in marine antibiotics bioprocessing National status of research in marine antibiotics bioprocessing 1 1.1.6 1.1 1.1.5 1.1.1.1.1.1.1.2.1.1.1.1.1.4 1.2.1.1.3 1.1.1 1.2 2 2.1.1 1.5 Marine biodiversity of bioactive compounds Marine enzymes International status of research in marine enzymes Microorganisms from sediment and sea water Hydrolases Oxidoreductases Lyases Ligases Other enzymes .2 1.1.2.1 1.1.2.1.5 1.1 2.2 1.1.

energy.1.2.1.1.2.4 1. materials and fine chemicals.2 1.1 2.1.1.1.2 2 2.2.1.1.2.6 1.1.2 1.1.1.2.2.2.2.1. The marine environment is a rich source of both biological and chemical .2 Extremophiles Marine symbiotic microorganisms National status of research in marine enzymes Microorganisms from sediment and sea water Marine symbiotic microorganisms Marine antibiotics International status of research in Marine antibiotics Microorganisms from sediment and sea water β -lactam antibiotics Aminoglycosides Tetracyclines Macrolides Polypeptides Other antibiotics Marine symbiotic microorganisms National status of research in marine antibiotics Bioprocessing Marine enzyme bioprocessing International status of research in marine enzyme bioprocessing National status of research in marine enzyme bioprocessing Marine antibiotics bioprocessing International status of research in marine antibiotics bioprocessing National status of research in marine antibiotics bioprocessing 1 Marine biodiversity of bioactive compounds The world’s expanding human population continually needs new sources of food.1.1 1.1 2.1.3 1.2.2.1.1 1.2 1.2 2.1.2.1.2 1.5 1.1. It has been realized by many that the marine realm is a rich and largely untapped resource of products that are of potential interest to mankind.2.2.1.1.1 1.3 1.1.2 1.1 2.1.2 2.2.1.1 1.1.2 1.

nutritional supplements.’’. marine biotechnology as a field is still in the realm of the future. The search for new biomedicals from marine organisms resulted in the isolation of more or less 10. Marine biotechnology encompasses pharmaceuticals. 1997). Each of these classes of marine bioproducts has a potential multi-billion dollar market value. antiviral. Unfortunately. since 1995. Although we are poised on the edge of a period of tremendous potential. 1995). The oceans represent a virtually untapped resource for discovery of even more novel compounds with useful activity. neurotoxic. insufficient quantities of materials to allow for study completion and difficulties of culturing marine organisms in the laboratories were cited. The oceans comprise more than 70% of the Earth’s surface and they contain microbial species of which only 10% is known to humans (Colwell. Marine biotechnology is defined as ‘‘the application of scientific and engineering principles to the processing of materials by marine biological agents to provide goods and services’’ (Zilinskas et al. antimitotic. for example: AIDS. Thousands of unique chemical compounds have been identified from a relatively small number of the ocean’s biological and chemical diversity (Pompony. cosmetics. Mebs (1995) of the University of Frankfurt observed ‘‘The sea is a hostile environment for the human intruder. antifungal. In a review of the biology of poisonous marine organisms. 1998). In more recent years. anti-inflammation. toxic. antineoplastic and cardio vascular activities. cytotoxic. and agrichemicals. molecular probes. fine chemicals. Alzheimer disease.diversity. The difficulties of retrieving a ‘‘sustained. ageing processes and some tropical diseases. However. The commercial success stories in biotechnology are familiar. reliable’’ harvest of marine organisms. Both the lack of accessibility to much of the oceans’ depths until recently and this perception of hostility may have resulted in the relatively tiny amount of knowledge we have of the ocean and sea creatures. A broad spectrum of biological activities has been detected. agricultural. immunosuppression. 1997). it . enzymes. But the commercial success stories in marine biotechnology are far less familiar and far fewer. new targets have been added to the general screening.000 metabolites many of which endowed of pharmacodynamic properties. such as: antibiotic. This diversity has been the source of unique chemical compounds with the potential for industrial development as pharmaceuticals. the same holds true today (Staley. industrial applications as well as bioremediation.

similarly. continuing to dominate the reports of new compounds. As a result. studies on coelenterates were also declining and the annual number of publications on sponge metabolites reached a maximum. over 120 of the most important medicines in use today (penicillins. 1999). Jensen & Fenical. It has been suggested that future partnerships should result in a higher level of technology transfer. one of the worlds leading pharmaceutical companies announced that it had entered into an agreement with the National Institute of Biodiversity in Costa Rica (INBio) to search for naturally occurring therapeutic agents produced by plants. Why marine microorganisms? The importance of terrestrial bacteria and fungi as sources of valuable bioactive metabolites has been very well established for more than half a century. Faulkner et al. 1993. Liberra & Lindquist. On September 21. Bernan et al. That morning.. 1995. Thomas Eisner. At first sight thus. 2000.became evident that all classical algal sources began to be much less studied than in the past. In return. commercial partners should be willing to compensate these organizations for their expertise (Garrity & Hunter-Cevera. The amazing increase in the number of reports on new metabolites from marine microorganisms has been emphasized by Kelecom (1999). 1997. Merck and Co. 1991. Kobayashi & Ishibashi. From all these literatures a very simple question arises. Davidson. 1997. 1996. Since then numerous articles have been published in scientific journals and the popular press detailed collaborations between commercial research laboratories of the developed world and various governmental and nongovernmental organizations in the developing world. in his philosophy of drug discovery said that various agencies in the species rich nations in the developing world could facilitate the drug discovery program through their knowledge of native species and local uses. Pietra. adriamycine. etc. 2000). 1994. natural product research saw a new sunrise. animals and microbes. 1993.) are obtained from terrestrial microorganisms. . Metabolites from marine microorganisms is a rapidly growing field as can be best observed from the number of reviews dedicated to this topic (Fenical. An additional possible explanation should be that marine microorganisms constitute the ultimate ‘‘inviolated’’ frontier for the search of marine natural products. cyclosporin A. 1995. the expectable enormous biodiversity of marine microorganisms might have been the reason for the interest in their study.

Man has indirectly used enzymes almost since the beginning of human history. Traditionally. 1996) and more recently the biologically active components of the Neem tree (Dickson & Jayaraam. enzymes were simply assigned names by the investigator who discovered the enzyme. Exploration of India’s rich natural resources for the search of pharmacologically active constituents dates back to several thousands of years as documented in the religious scriptures of that period. This thesis embodies the results of the explorations. India never gained any monetary profit from the commercialization of her natural resources by the western countries.) which is given in the following table (Table No. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I. 01).B. systems of enzyme classification became more complex. 1998) are being tested as candidate drugs in modern drug discovery programs by US American Pharmaceutical companies. Unfortunately. Some of the discoveries of those days have found their application in modern western medicines for example. the alkaloids extracted from Rouwolfia serpentina used as antihypertensive drugs (Pieter et al. 1. Such a strategy would have relatively little impact on most major pharmaceutical companies and would provide developing countries an opportunity to build their own manufacturing and discovery capacity. Table No.1 Marine enzymes Most of the reactions in living organisms are catalysed by protein molecules called enzymes. Against this background. 1998) and a novel macrolactam-trisaccharide antifungal antibiotic (Hegde et al.with a focus on off-patent compounds in developing countries. As the knowledge expanded. New quinolone compounds Pseudonocardia Species (Dekker et al. Enzymes can rightly be called the catalytic machinery of living systems. 01 : Classifications of enzymes . a research program has been initiated by the Department of Life Sciences and Biotechnology and the Environmental Science Programme of Jadavpur University for a systematic search for novel bioactive compounds from the Bay of Bengal.U. 1995).

No. 1 2

Classifications Oxidoreductases Transferases

3 4 5 6

Hydrolases Lyases Isomerases Ligases

Biochemical Properties Act on many chemical groupings to add or remove hydrogen atoms Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules. Add water across a bond, hydrolyzing it. Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds. Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others. Catalyze reactions in which two chemical groups are joined (or ligated) with the use of use of energy from ATP.

Enzymes are responsible for the biocatalytic fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing. Enzymes oxidise ethanol to acetic acid. This reaction has been used in vinegar production for thousands of years. Similar microbial enzyme reactions of acid forming bacteria and yeasts are responsible for aroma forming activities in bread making and in preserving activities in sauerkraut preparation. Presently the industrial enzyme companies sell enzymes for a wide variety of applications. The estimated value of world enzyme market is presently about US $ 1.3 billion and it has been forecasted to grow to almost US $ 2 billion by 2005. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially, produced enzymes. Enzymes are also indirectly used in biocatalytic processes involving living or dead and permeabilised microorganisms. Below are given the general ideas of some of the commercially important enzymes with which this doctoral thesis has been dealt. Amylase: Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaves polysaccharides). The primary function of the enzyme amylase is the hydrolysis of α -1, 4-glycosidic linkages of polysaccharides to yield dextrins, oligosaccharides, maltose and D-glucose. Amylase is also synthesized in the fruit of plants during ripening, causing them to become sweeter. It is commercially used in food industry.

L-asparaginase: L-asparagine

+ H2O = L-aspartate + NH3. This reaction is mediated by

the enzyme L-Asparaginase. Tumor cells, especially Acute Lymphoblastic Leukemia (ALL) cells, have low levels of asparagine synthetase, the enzyme that catalyzes the synthesis of asparagine. Therefore, tumor cells require an external source of asparagine. Asparaginase serves to destroy all the asparagine that does manage to get synthesized in a tumor cell or that comes in from other sources, thus, the cells die because they do not have the asparagine needed to build proteins and the tumor cell dies. Asparaginase does not occur naturally in humans, but it is found in bacteria, plants, and many animals, including guinea pigs. It is used in the treatment of lymphoblastic leukemia.

L-glutaminase: L-glutamine

+ H2O = L-glutamate + NH3. This reaction is mediated by

the enzyme L-glutaminase.
L-glutaminase

is considered a potent anti-leukamic drug and has found

application as a flavour-enhancing agent in food industry. Protease: Proteases are enzymes that hydrolyse peptide bonds and therefore, lead to the disassembly of proteins. Commercially they are extremely important as more than 60% of the total enzyme market is made up of proteases, they are isolated from plants, animals, bacteria and fungi and there are very many available commercially. They all catalyse the hydrolysis of proteins but there are many differences in the method of catalysis.

Cellulase and Xylanase: Cellulases are a complex enzyme system, comprising endo-1,4-

β -D-glucanase, exo-1,4-β -glucanase (exocellobiohydrolase) and β -D-glucosidase
(β -D-glucoside glucanhydrolase). These enzymes, together with other related enzymes, viz. hemicellulases and pectinases, are among the most important group of enzymes that are employed in the processing of ligno-cellulosic materials for the production of feed, fuel, and chemical feedstocks. Cellulases and xylanases (endo-1,4-β -D-xylanase) however find applications in several other areas, like in textile industry for fibre treatment and in retting process. Xylanases find specific application in jute fibre

upgradation also.

DNase: Deoxyribonuclease is an endonuclease, splitting phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The average chain of limit digest is a tetranucleotide. DNase acts upon single chain DNA, double-stranded DNA and chromatin. In the latter case, although histones restrict susceptibility to nuclease action, over a period of time nearly all chromatin DNA is acted upon. It is mainly used in research work. It is mainly used in the identification of regions of DNA that are bound by protein and thereby protected from DNase I digestion, also used to identify transcriptionally active regions of chromatin since they are more susceptible to DNase I digestion

RNase: A Ribonuclease (RNase) is an enzyme that catalyzes the breakdown of RNA molecules into smaller components. RNases are extremely common in the modern world, resulting in very short life spans for any RNA that is not in a protected environment. An RNase that is commonly used in research is RNase A. RNase A (bovine pancreatic ribonuclease A) is one of the hardiest enzymes in common laboratory usage; one method of isolating it is to boil a crude cellular extract until all enzymes other than RNase A are denatured. Esterase: A carboxylic ester + H2O = an alcohol + a carboxylate. This reaction is mediated by esterase. The enzyme is mainly used in aroma industry and in organic compound synthesis.

Nitrilase: These are a group of enzymes that have good potential in the chemical process industry for conversion of nitriles to a wide range of useful products and intermediates. The enzymes are mostly bacterial and there are two main groups nitrilases and hydratases with the latter being more readily available than the former. Traditional chemical processes for the hydrolysis of nitriles are expensive requiring high

soil remediation. fungi and actinomycetes many other marine organisms such as fishes. . Apart form this the enzyme is also useful in pulp and paper bleaching (substitute for chlorine bleach). prawns. By manipulation of the reaction conditions it is possible to synthesize amino acids pure and optically active. In addition many other important compounds can be produced depending on the starting nitrile material. A marine enzyme may be a unique protein molecule not found in any terrestrial organism or it may be a known enzyme of a terrestrial source but with novel properties. Samples containing greater than 0. So the enzyme nitrilase is mainly used in biodegradation. which means that the isolation of the commercial product is more difficult and there is a disposal problem for the unwanted by-products. oceanic caves and some areas where high pressure and absence of light are obvious. cold adaptivity.2 mg. plants and algae had also been studied to tap the arsenal of the marine world.L-1 may be diluted with distilled water to a concentration suitable for this method. Enzymes produce a pure single product under gentle conditions.temperature. Properties like high salt tolerance. hyperthermostability. This method is suitable for these and similar applications where the water matrix is clear and free of turbidity. snakes. The absorbance of λ 596 light by the sample is compared to a reference curve generated by standard H2O2 solutions. Besides microorganisms like bacteria. New applications for H2O2 within the food processing and drinking water industries require the accurate measurement of residual H2O2 to 0. These properties may not be expected in terrestrial sources as the marine organisms thrive in habitats such as hydrothermal vents. Peroxidase: Peroxidase enzyme catalyzes the transfer of electrons from H2O2 to a colorimetric indicator.L-1. Hydratase is a mixture of enzymes. piezophilicity and ease in large scale cultivation are the key interests of scientists.1 mg. crabs. In the last decade there has been a continuous effort to learn more about the still largely unexplored realm of marine enzymes. on-site waste destruction and medical diagnostics. also inefficient as many by-products are produced. Nitrilase is a multi-enzyme complex capable of converting nitriles directly to carboxylic acid.

deep sea sediments and sea water have been easily reachable to marine biotechnologists and therefore reports of marine enzymes from these sources have been numerous over the past decade. alkaline serine exoprotease.1.1 Microorganisms from sediment and sea water Near shore sediments. 1. corals and with other species) and their occurrence in extreme environments (extremophiles) like hydrothermal vents have also been the areas of recent research and are therefore reviewed. identify (by molecular phylogenetic method) and bioprocessed. 1995). As marine microorganisms are very easy to tap. they are of major interest to researchers worldwide. Vibrio species have been found to produce a variety of extracellular proteases.1 International status of research in marine enzymes The marine enzyme research in the developed world has been described first in the following section.1. 2004). This marine bacterium also produces collagenase. BAL 31 Nuclease manufactured by a Japanese . an enzyme with a variety of industrial and commercial applications (Biotechnology for the 21st century: new horizons. cultivate.1. Portions of this section has been published as a review (Ghosh et al. including an unusual detergent resistant. marine animals and plants. The symbiotic nature (microorganisms found associated with various marine sponges. carbohydrases and peroxidases have been the most cited ones. Among them proteases.A survey of literature of the past ten years shows that the occurrence of marine enzymes has been most widely studied in various sources classified as marine microorganisms. 1. Vibrio alginolyticus produces six proteases. In this chapter only the microbial sources have been highlighted. one being the international status of research and the other being the national status of research. The extracellular proteases are of particular importance and can be used in detergents and industrial cleaning applications. The entire literature survey has been distributed under to major headings. Some of them have found commercial applications. such as in cleaning reverse osmosis membranes.

In order to identify strains with α -1. A novel enzyme. A marine bacterial strain of genus Vibrio that decomposes the cell walls of some seaweed.1 Hydrolases Shibata et al discovered a novel metalloproteinase. is produced by the marine bacterium Alteromonas espejiana BAL 31. Chile. Bacillus subtilis was isolated from marine environments by ArellanoCarbajal and Olmos-Soto (2002). A protamine-degrading marine bacterium was isolated from marine soil and identified as Aeromonas salmonicida subsp.1.1. almelysin which has high activity at low temperatures and another proteinase from the culture supernatant of a marine bacterium.3-glucanase were determined by Burtseva et al (2003). 1997).3 linkage of neoagarooligosaccharides to yield agaropentaose and D-galactose was isolated from the . Results obtained by Arrieta and Herndl (2001) with natural bacterial communities analyzed by capillary electrophoresis zymography from the coastal North Sea suggest that the diversity of β -glucosidases in the marine environment might be much higher than previously observed. 1992). an endonuclease specific for single-stranded deoxyribonucleic acid (DNA) (activity I) and also has exonuclease activity (activity II). The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan was studied by Arnosti and Repeta (1994). secretes a metalloprotease involved in the chitin degradation system of the strain (Miyamoto et al. (Shibata et al. produced high levels of an extracellular agarase in the presence of agar (Leon et al.1. and Undaria pinnatifida. A marine bacterial strain isolated from the Bay of San Vicente. Optimal conditions for growth of a marine fungus Chaetomium indicum and for biosynthesis of β -1. TaKaRa. Alteromonas sp. 1. (Obata et al. 1997). α neoagarooligosaccharide hydrolase which hydrolyzes the α -1.. identified as Alteromonas sp. including a Laminaria sp.firm. 2002).and 1. Alteromonas sp.4. has been isolated from seawater by Sugano et al (1993). A β -mannanase from Vibrio sp.6-glucosidase enzymes with potential uses in shrimp feed production. was purified by Tamaru et al (1995). Rath and Herndl (1994) reported that marine snow of the northern Adriatic Sea showed β -D-glucosidase activity.

1998). producing a particularly heat-labile alkaline phosphatase was isolated by Hauksson et al (2000) from North Atlantic coastal waters. 2000). the most abundant polymer in marine environment was studied. α -agarase II and α -agarase. 1992). Chitinase from the marine bacterium.6-anhydro-L-galactose (Whitehead et al. Marine bacterial strains of Cytophaga diffluens. The phenotypic and agarolytic features of a marine bacterium Pseudoalteromonas antarctica that was isolated from the southern Pacific coast was investigated.marine bacterium Vibrio sp.1. When exposed to different types of chitin.1. Constitutive amidase with broad substrate specificity from a number of deep sea Actinomycetes were reported by Brandão and Bull (2003). hutchinsonii. Vibrio harveyi was found to have a higher growth rate and more chitinase activity when grown on β -chitin (isolated from squid pen) than on α -chitin (isolated from snow crab) probably because of the more open structure of β -chitin. 1. 2001). and characterized by Sugano et al (1994). were found to be highly adaptive and therefore. can significantly contribute to the phosphate economy of the marine environment (D’Souza et al. Phosphate solubilizing bacteria belonging to Bacillus sp. (fluorescent) and Vibrio fluvialis were examined for cellulase production using four media by Vaidya et al (2000) and Araki et al (1999) reported that β -1. A marine Vibrio sp. harveyi excreted several chitin-degrading proteins into the culture media (Svitil et al. such as agar.2 Oxidoreductases Chloroperoxidase isolated from the marine fungus Caldariomyces fumago is unique . The degradation of chitin. α agarase I. Another marine bacterium degraded numerous complex carbohydrates. chitin and alginate with an agarase system that consisted of at least three enzymes. from marine bacterium Bacillus cereus was purified and characterized gave a single band on polyacrylamide gel electrophoresis (PAGE) with activity staining (Kim et al.1. C. which acted in concert to degrade polymeric agar to D-galactose and 3. 1999). V. Alteromonas sp. β -agarase.3-xylanase was purified to gel electrophoretic homogeneity from a cell free culture fluid of Vibrio sp. 1997). It produced a diffusible agarase that caused agar softening around the colonies (Vera et al. Pseudomonas sp. was purified (Tsujibo et al.

Candida tropicalis and Umbelopsis isabellina. The GDH can react under high salinity. were responsible for colour removal of synthetic dyes by enzymatic biodegradation (Yang et al. an algal osmolyte. IV) oxides. Nitrite reductase was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica (Besson et al. the most abundant volatile sulfur compound emitted from oceans is formed primarily by the action of dimethylsulfoniopropionate (DMSP) lyase which cleaves DMSP. This enzyme is unusually versatile: it catalyses not only the reactions typical of peroxidases but also those of catalases and monooxygenases and it is also almost unique in catalysing halogenation reactions (except fluorination) in the presence of halide ions and H2O2 (Colonna et al.1. 1998) from a soluble fraction of a marine Gram-negative bacterium identified as a Deleya sp. The soluble enzyme was purified to electrophoretic homogeneity from a facultatively anaerobic Gram-negative rod-shaped marine bacterium identified as an Alcaligenes . 2003).among the peroxidases because it contains a cysteinic thiolate as the fifth axial ligand of the heme instead of the imidazole ligand.3 Lyases Dimethyl sulfide (DMS).1. 1. Three constitutive forms of superoxide dismutase (iron. by Chadd et al (1996). Another novel GDH which reacts with a clinical marker of diabetes was purified by the same group (Tsugawa et al. A yellow pigmented marine bacterium was isolated by Francis et al (2001) from surface sediments of San Diego Bay based on its ability to oxidize soluble Mn (II) to insoluble Mn (III. 1995).1. copper/zinc and an unidentified) activity have been demonstrated in the marine cyanobacterium Synechococcus sp. 1999). Manganese-dependent peroxidase activities detected in culture supernatants of Debaryomyces polymorphus. 1999). D’Souza and Yoch (1995) reported the isolation and purification of DMSP lyase. to equimolar amounts of DMS and acrylate. A novel glucose dehydrogenase (GDH) from a marine bacterium Cytophaga marinoflava was isolated by Tsugawa et al (1996) from its membrane fraction. The cytosolic form of Cu-Zn superoxide dismutase has been isolated from the marine yeast Debaryomyces hansenii (Hernandez-Saavedra & Ochoa.

1.species a salt marsh bacterial isolate. L-serine dehydratase (Laroche et al. Highly active constitutive nitrile hydratase with broad substrate specificity from several deep sea actinomycetes were reported by Brandão and Bull (2003). and β -HP all induced DMSP lyase activity (Ansede et al. It was demonstrated in α -subclass of Proteobacteria in marine bacterioplankton community that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase.5 Other enzymes Recent reports of other marine microbial enzymes belonging to the above categories obtained from sediment and sea water include lycopene k-cyclase (Stickforth et al. 1. which accumulated briefly and was then taken up by cells. for coping with a light and nutrient limited environment (Alaoui et al. DMSP. The production of DMSP lyase from this organism and a marine strain. The physiological regulation of glutamine synthetase in the axenic cyanobacterium Prochlorococcus sp. 2002). was studied and the unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus sp.1. 1997) and arylsulphatase (Barbeyron et al. Added acrylate was β -hydroxylated on (or near) the cell surface to β -hydroxypropionate (β -HP).4 Ligases Glutamine synthetase in cells of the marine diazotrophic cyanobacterium Trichodesmium thiebautii was investigated by Carpenter et al (1992). 2003). quinol oxidase (Simpson et al. 1995). 1995).1. 1995). a marine bacterial strain isolated by Sakai et al (2003). 2001). Fucobacter marina.1. DMSP lyase was also isolated from the sulfate-reducing bacterium by Jansen and Hansen (2000).1. produced extracellular sulfated fucoglucuronomannan lyase. 1. acrylate. . polyhydroxybutyrate depolymerase (Kita et al. Pseudomonas doudoroffii were induced at optimum rates by DMSP and vigorous aeration (D’Souza and Yoch.1. 2001).

2 Extremophiles Diversa. psychrophilic enzymes can be useful for commercial laundry detergents as consumers can wash clothes in cold. 1999.1. describing enzymes from thermophiles. A second generation of thermostable polymerase chain reaction (PCR) enzymes has already been harvested from bacteria living near thermal vents on the ocean floor. Although such applications have been projected. Even if they are to be used at mild temperatures. 1998). A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria Pyrococcus abyssi by Dib et al (1998). psychrophiles and piezophiles. a market that was expected to grow about 10% each year. current literature. with unexpected genetic diversity and new natural products including enzymes of potential relevance to human health or environmental bioremediation (Deming. the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. Major recent advances in cold deep sea biotechnology have come in the form of continuing discoveries of novel microorganisms. A novel intracellular serine . rather than hot water which could significantly reduce power consumption. barophilic protease from Methanococcus jannaschii. however.1.1. an American company engaged in the application of microbial biodiversity in the biotechnology industry recognized that thermophilic enzymes from vents with temperatures of 3504000C are stable protein molecules. they remain active far longer than regular enzymes. Enzymes from extremophiles will constitute a part of this market (Colwell. Michels and Clark (1997) reported the properties of a hyperthermophilic. Deming. They also resist the destabilizing effects of organic chemicals used in industrial downstream processes. 2002). Similarly. 2002). Horikoshi. Continuing explorations of submarine hydrothermal vent environments have yielded new hyperthermophiles and more evidences of pressure regulated operons and elevated hydrostatic pressure stabilization of cells and enzymes at high temperature. This section is broadly divided into three parts. A recent US National Academy of Sciences report noted that in 1993. 1997). 1998. and is marketed as Vent® and Deep Vent® Polymerases (Grace. world enzyme sales equaled US$1 billion. is limited to the occurrence of metabolic enzymes in psychrophilic organisms (Wells.

Gantelet and Duchiron (1998) isolated the extremely thermophilic archaeon Thermococcus hydrothermalis from a deep sea hydrothermal vent in the East Pacific Rise which produced an extracellular pullulanase. Microorganisms growing above 60°C isolated from deep sea hydrothermal vents were screened for amylolytic activity. 2002). Pullulanase. Brown and Kelly (1993) purified extracellular pullulanases from cell free culture supernatants of the marine thermophilic archaea Thermococcus litoralis and Pyrococcus furiosus. The pyruvate carboxylase of Methanococcus jannaschii was purified and expressed in cells grown without an external source of biotin (Mukhopadhyay et al. and Thermococcus sp. . versions of which however. Nine archaea and one thermophilic bacterium were selected for the determination of thermostability and pH optima. glucosidase and amylase activities were detected in four archaeal strains related to the genus Thermococcus (Legin et al.proteinase (pernilase) was identified from a marine aerobic hyperthermophilic archaeon Aeropyrum pernix having enzyme half-lives of 85 min at 1000C and 12 min at 1100C by Chavez et al (1999). 1995). litoralis and P. Optimal growth conditions were analyzed to determine final yields and metabolic rates. thermostable hydrolytic enzymes were characterized for potential applications. The membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus was characterized by Nishihara et al (1997) as highly oxygentolerant. extremely thermophilic and thermostable in its membrane-bound form. Four different tungsten-containing enzymes have been purified from Pyrococcus furiosus which oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids (Roy and Adams. 2002). The chitinoclastic enzyme system of Thermococcus chitonophagus. furiosus appeared to represent highly thermostable amylopullulanases. cellassociated. have been isolated from less thermophilic organisms. and inducible by chitin (Huber et al. the effects of temperature and hydrostatic pressure on cell replication and enzymatic activities were also investigated (Francesco. 1998). 2000). mostly Pyrococcus sp. isolated from a hydrothermal vent site off the Mexican west coast was oxygen-stable. After extensive investigation of shallow water and deep sea hydrothermal vents for the isolation of hyperthermophilic microorganisms. The enzymes from T..

enzymes studied so far are heat stable. Kazuoka et al (2003) reported that Cytophaga sp. 2001). a mesophile are that cold-active enzymes are more broadly distributed and that lateral transfer of the gene from a psychrophile occurred or that F. 2001).3-glucanase activities have been isolated and the strain with the highest glucanase activity has been taxonomically identified and the enzyme was purified (Kammel et al. It was a remarkably stable enzyme from a psychrophilic microorganism. isocitrate lyase having maximum activity at 200C but rapidly inactivated at temperatures above 300C and malate synthase having optimum temperature of activity of 450C were discovered (Watanabe et al. 2002). but cannot grow above 30°C produces a variety of NAD(P)dependent dehydrogenases among which alcohol dehydrogenase and aldehyde dehydrogenase are thermostable have been reported by Soda et al (2002) Alanine dehydrogenase.Turning to psychrophiles and psychrotolerant organisms. succinogenes originated from the marine environment (Iyo & Forsberg. Possible explanations for the presence of a cold-active enzyme in Fibrobacter succinogenes. which grows optimally at 15°C. Marine psychrophilic bacteria with β -1. A few studies have been reported on enzymes from piezophilic microorganisms. An extracellular serine peptidase was purified from the culture supernatant of a sub-Arctic psychrophilic bacterium. recent studies have elucidated that although microorganisms produce various psychrophilic enzymes in order to carry out metabolism efficiently under the cold conditions. The enzyme showed higher pH and thermal stabilities than that from mesophiles. closest to Shewanella isolated from a sea urchin off the Icelandic coast by Irwin et al (2001). 1999). A psychrophile from Antarctic seawater identified as Cytophaga sp. Two enzymes from Colwellia maris. remaining active after one week at 20°C and after five freeze–thaw cycles (Irwin et al. produces aspartase abundantly. malate dehydrogenase and glutamate dehydrogenase were detected in extracts from a psychrophilic bacterium. Konisky et al (1995) noted that the application of 50 MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens in contrast to the evidence on hyperbaric stabilization . A heatlabile β -lactamase similar to highly specialized cephalosporinases from pathogenic mesophilic bacteria has been purified by Feller et al (1997) from the cold-adapted psychrophile Psychrobacter immobilis.

Marine microorganisms often survive as intracellular or extracellular symbionts and their hosts are mostly marine animals (vertebrates or invertebrates) which are first described in this section. Vibrio fischeri. The arginine biosynthetic enzymes are present in all the tissues of the worm and in the bacteria (Minic et al. isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura. exhibits the unusual attribute of . 2001). a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids.3 Marine symbiotic microorganisms Marine invertebrates and their cultivatable bacterial symbionts have become a focal point of marine natural product research. 1992). Basic research on some of these associations has resulted in the biotechnological development of these valuable resources. Pseudoalteromonas sp. 1. 2003).1. Euphausia superba Dana was purified and characterized (Turkiewicz et al. synthesizes an intracellular cold-adapted β -galactosidase (Turkiewicz et al. 1995). Scientists present microscopical and enzymatic (presence of methanol dehydrogenase) evidence that methylotrophic bacteria occur as intracellular symbionts in a new species of mytilid mussel discovered at the mid-Atlantic ridge hydrothermal vents (Cavanaugh et al. 2003). 1999). 1999).of enzymes from deep sea thermophiles. two classes of enzymes important to the industry (Wells. A study of its properties by Qureshi et al (1998) suggested the presence of two kinds of respiratory chains regulated in response to pressure in this bacterium.1. An extracellular protease from the marine bacterium Sphingomonas paucimobilis isolated from the stomach of Antarctic krill. followed by one example of a plant host. Researchers investigated the respiratory chain system of a deep sea barophilic bacterium Shewanella sp. Bacterial mats that grow on whales have been found to be a rich source of the lipases and esterases. The deep sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium and investigations indicate that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines (Minic et al. A bacterial protease was isolated from a marine shipworm and was tested in cleansing formulations (Greene et al. and a novel heme c containing quinol oxidase was purified from cells grown at 60 MPa pressure.

5'-cyclic adenosine monophosphate (c-AMP).2.5'-cyclic nucleotide phosphodiesterase (3'.5 km coast line and the Arabian Sea and the Bay of Bengal have been the focus of scientists and following are some recent reports. 1. 1999). produced three major classes of extracellular ligninmodifying enzymes: manganese-dependent peroxidase. apparently through the activity of a 3'. 1995). Phylogenetic analysis grouped 132 nirS Arabian Sea sequences into 12 major clusters.1. The only example of a plant host is Laminaria fronds that house a bacterium Alteromonas sp. 1. allows this symbiotic bacterium to utilize extracellular 3'. The dominant sequence type from one surface sample showed 99% identity to the nirS sequence of the cultivated denitrifier Pseudomonas aeruginosa.1 Microorganisms from sediment and sea water A basidiomycetes fungus Flavodon flavus isolated from decaying sea grass from a coral lagoon off the west coast of India. nitrogen and phosphorus (Callahan et al. India has a 7516.1. lignin peroxidase. which due to its unusual location in the periplasm. 1. 1993). The diversity and distribution of nirS (nitrite reductase) genes in relation to nitrite and nitrate distribution in the Arabian Sea coastal denitrifying region (South west coast of India) has been reported (Jayakumar et al. nirS gene fragments were PCR-amplified.growth on 3'. and sequenced from DNA extracted from the water column. 1997).2 Marine symbiotic microorganisms . Most of the nirS sequences from the coastal water column did not show a high level of identity with other nirS sequences previously reported from marine and estuarine sediments. and laccase (Raghukumar et al.2 National status of research in marine enzymes Our country.1. cloned. c-AMP) as sole sources of carbon.5'-CNP) with exceptionally high activity (Dunlap & Callahan. producing extra and intra cellular alginate lyases and utilizes alginate as its sole carbon source (Sawabe et al.5'-cyclic nucleotides (e.2.g. 2000).

02 :Classification of antibiotics Class Characteristics Process interrupted β-lactam antibiotics Contains β -lactam ring Composed of aminosugars linked by glycosidic bonds to various bases Have a rigid structure composed of 4 fused benzenelike rings Cell wall cross linking Mode of Action Site of action Cell wall Ribosomes 30S subunits Type of action Bactericidal Bactericidal Aminoglycosides Protein synthesis and fidelity Tetracyclines Protein synthesis Ribosomes 30S subunits Bacteriostatic . (Mohapatra et al. also were found to be L-asparaginase producers. 1997) and an acetylcholinesterase producing bacterium Arthrobactor ilicis (Mohapatra & Bapuji. In the following table (Table No. which are produced by microorganisms and at a low concentration. A Bacillus sp. a Micrococcus sp. 1998) associated with the intertidal marine alga Sargassum sp. A Streptomycetes sp.2 Marine antibiotics Antibiotics are the chemotherapeutic agents. 1998). isolated from the prawn Penaeus indicus showed good L-asparaginase activity (Dhevendaran & Annie. 1996) and a Mucor sp. The marine sponge Spirastrella sp. For many years source of antibiotics was limited to the terrestrial microorganisms and after 1940 scientists started looking for new antibiotics from the marine source. producing urethenase (Mohapatra & Bapuji.Mohapatra (1997) reported the isolation of carboxymethylcellulase from a Bacillus sp associated with the marine sponge Axinella sp.02) a general classification of antibiotics is given. (Mohapatra et al. 1998). 1998). Table No. was found to be an interesting host organism as it harboured a Mucor sp. producing a novel amylase (Mohapatra et al. are capable of killing or inhibiting the growth of a microorganism without causing damage to the host. 1.

Macrolides

Have a large ring structure Amino acids linked by peptide bonds form a major component of the structure

Protein synthesis

Ribosomes 50S subunits Cell wall and cell membrane

Bactericidal/ Bacteriostatic Bactericidal

Polypeptides

Membrane integrity and mucopeptide synthesis

Miscellaneous

Includes many drugs, such as chloramphenicol, vancomycin, nitrofurantoin and isoniazid that Protein synthesis have and mucopeptide only one or two synthesis representatives of the class and are seldom referred to by their chemical nature in clinical practice.

Ribosomes 50S subunits and cell wall

Bactericidal

The isolation by Brotzu, in the late forties, of the antibiotics cephalosporins with other metabolites, from the fungi Cephalosporium sp. cultivated from seawater collected near a sewage outlet off the coast of Sardinia seems well to be the first conclusive work in this area, but it remained an isolated fact and the marine ancestry of such compounds was even claimed to be ‘‘dubious’’. Undoubtedly more important was the suspicion that a number of metabolites obtained from algae and invertebrates could be produced by associated microorganisms. Indeed, it has been frequently suggested, but seldom demonstrated, that microorganisms should be in some instances the true producers of a number of secondary marine metabolites. Dibromotyrosines from Aplysia sponges, halogenated metabolites from Dysidea sp., macrolactones and sulfur containing compounds were claimed to be probably produced by associated organisms. Aryl carotenoids in sponges were suspected to be originated from inhabiting bacteria. Similarly, it was stated that ‘‘there is strong circumstantial evidence that the alkaloids from a species of the genus Reniera may be fabricated by a symbiotic microorganism’’, since mimosamycine obtained from the sponges Reniera sp. and Xestospongia sp. had previously been isolated from the fungi Streptomyces lavendulae No. 314. Such

considerations stimulated some very interesting works and resulted in important contributions (Kelecom, 2000) and the waterworld has become a happy hunting ground for the marine biotechnologists to scour new antibiotics with an idea that they may be more efficient at fighting infections because the terrestrial bacteria have not developed any resistance against them.

1.2.1

International status of research in marine antibiotics

In the following section research work on marine derived antibiotics accomplished in foreign countries has been described.

1.2.1.1

Microorganisms from sediment and sea water

Along with marine microorganisms, various marine plants and animals were found to produce novel antibiotics. But this literature survey has been restricted only to marine microorganisms found in marine sediment and sea water as well as associated with various marine animals and plants in symbiosis. Marine microorganisms belonging to the genus of Actinomycetes, Bacillus, Pseudomonus were also found to be potent producers of antibiotic lead compounds as in the cases of the terrestrials. Numerous published literature show that many novel antibiotics were isolated from various Streptomycetes sp.

1.2.1.1.1

β

-lactam antibiotics

Kim et al (2003) identified β -lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys.

1.2.1.1.2

Aminoglycosides

Plasmid profiles were used to screen Streptomycetes for production of new antibiotics.

Among about numerous strains isolated from sea muds, an isolate was revealed to harbor several plasmids of different sizes, and to produce istamycins, new aminoglycoside antibiotics. Based on the characteristics of the strain, Hotta et al (1980) proposed for a new Streptomyces species S. tenjimariensis. They examined the production rate of the antibiotic in presence of various chemicals and the possible involvement of plasmids in istamycin production (Hotta et al, 1980). Nutritional effects as well as other biological and chemical properties of istamycin were also studied by the same group (Hotta et al, 1980). Slattery et al (2001) reported that the marine bacterium Streptomyces tenjimariensis produced the antibiotics istamycin A and B under select laboratory culture conditions. Presumably these compounds served an ecological role under natural conditions.

1.2.1.1.3

Tetracyclines

Maskey et al (2003) found two new anthracycline antibiotics designated as himalomycin A and B, isolated from the culture broth of the marine Streptomyces sp. The structure of the new antibiotics was determined by detailed interpretation of mass and 1D and 2D NMR spectra.

1.2.1.1.4

Macrolides

Asolkar et al (2000) found a marine Streptomycetes isolate which produced a new macrolide antibiotic designated as chalcomycin B. A new antibiotic, aplasmomycin, which inhibited the growth of Gram-positive bacteria including myobacteria in vitro, and plasmodia in vivo had been obtained from a strain of Streptomyces griseus isolated from shallow sea sediment in Sagami Bay (Okami et al , 1976).

1.2.1.1.5

Polypeptides

A few reports of antibiotics producing marine Bacillus were found with in past three

Three novel antibiotics designated as chandrananimycin A. to have strong insecticidal activity against both brine shrimp and H. Hawaii.years. C and D a family of new cyclic decapeptide antibiotics. Bogorol A illustrated a new structural template for “cationic peptide antibiotics”. collected in Papua New Guinea by Barsby et al (2001). vancomycin-resistant Enterococci. has been isolated from cultures of a marine Bacillus sp. Two new indole nucleosides Kahakamides A and B were isolated from the actinomycete Nocardiopsis dassonvillei. obtained from a shallow water sediment sample collected on the island of Kauai. Xiong et al (2004) discovered a Streptomyces sp. Loloatin B was previously reported by the same group in 1996 to be produced in culture by a Bacillus sp. Bioassayguided fractionation of the ethyl acetate extract of a marine Bacillus sp. 2001). B. 1996).2. B and C were isolated from the culture broth of a marine actinomycetes Actinomadura sp. They exhibited in vitro antimicrobial activity against MRSA. 1D and 2D NMR spectra by Maskey et al (2003). A survey of literatures also . and drug-resistant Streptococcus pneumoniae.1. Loloatins A.6 Other antibiotics With the aim to find new insecticidal antibiotics from marine microorganisms from seawater and sea sediments from Beidiahe and Dagang of the east coast of China. Microbial competition for limiting natural resources within a community was thought to be the selective force that promoted biosynthesis of antimicrobial compounds. armigera similar to that of avermectin B1. Kahakamides A exhibited antimicrobial activity toward the Gram-positive bacterium Bacillus subtilis (Schumacher et al. a novel peptide antibiotic active against methicillin-resistant Staphylococcus aureus (MRSA) and VRE. culture broth has led to the isolation of two new compounds. 7-O-succinyl macrolactin F and 7-O-succinyl macrolactin A. have been isolated from laboratory cultures of a tropical marine bacterium recovered from the Great Barrier Reef in Papua New Guinea by Gerard et al (1999). The structures of the new antibiotics were determined by detailed interpretation of mass. Compounds exhibited antibacterial activity against Bacillus subtilis and Staphylococcus aureus (Jaruchoktaweechai et al. Bogorol A. 2000).1. isolated from the tissues of a marine worm collected in Papua New Guinea (Gerard et al. 1.

P. was produced by the fermentation of Agrobacterium sp. Onyshchenko et al (2002) isolated strains of Alteromonas-like bacteria from the Black Sea water having high antagonistic activity against phytopathogenic fungi as well as against Bacillus subtilis. Pseudoalteromonus etc. aurantia. Agrochelin. 1999). They reported the isolation and taxonomy of the producing microorganisms as well as fermentation and isolation of sesbanimides. Two new antiinflammatory macrolides. Strains producing the wide spectrum of antimicrobial substances (Alteromonas macleodii. Pseudoalteromonas phenolica which produced a bactericidal antibiotic against MRSA.2. 1. fungicidal and algocidal substances were found.and intracellular metabolities of marine bacteria (including the pigments) were active. The purified anti-MRSA substance from the methanol extract of the cells of P. Acebal et al again (1998) described the bacterial production of sesbanimides by two marine Agrobacteria which produce Sesbanimide-A and Sesbanimide-C.1. The compound was obtained from the bacterial cells by solvent extraction and purified by silica gel chromatography. Pseudoalteromonas citrea. P. a moderately active antibiotic. 1999). Isnansetyo and Kamei (2003) reported a new marine bacterium. Both extra.) as well as fungal source.highlight the novel antibiotics from various marine bacterial (Pseudomonus. Enterococcus faecium. Kupka et al (1981) isolated siccayne.2 Marine symbiotic microorganisms Many novel antibiotics were discovered from the microorganisms associated with . a new alkaloid cytotoxic substance. Agrochelin and its acetyl derivative exhibited cytotoxic activity (Acebal et al. haloplanktis. Proteus vulgaris and Candida albicans.From submerged cultures of the marine basidiomycete Halocyphina villosa. Taranzo et al (1982) found antiviral activity of antibiotic producing marine bacteria isolated from the estuarine water and sediment samples collected from Atlantic ocean. and Enterococcus faecalis but was less active against Streptococcus sp.). phenolica was highly active against Enterococcus serolicida. which inhibits Gram-positive bacteria and some fungi. lobophorins A and B have been isolated from fermentation broths of a marine bacterium isolated from the surface the Caribbean brown alga Lobophora variegate (Jiang et al. Pseudoalteromonas sp.

This was the first time a series of dd-diketopiperazines has been isolated from a single natural source. urauchimycins A and B. 2003). and the octocoral Plexaura flexuosa and various bivalve mollusks) were reported by Castillo et al (2001) to produce antibacterial substances against human pathogenic strain of Staphylococcus aureus. Fdhila et al (2003) isolated bacterial strains from cultures of larvae of mollusks produced dd-diketopiperazines with strong antibiotic activity against Vibrio anguillarum. The Actinomycetes of the genus Micromonospora having antitumor and the Streptomycetes with antimicrobial activity were isolated from the surface. The strain was isolated from an unidentified sponge. Imamura et al (1993) described them to be the first antimycin antibiotics which possess a branched side chain moiety. the sponges Cliona sp. were isolated from a fermentation broth of a Streptomyces sp.4-diacetylphloroglucinol (DAPG) was evaluated by Isnansetyo et al (2003) against Vancomycin-resistant Staphylococcus aureus (VRSA) strains isolated from marine alga from several Asian and European countries. Dhuvendaran & Annie .2 National status of research in marine antibiotics Survey of literature shows that national status of research in marine antibiotics from microbial source is almost nil. and Vibrio sp. Only one report has been found.2. isolated from several groups of marine organisms (hard coral Madracis decactis. Brazil. 1. Antibacterial activity of 2. Bacteria of Aeromonas sp. Two novel antimycin antibiotics. South Africa and USA. A new cyclotetrapeptide. isolated from the exocellular extract of Pseudomonas sp. They exhibited inhibitory activity against morphological differentiation of Candida albicans. Sugita et al (1998) isolated intestinal bacteria from seven coastal fishes were examined for their antibacterial ability against Vibrio vulnificus. China by Zheng et al (2000). epidermis and intestines of sea plants and animals collected from the Taiwan Strait.various marine animals and plants. Three new chlorine containing compounds were obtained from a marine-derived fungus Aspergillus ostianus isolated from a marine sponge at Pohnpei as antibacterial components against a marine bacterium Ruegeria atlantica isolated from a glass plate submerged in the coastal water (Namikoshi et al. a bacterium associated with the sponge Ircinia muscarum was reported by Mitova et al (2003).

MarBEC (Marine Bioproducts Engineering Center) is a recently established multi-disciplinary engineering-science cooperative effort of the University of Hawaii and the University of California at Berkeley (Zaborsky. shellfish and sediment of veli estuarine lake along Kerala coast. chemists and engineers venture jointly to integrate their research disciplines in order to develop bioprocess technology for the production of marine natural compounds. bioprocess engineering represents the key. 2 Bioprocessing There is always been a gap between discovery and commercialization and this gap may be bridged when biologists. Success of now-a-days biotechnology has been and continues to be dependent on new discoveries and their timely transformation into useful products through bioprocess engineering and a systems approach. industry and the government to develop next-generation technology through cutting-edge research. bioactive structural analogs of known compounds via manipulation of culture conditions presents marine biotechnologists with a unique challenge for new bioproduct discovery.(1998) successfully isolated Streptomycetes with antibiotic activity from fish. For marine biotechnology. The area in which marine biotechnology in general and marine bioprocess engineering in particular has the greatest potential is in the design and optimization of bioreactors for marine metabolite production. A variety of bioreactor designs have been implemented. India. relevant education and innovative technology transfer. the National Science Foundation launched the Engineering Research Centers Program in the mid 1980s. bioreactor design and transgenic production coupled with efficient downstream processing and product recovery will be necessary to meet the needs of both discovery and bulk production of . with varying degrees of success. To address international competitiveness and the revitalization of key US industries. like biopharmaceutical biotechnology. 1999). The essential feature of this program is a partnership among academia. The opportunity to produce new. The many hundreds of tantalizing bioactive compounds discovered and isolated from varied marine organisms over the past decades have led to only minimal commercialization due to the limited availability of the compounds in question. Innovations in media development.

1998). The addition of skimmed milk to Zobell medium enhanced the extracellular enzyme production fivefold.2 National status of research in marine enzyme bioprocessing In this area significant contributions have been made by the researchers of Cochin University. 1998).1 2. 2003). high temperature bioreactor operation and corrosion of materials. 1999). They may also require an entirely different kind of .1 Marine enzyme bioprocessing International status of research in marine enzyme bioprocessing With the discovery and increased research into extremophiles. Kerala. 2000). the conventional restrictions of low to moderate bioreactor temperatures and pressures faced by engineers may be circumvented (Wright et al. The use of sea water in the production medium enhanced the production of this activity by 150% (Manachini & Fortina. 2. Research in these areas examines the biotechnological potential of marine extremophiles from a biochemical engineering perspective (Bustard et al. Running bioprocess systems using marine hyperthermophiles. supplemented with skimmed milk under different hydrodynamic conditions. Marine microorganisms often require special culture conditions. such as high hydrostatic pressure in the case of deep sea bacteria and an optimized production medium for increased enzyme yield. 2. A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell based medium. A halotolerant strain of Bacillus licheniformis produced high protease activity during the early stationary phase of growth.1.1. India. Specific growth rate increased as a consequence of increasing agitation rates (César & Facunda.novel marine bioproducts (Manachini & Fortina. poses interesting challenges such as bioreactor design and manipulation of their products.

unlike traditional sources such as soybean meal. In general. a chitinous solid waste of the shellfish processing industry was used as a substrate for chitinase production by the marine fungus Beauveria bassiana in a SSF culture. 1997). sodium chloride and methionine. Pseudomonas pseudomelli produced maximal chitinase activities during the late exponential and stationary phase under submerged fermentation. immobilized by Ca-alginate gel entrapment was used for the production of extracellular L-glutaminase under repeated batch process and continuous process employing a packed bed reactor by Kumar and Chandrasekaran (2003).. Results indicate scope for production of salt tolerant L-glutaminase using this marine fungus. 1999). The results indicate scope for the utilization of shellfish processing (prawn) waste for the industrial production of chitinase by using SSF (Suresh & Chandrasekaran. Keerthi et al (1999) reported Beauveria sp. 1997). 1998). corn steep liquor or molasses or chemically defined medium with known inducers (Chandrasekaran. 1995). The process parameters influencing SSF were optimized. was observed during SSF using polystyrene as an inert support by Sabu et al (2000). Recently some investigations have been reported on the applications of solid state fermentation (SSF) in marine bioprocessing. Maximal L-glutaminase yield was obtained in a medium supplemented with yeast extract and sorbitol. isolated from marine sediment also produced extracellular L-glutaminase. . Manganese significantly enhanced chitinase production (Suresh et al. Prawn waste. A chitinolytic fungus Beauveria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in SSF using wheat bran were optimised (Suresh & Chandrasekaran. isolated from marine sediment. which may be closer to the type of complex substances they are familiar with. This enzyme was inducible and growth associated. A marine Pseudomonas sp. Extracellular L-glutaminase production by Beauveria sp. Process parameters influencing L-glutaminase production by marine Vibrio costicola in SSF using polystyrene as an inert support were optimized (Prabhu & Chandrasekaran. the volumetric productivity increased with increased dilution rate and substrate concentrations and the substrate conversion efficiency declined.complex nutrients in the production medium.

bioprocess also provides the achievement of better chemotherapeutic profiles of the antibiotic lead compounds as various growth parameters are best accessed in fermentors. in order to assess their suitability as chemotherapeutic drugs. In the context of marine biochemical systems. with process scale-up in mind. offering poor prospects for successful scale-up. Most production strategies are carried out at the shake-flask level and lack a mechanistic understanding of the antibiotic production process.2. A number of literature published in the last decade gave the world some promising antibiotics ready for commercial trials. Not only do they boost up in the production rates. along with investigations into the control mechanisms for biosynthesis. 1999) showed that data need to be collated on media and physical optima differences between the trophophase and idiophase. A limiting factor in the widespread commercial acceptance of a large range of marine metabolites is the efficient production . 2. through again this area is understudied.2. In a review (Marwick et al. to allow implementation of novel fermentation protocols. Similarly. with optimization and conversion of current technologies having the potential to yield more efficient units.2 Marine antibiotics bioprocessing It has always been cited that secondary metabolites are best produced in bioreactors. opportunities exist for the development of novel reactors.1 International status of research in marine antibiotics bioprocessing There is a lack of research into bioreactor engineering and fermentation protocol design in the field of marine bacterial antibiotic production. mass transfer and shear stress data of fermentations are needed to provide the bioreactor design requirements to intensify antibiotic biosynthesis. The application of bioprocess intensification methods to the production of antibiotics (and other metabolites) from marine microbes will become an important strategy for improving supply of natural products. Immobilization may play a part in bioprocess intensification of marine bacterial antibiotic production.

An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10T and Bacillus pumilus . Excellent antimicrobial activity against Staphylococcus sp. An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. A series of new antibiotics called the bioxalomycins was identified as the antibacterial products from fermentations of this culture. Conventional methods utilised for physical and chemical process intensification require careful analysis of their potential application to shear-sensitive bioprocess systems. Stress induction. during which time glucose was more rapidly assimilated than dextrin. Use of high pressure as a stressing agent and:or intensification tool is discussed. for example. provides one route to marine bioprocess intensification due to the expression of metabolites not otherwise possible. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. naphthyridinomycin. Florida. sufficient quantities of antibiotics and nutraceuticals to allow for structural analysis and clinical testing. Release of these secondary metabolites was not due to the onset of sporulation.5 m deep). for example. Fermentation conditions for production of bioxalomycin α differed substantially from those required for production of a related compound. and its potential. When Bacillus licheniformis strain EI-34-6. An actinomycete strain was isolated from an intertidal sediment sample collected in Key West. and Enterococcus sp. cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. is shown. demonstrated by showing the existence of barotolerant (at 120 MPa) marine microorganisms obtained from shallow surface waters (1. Neither glycerol nor ferric iron was required for production of these inducer compounds. for example. were detected in both the supernatant and cell extract samples from fermentations by Bernan et al (1994). Microorganisms associated with the surface of. by the reference culture Streptomyces lusitanus. Production of antibiotic activity peaked at 48-50 h and closely paralleled cell growth. seaweed show a greater likelihood of being barotolerant (Wright et al. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. was grown in this reactor. 1999).of. isolated from the surface of a marine alga.

Although a number of researchers have touched on aspects dealing with bioprocess intensification to improve the process efficiency and effectiveness. It is hoped that common fermentation principles can be developed for bacteria isolated from similar ecological niches. licheniformis isolate EI-34-6 grown in shake flask cultures. .2 National status of research in marine antibiotics bioprocessing No noteworthy reports were found in the literatures. 2001). A modified roller bottle culture method elicited the production of antimicrobial compounds from two epibiotic marine Bacillus strains isolated from the surface of the marine alga Palmaria palmate (Yan et al. biochemically active natural products. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds. A number of antibiotic lead compounds have already been isolated and reviewed (Burja et al. no programme has tried to increase product yield of cultured cyanobacetria via culture and reactor configuration manipulation. 2002). through the commercialization stage.2. the corresponding spent medium from shake flask cultures of DSM10T and EI-25-8 could not. Along with other marine microorganisms cyanobacteria (blue-green algae) have also been identified as one of the most promising groups of organisms from which to isolate novel. (Yan et al. 2003). leading to the design of an intensification process. 2. The authors believed that the application of bioprocess intensification methods to the production of cyanobacterial natural products is likely to become an important strategy for improving supply of natural products. however.strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B.

SCHEME OF WORK .

SCHEME OF WORK
Bioprospecting for commercial bioactive compounds needs extensive sampling which is followed by a thorough and methodical screening program. The entire scheme of work has been classified under the following headings. 1 1.1 1.1.1 1.1.2 1.1.3 1.2 1.2.1 1.2.2 1.2.3 1.2.4 Bioprospecting for enzyme Sampling. Field survey. Sampling techniques Sampling data. Isolation of “true” marine microorganisms Medium preparation Sample preparation and spreading. Primary stock cultures preparation. Identifications of true marine microorganisms.

1.2.5 1.3 1.3.1 1.3.1.1 1.3.1.2 1.3.1.3 1.3.1.4 1.3.1.5 1.3.1.6 1.3.1.7 1.3.1.8 1.3.1.9 1.3.1.10 1.3.1.11 1.3.2 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.1.2.1 1.4.1.2.2 1.4.1.2.3 1.4.1.2.4 1.4.1.2.5 1.4.2 1.4.2.1 1.4.2.2 1.4.2.2.1 1.4.2.2.2 1.4.3 1.4.3.1 1.4.3.2 1.4.3.2.1 1.4.3.2.2 1.4.3.2.3 1.4.3.2.4 1.4.3.2.5 1.4.4 1.4.4.1 1.4.4.2 1.4.4.3 1.4.4.3.1 1.4.5 1.4.5.1 1.4.5.2 1.4.5.3 1.4.5.3.1 1.4.6 1.4.6.1 1.4.6.2 1.4.6.2.1 1.4.6.2.2

Results. Enzyme screening Assay techniques Amylase Protease L-asparaginase L-glutaminase Cellulase Xylanase DNase (Deoxyribonuclease) RNase (Ribonuclease) Esterase Nitrilase Peroxidase Results Screening for special enzyme properties L-asparaginase Reaction conditions Results Asp-01 (L-asparaginase producer) Asp-02 (L-asparaginase producer) Asp-03 (L-asparaginase producer) Asp-04 (L-asparaginase producer) Asp-05 (L-asparaginase producer) L-glutaminase Reaction conditions Results Glut-01 (L-glutaminase producer) Glut-02 (L-glutaminase producer) Protease Reaction conditions Results Prot-101 (protease producer) Prot-102 (protease producer) Prot-103 (protease producer) Prot-104 (protease producer) Prot-105 (protease producer) Cellulase Bioreactor specifications Reaction conditions Result Cel-2/03 (Cellulase producer) Xylanase Bioreactor specifications Reaction conditions Result Microorganism 2/47 (xylanase producer) DNase Reaction conditions Results Microorganism 2/01 (DNase producer) Microorganism 2/22 (DNase producer)

1.4.7 1.4.7.1 1.4.7.2 1.4.7.2.1 1.4.7.2.2 1.4.7.2.3 1.4.7.2.4 1.4.7.2.5 1.4.7.2.6 1.4.7.2.7 1.4.7.2.8 1.4.7.2.9 1.4.8 1.4.8.1 1.4.8.2 1.4.8.2.1 1.4.8.2.2 1.4.8.2.3 1.4.8.2.4 1.4.8.2.5 1.4.9 1.4.9.1 1.4.9.2 1.4.9.2.1 1.4.9.2.2 1.4.9.2.3 1.4.9.2.4 1.4.9.2.5 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 3 3.1 3.1.1 3.1.1.1 3.1.1.2 3.1.1.3 3.1.2 3.1.2.1 3.1.2.2 3.1.2.3 3.1.3

RNase. Reaction conditions Results R-01 (RNase producer) R-02 (RNase producer) R-03 (RNase producer) R-04 (RNase producer) R-05 (RNase producer) R-06 (RNase producer) R-07 (RNase producer) R-08 (RNase producer) R-09 (RNase producer) Esterase Reaction conditions Results Es-01 (esterase producer) Es-02 (esterase producer) Es-03 (esterase producer) Es-04 (esterase producer) Es-05 (esterase producer) Nitrilase Reaction conditions Results N-01 ( nitrilase producer) N-02 ( nitrilase producer) N-03 ( nitrilase producer) N-04 ( nitrilase producer) N-05 ( nitrilase producer) Bioprospecting for antibiotics Screening Medium design for optimum antibiotic activity Media standardization and day wise study Results for media standardization Antifoam activity Results for antifoam activity Extraction of antibiotic into organic solvent Culture preparation Extraction procedure Test culture preparation Cup-plate method Results Bioprocessing Bioprocessing of enzymes Bioreactor Run No. 1 Operating conditions Offline analysis Result Bioreactor Run No. 2 Operating conditions Offline analysis Results Bioreactor Run No. 3

2.3 3.2.2 3.1.3.3 3. Scientists from all over the world have kept themselves engaged in this arena.2.4.1 Sampling .2.1.3 3. 4 Operating conditions Off line analysis Result 1 Bioprospecting for enzyme Bioprospecting for enzymes from the marine source has always been a challenge for any marine biologist. But of course it goes without saying that choosing a proper sampling site is very critical and the outcome of the entire project depends on this first step.4 3.6.2.2.2.2 3.4.2.2.1 3. 3 Operating conditions Off line analysis Result Bioreactor Run No.4.4.1.1.3.1.3 3.1.1 3.1 3. 4 Operating conditions Offline analysis Results Bioreactor Run No.3 3.2 3.2.1.2 3.1.1.1.1.1. This was more difficult in our case as absolutely no published data exists on the occurrence of microorganisms in the Sundarbans region.1.2.3. 1.1. 5 Operating conditions Offline analysis Results Bioreactor Run No.2.2 3.1 3.3 3.2 3.3.2 3.1 3.1 3.2.5.3 Operating conditions Offline analysis Results Bioreactor Run No.6.3 3.1.5. 6 Operating conditions Offline analysis Results Bioprocessing of antibiotics Bioreactor Run No.4.6.6 3. 2 Operating conditions Off line analysis Result Bioreactor Run No.2.2 3.2 3.5.2.3. 1 Operating conditions Off line analysis Result Bioreactor Run No.3.2 3.1 3.2.1.1 3.3 3.5 3.4.1 3.1.4 3.2.2.1.2.3.

The whole Sundarbans area is inundated twice daily by high tidal water Hooghly-Matla . monsoon period and the post monsoon period. For this survey the post monsoon period (winter season in India) was chosen considering the higher water salinity than the other seasons. that this work is the first ever biotechnological survey in the deltaic Sundarbans area of Bay of Bengal. West Bengal is a maritime state in the north eastern part of the country adjacent to Bangladesh and is indented at its southern parts by numerous river openings.1. But on the way encompass about 54 islands crisscrossed and intersected by various creeks and delta distributaries (Figure 01). The important rivers from east to west are Harin-Bhanga. The sampling has been done in the areas which are devoid of human inhabitation to avoid the occurrence of the human fecal coliform bacteria. western and southern coasts of peninsular India are repleted by numerous river openings and highly productive estuarine areas. which is intersected from north to south by several wide channeled rivers and sluggish. Sampling can be done in three times a year. The eastern. The best times for soil and water sampling are the full moon days as the low tide exposes large offshore sampling areas and many new creeks formed during the monsoon periods become surfaced during that period facilitate to penetrate them for inland surveys with small boats. the world’s largest tidal mangrove forest located in the alluvial flats of the Brahmaputra. Matla. In consecutive four fieldwork the first two field surveys were done in the Lothian Island and Sajnakhali surroundings had been focused in the last two fields. The geographic location of the study area lies in the brackish water complex of deltaic Sundarbans of West Bengal. Saptamukhi. 1. Deltaic Sundarbans. Meghna and the Gangetic delta on the Bay of Bengal.As we mentioned above.pre monsoon period. weather as well as the lesser chances of poisonous snake attacks in the deltaic Sundarbans. Thakuran. winding creeks interspersed with lagoons. Gosaba. Mooriganga and Hooghly which ultimately end up at the Bay of Bengal. two different locations had been targeted for the survey.1 Field survey The Indian subcontinent lies entirely to the north of equator with the Tropic of Cancer cutting the country roughly into two equal halves.

km has got the status of ‘Sundarbans National Park’ since 1984 and ‘World Heritage Site’ as designated by the International Union for Conservation of Nature and Natural Resources (IUCN) in the year 1989 for unique biodiversity of both flora and fauna as well as characteristic adaptability of species like mangroves. fishing cat. km. the Indian part was partitioned from the Bangladesh part on December 18. The area of about 1255 sq. Considering the existence of unique flora and fauna (except tiger) of the Sundarbans-‘The Lothian Island’ has been declared as Wildlife Sanctuaries in the year 1948 and renotified through Notification No. 2002). (i) pre-monsoon (March–June with intermittent rain and prevalence of higher ambient temperature (ii) monsoon (July–October with heavy rain fall) and (iii) post-monsoon (November–February. This Island covers an area of 38 sq. Expanded over two countries. the Royal Bengal tiger. The air temperature of Sundarbans area varies from 190C to 340C and velocity of wind from 0. chitals. (Ghosh. This tropical eco-system is well recognized by three seasons viz.s -1. after then it changes its direction and commences to blow from south east which gradually subsides in September. km and the rest area is under the jurisdiction of South 24 Parganas Division. 1947 with an area of approximately 4264 sq. sea turtles.estuarine complex. the ground level is low. In the southern part of the island the ground is high. km. The wind blows from north and north–west from end of October to the mid of March. The estuary is a well mixed type and has a funnel shaped mouth. while in the northern part. In the first two years Lothian Island was targeted. In the Project Tiger area of ‘Sundarban Tiger Reserve’ the core area of 1331. km has been designated as ‘Buffer Zone’ (Singh.54 m. 1976 as it is endowed with rich biodiversity like mangroves. chital. dated June 24. snakes and birds. 2001) (Figure 02) . It experiences bidiurnal types with a period of the order of 12 h 40 min. which is accompanied by almost no rain and low temperature). fishing cats. dolphins. Sampling had been done in the Buffer Zones in two wild life sanctuaries. estuarine crocodile. sea turtle and birds.85 to 4. dolphin.12 sq. Since ‘Project Tiger’ was initiated with an area of 2585 sq. India and Bangladesh. estuarine crocodiles. estuaries. It is situated at the confluence of Saptamukhi River and Bay of Bengal. 5392-For.

2 Sampling techniques The parameters to be recorded include water temperature. fishing cat. km.1. Similarly sterile plastic bottles (500 ml) were used to contain the sea water collected and . refractometer and conductivity meter. Saptamukhi river in the east and Bay of Bengal in the south with a length of 25 km in the north-south direction and maximum width around 14 km. sea turtle and birds are the pinnacle of both terrestrial and the aquatic web (Figure 04) 1. The southern part of the island faces the open sea-Bay of Bengal. (Bhattacharyya.5 m above the sea level. The station Fraserganj lies in the southern tip of Namkhana delta lobe. After collection temperature. This island is partially reclaimed and crisscrossed by numerous large and small creeks bordered reach mangrove vegetation. soil and water samples from Fraserganj and Sagar Island were also collected. the largest delta in the Hooghly-Matla estuarine complex is surrounded by large water bodies. chital. The island covers an area of about 235 sq. (Figure 02) Sagar Island. estuaries.4 sq. which is surrounded by Hatanya-Doanya canal in the north. the river Hooghly in the north and north-western side and the Mooriganga in the eastern side with a length of 30 km in the north-south direction and maximum width of around 12 km. and salinity were measured of that area of collection. estuarine crocodile. Sediment samples were collected by sterile spatula or by a corer. Mooriganga river in the west. the cap was sealed with Parafilm. pH meter. Sediment Samples were collected in sterile glass tubes having plastic caps and after collection. pH. water pH and salinity which were recorded by thermometer. 2002) (Figure 03) The third and fourth field surveys were done in the ‘Sajnakhali Wildlife Sanctuary’ which belongs to the ‘Sundarban Tiger Reserve’ forest and has an area of 362. km Mangroves.During the first field. swampy islands. dolphin. tiger. The island is partially reclaimed and crisscrossed by 12 large and small tidal creeks strewn with mangrove vegetation all connected with the principal estuarine water bodies either on the East or on the West coast. Water sample was also collected by Nansen Water Sampler. This is a tide dominated deltaic island and it is only 6.

1 1.1.3 Sampling data The following tables represent the sampling data of the four field surveys. . Field survey. swampy islands so as to prevent damage to the fragile biodiversity of the largest tidal mangrove ecosystem of the world. India Figure 02: Map of Lothian and Sagar island Figure 03: Sundarban Tiger Reserve Forest. 3rd and 4th field.1 Bioprospecting for enzyme Sampling.after collection. The collected samples were brought to Kolkata under refrigeration and were preserved at -200C. 06) surveys were conducted in the Sajnakhali regions where ten samples were collected in both cases. 03) and another seven samples were collected in the second field (Table No. 1. The entire scheme of work has been classified under the following headings. the cap was sealed with Parafilm. Sajnakhali Table No.1 1. In the first field seven samples were collected (Table No. 04: Field data of second field Table No. Necessary precautions were taken during sampling from the estuaries. Sajnakhali Figure 04: Survey area.1.04). Figure 01: Location of Sundarbans in West Bengal. 05: Field data of third field Table No. 05) and the fourth field (Table No. The third (Table No. 06: Field data of fourth field SCHEME OF WORK Bioprospecting for commercial bioactive compounds needs extensive sampling which is followed by a thorough and methodical screening program. 03 : Field data of first field Table No.

3 1.4.2.4 1.1.3.4.4.4.4.6 1.3.5 1.2 1.4.1.1.4.4.2.3.4 1.1 1.3.4 1.4.3 1.2.4. Results.2.2.2 1.1.4 1.4.2 1.3.1.3.1 1.4.5.1.3. Primary stock cultures preparation.1 1.1 1.4 1. Enzyme screening Assay techniques Amylase Protease L-asparaginase L-glutaminase Cellulase Xylanase DNase (Deoxyribonuclease) RNase (Ribonuclease) Esterase Nitrilase Peroxidase Results Screening for special enzyme properties L-asparaginase Reaction conditions Results Asp-01 (L-asparaginase producer) Asp-02 (L-asparaginase producer) Asp-03 (L-asparaginase producer) Asp-04 (L-asparaginase producer) Asp-05 (L-asparaginase producer) L-glutaminase Reaction conditions Results Glut-01 (L-glutaminase producer) Glut-02 (L-glutaminase producer) Protease Reaction conditions Results Prot-101 (protease producer) Prot-102 (protease producer) Prot-103 (protease producer) Prot-104 (protease producer) Prot-105 (protease producer) Cellulase Bioreactor specifications Reaction conditions Result Cel-2/03 (Cellulase producer) Xylanase Bioreactor specifications Reaction conditions .2.5 1.1.4.2.1 1.2.2 Sampling techniques Sampling data.2.4.3.3.3.4.1.8 1.4 1.1.2.3.2 1.1.2 1.3.1 1.1.3 1.2 1.3.4.5 1.2 1.2.3.1 1.1 1.3.4.2.4.1 1.1.2.1 1.3 1.5 1.4.4.4.2.4.1.2.4. Identifications of true marine microorganisms.4.5 1.1.3.2 1.1 1.2 1.1.7 1.5.2.2 1.2.11 1.3.4.1.2 1.1.2.3.1.3 1.1.4.4.4.3 1.2 1.1 1.3.1 1.4.10 1.4.4.1.3 1.2.1.3.2.9 1. Isolation of “true” marine microorganisms Medium preparation Sample preparation and spreading.2.3.3 1.4.4.2 1.

1.3 1.7.5.9.4.4.1 1.5 3 3.8 1.4.9.1 1.2.2.2 1.1 1.3 1.9.2.3.7.9.2 2.6 1.1.6 1.4.1 1.9.2.4.5 2 2.6.4.4.4.3.1 Result Microorganism 2/47 (xylanase producer) DNase Reaction conditions Results Microorganism 2/01 (DNase producer) Microorganism 2/22 (DNase producer) RNase.2.4.8.2.2 1.4.3.4.4.4.3.4.4.8 1.4 1.2.6.5 1.4.4.1 1.1 3.2.5 1.5.4.6.4.7.4.2. 1 Operating conditions .4.4 2.2 1.4.7 1.7.2.1 3.3.2.1.2 1.1 1.1 1.2 1.3 1.8.7.4.3.2.2.9.2.8.1.7.2.2 1.6.2.2 1.4.2.4.4 2.1 1.8.3 1.2 2.4 1.2 2.2.9.7.8.4.4.4.2.2.2.3 2.1 2.4.2 1.1 2.1 1.9 1.4.3 2.9 1.4.7.2.3 2.2.4.4.2.1 2.7.7. Reaction conditions Results R-01 (RNase producer) R-02 (RNase producer) R-03 (RNase producer) R-04 (RNase producer) R-05 (RNase producer) R-06 (RNase producer) R-07 (RNase producer) R-08 (RNase producer) R-09 (RNase producer) Esterase Reaction conditions Results Es-01 (esterase producer) Es-02 (esterase producer) Es-03 (esterase producer) Es-04 (esterase producer) Es-05 (esterase producer) Nitrilase Reaction conditions Results N-01 ( nitrilase producer) N-02 ( nitrilase producer) N-03 ( nitrilase producer) N-04 ( nitrilase producer) N-05 ( nitrilase producer) Bioprospecting for antibiotics Screening Medium design for optimum antibiotic activity Media standardization and day wise study Results for media standardization Antifoam activity Results for antifoam activity Extraction of antibiotic into organic solvent Culture preparation Extraction procedure Test culture preparation Cup-plate method Results Bioprocessing Bioprocessing of enzymes Bioreactor Run No.4 1.4.2.8.7 1.8.4.7.

1.3 3.2. 6 Operating conditions Offline analysis Results Bioprocessing of antibiotics Bioreactor Run No. 3 Operating conditions Off line analysis Result Bioreactor Run No.6 3.4.3 3.1.1.1.2 3. 2 Operating conditions Offline analysis Results Bioreactor Run No.1 3.2. 5 Operating conditions Offline analysis Results Bioreactor Run No.2.2.4.1. 4 Operating conditions Offline analysis Results Bioreactor Run No.1.3.5.1.2. 2 Operating conditions Off line analysis Result Bioreactor Run No.2.1.2.1.1.3.5.3 3.1.1.1.2 3.4.5 3.2.1.1.1.1 3.2 3.1.2 3.1.1.2. This was more .2 3.2.2 3.5.4.1.1 3. 1 Operating conditions Off line analysis Result Bioreactor Run No.2.3 3.2 3.1.3 3.3 3.2 3.1 3.2 3. 4 Operating conditions Off line analysis Result 1 Bioprospecting for enzyme Bioprospecting for enzymes from the marine source has always been a challenge for any marine biologist.1. But of course it goes without saying that choosing a proper sampling site is very critical and the outcome of the entire project depends on this first step.3.1 3.3 3.4 3.1.1 3. Scientists from all over the world have kept themselves engaged in this arena.2.2 3.4.3.2.1.3 3.3.3.2 3.1 3.1 3.2.3 Offline analysis Result Bioreactor Run No.6.2.2 3.3 3.4.2.2.3.3 3.3 3.2 3.2.1.6.1 3.1 3.2. 3 Operating conditions Offline analysis Results Bioreactor Run No.4 3.1.2.2.2.6.1.

1.pre monsoon period. The important rivers from east to west are Harin-Bhanga. The best times for soil and water sampling are the full moon days as the low tide exposes large offshore sampling areas and many new creeks formed during the monsoon periods become surfaced during that period facilitate to penetrate them for inland surveys with small boats. western and southern coasts of peninsular India are repleted by numerous river openings and highly productive estuarine areas.1 Sampling As we mentioned above. 1. Thakuran. But on the way encompass about 54 islands crisscrossed and intersected by various creeks and delta distributaries (Figure 01). Mooriganga and Hooghly which ultimately end up at the Bay of Bengal.1 Field survey The Indian subcontinent lies entirely to the north of equator with the Tropic of Cancer cutting the country roughly into two equal halves. Matla.difficult in our case as absolutely no published data exists on the occurrence of microorganisms in the Sundarbans region. The eastern. Gosaba. For this survey the post monsoon period (winter season in India) was chosen considering the higher water salinity than the other seasons. weather as well as the lesser chances of poisonous snake attacks in the deltaic Sundarbans. Sampling can be done in three times a year. monsoon period and the post monsoon period. In consecutive four fieldwork the first two field surveys were done in the Lothian Island and Sajnakhali surroundings had been focused in the last two fields. The sampling has been done in the areas which are devoid of human inhabitation to avoid the occurrence of the human fecal coliform bacteria. The geographic location of the study area lies in the brackish water complex of . West Bengal is a maritime state in the north eastern part of the country adjacent to Bangladesh and is indented at its southern parts by numerous river openings. Saptamukhi. that this work is the first ever biotechnological survey in the deltaic Sundarbans area of Bay of Bengal. 1. two different locations had been targeted for the survey.

which is accompanied by almost no rain and low temperature). The estuary is a well mixed type and has a funnel shaped mouth. Considering the existence of unique flora and fauna (except tiger) of the Sundarbans-‘The Lothian Island’ has been declared as Wildlife Sanctuaries in the year 1948 and renotified through Notification No. km and the rest area is under the jurisdiction of South 24 Parganas Division. after then it changes its direction and commences to blow from south east which gradually subsides in September. the world’s largest tidal mangrove forest located in the alluvial flats of the Brahmaputra. The wind blows from north and north–west from end of October to the mid of March. estuarine crocodile. sea turtle and birds. Deltaic Sundarbans. fishing cat. It experiences bidiurnal types with a period of the order of 12 h 40 min. winding creeks interspersed with lagoons. The whole Sundarbans area is inundated twice daily by high tidal water Hooghly-Matla estuarine complex. In the first two years Lothian Island was targeted.s -1. The area of about 1255 sq. the Royal Bengal tiger. km has been designated as ‘Buffer Zone’ (Singh. chital. Meghna and the Gangetic delta on the Bay of Bengal. which is intersected from north to south by several wide channeled rivers and sluggish.85 to 4. dolphin. 1947 with an area of approximately 4264 sq. India and Bangladesh. . This tropical eco-system is well recognized by three seasons viz. (i) pre-monsoon (March–June with intermittent rain and prevalence of higher ambient temperature (ii) monsoon (July–October with heavy rain fall) and (iii) post-monsoon (November–February.54 m. Since ‘Project Tiger’ was initiated with an area of 2585 sq. km has got the status of ‘Sundarbans National Park’ since 1984 and ‘World Heritage Site’ as designated by the International Union for Conservation of Nature and Natural Resources (IUCN) in the year 1989 for unique biodiversity of both flora and fauna as well as characteristic adaptability of species like mangroves.12 sq. 5392-For. the Indian part was partitioned from the Bangladesh part on December 18. In the Project Tiger area of ‘Sundarban Tiger Reserve’ the core area of 1331.deltaic Sundarbans of West Bengal. 2002). The air temperature of Sundarbans area varies from 190C to 340C and velocity of wind from 0. km. Sampling had been done in the Buffer Zones in two wild life sanctuaries. Expanded over two countries.

the ground level is low. 2001) (Figure 02) During the first field. (Figure 02) Sagar Island. the largest delta in the Hooghly-Matla estuarine complex is surrounded by large water bodies. This Island covers an area of 38 sq. chital. snakes and birds. fishing cat. The southern part of the island faces the open sea-Bay of Bengal. This is a tide dominated deltaic island and it is only 6. chitals. water pH and salinity which were recorded by thermometer. while in the northern part. which is surrounded by Hatanya-Doanya canal in the north. the river Hooghly in the north and north-western side and the Mooriganga in the eastern side with a length of 30 km in the north-south direction and maximum width of around 12 km. swampy islands. sea turtles. Sediment samples were collected by sterile spatula or by a corer. estuaries.5 m above the sea level. dolphins. In the southern part of the island the ground is high. estuarine crocodile. Mooriganga river in the west. tiger. Saptamukhi river in the east and Bay of Bengal in the south with a length of 25 km in the north-south direction and maximum width around 14 km.4 sq. dolphin. soil and water samples from Fraserganj and Sagar Island were also collected. (Bhattacharyya.2 Sampling techniques The parameters to be recorded include water temperature. 2002) (Figure 03) The third and fourth field surveys were done in the ‘Sajnakhali Wildlife Sanctuary’ which belongs to the ‘Sundarban Tiger Reserve’ forest and has an area of 362. (Ghosh. This island is partially reclaimed and crisscrossed by numerous large and small creeks bordered reach mangrove vegetation. estuaries. km.dated June 24. The island covers an area of about 235 sq.1. estuarine crocodiles. sea turtle and birds are the pinnacle of both terrestrial and the aquatic web (Figure 04) 1. km Mangroves. refractometer and conductivity meter. The island is partially reclaimed and crisscrossed by 12 large and small tidal creeks strewn with mangrove vegetation all connected with the principal estuarine water bodies either on the East or on the West coast. It is situated at the confluence of Saptamukhi River and Bay of Bengal. pH meter. The station Fraserganj lies in the southern tip of Namkhana delta lobe. Water sample was also . km. fishing cats. 1976 as it is endowed with rich biodiversity like mangroves.

04).1. Sediment Samples were collected in sterile glass tubes having plastic caps and after collection. and salinity were measured of that area of collection. Tech.collected by Nansen Water Sampler.3 Sampling data The following tables represent the sampling data of the four field surveys. 06) surveys were conducted in the Sajnakhali regions where ten samples were collected in both cases. 1.2 Isolation of “true” marine microorganisms As the aim of this project was to study enzymes from marine microorganisms. Similarly sterile plastic bottles (500 ml) were used to contain the sea water collected and after collection. Data are given in the M. pH. In the first field seven samples were collected (Table No. 05) and the fourth field (Table No. 1974) and their isolation was the key of this investigation. the cap was sealed with Parafilm. (Biotechnology) dissertation work . After collection temperature. 1. Necessary precautions were taken during sampling from the estuaries.2. Scientific evidence suggests that terrestrial microorganisms may be “washed off” to the coastal regions. the isolation of true marine microorganisms was obvious. swampy islands so as to prevent damage to the fragile biodiversity of the largest tidal mangrove ecosystem of the world. The collected samples were brought to Kolkata under refrigeration and were preserved at -200C. The third (Table No.1 Medium preparation Experiments were done to standardize different growth media with the samples collected in the first field. Hence to obtain “true” marine microorganisms [defined as those which have an obligatory requirement of 3% (w/v) sodium chloride (NaCl) for growth] (Reichelt & Baumann. 1. the cap was sealed with Parafilm. 03) and another seven samples were collected in the second field (Table No.

conical flask.8 g CaCl2.0 g sucrose. 1. 5.0024 g NaF. . 2001) done by the author.2. 0.2 Sample preparation and spreading Samples were taken out of –200C freezer and kept at 40C for 1-2 h.0 g peptone. pH 8.24 g Na2SO4. 0. 15. 15.s.0 g peeled & mashed potato. pH 7.034 g SrCl2.0 g agar agar.022 g H3BO3.004 g Na2SiO3. 8.0 g agar agar. 10. 1.55 g KCl.0 g K2HPO4..50) were selected for the growth of marine bacteria and fungi.0 g yeast extract.s. 0. 1000 ml single distilled water q.8 g MgCl2. 0. 19. A definite amount of soil sample was taken out with a sterile spatula and mixed with 25 ml of sterile natural seawater in a 100 ml..0016 g NH4NO3. ↓ Microorganisms in the water phase ↓ Various dilutions were made (1:1000.1 g FeCl3. From different 13 media only one bacterial growth medium Marine Difco 2216 medium (5. 0. 0. 0. Then they were kept at room temperature (RT) for 12 h to avoid the heat shock. 3. 100.0 g peptone. 0. 1. The following flow-chart shows the procedure of isolating microorganisms from soil and water samples.2) and one fungal F-III medium (20.6H2O.45 g NaCl. 0.08 g KBr. 1000 ml natural sea water q.008 g Na2HPO4. It was stirred and allowed to settle.(Ghosh. 1:10000 and 1:1000000) with serial dilution method ↓ 200 µ l of each dilution was plated in marine bacterial and fungal media ↓ The plates were incubated in 300C for 4 to 5 days and in cases of the fungal plates they were kept at 230C for 7 days Same procedures were followed in case of water samples but they were spread without dilution.16 g NaHCO3. 0.

Eight different colonies were streaked in one single Petri plate of desired growth medium by dividing the plates into eight equal quadrants in order to reduce the number of Petri dishes. The selected microorganisms were streaked onto solid Difco agar medium (one organism in one plate) in order to make final and pure working cultures and at the same time glycerol stock [850 µL culture broth (microorganisms grown individually in liquid Difco medium) and 150 µL sterile glycerol..0 g yeast extract.0 g agar agar. They were then picked and streaked again in the Marine Difco or F-III plates (Marine Difco for bacteria and F-III for fungi) with sterile tooth picks keeping in mind to get single colonies. vortexed] were prepared for the final selected bacteria for long term preservation and preserved in -700C freezer. 1000 ml single distilled water q. this test was limited to bacteria only.2.2. 15.s. 1. After two days only those microorganisms were selected which did not grow in the LB agar plates as it was considered that requirement of NaCl was obligatory for the growth of “true” marine microorganisms.1.2.5 Results . The entire procedure was repeated until a certain number of true marine microorganisms were found to carry on the further experiments.2) were taken. pH 7. Each plate was divided into 50 squares and the selected colonies were picked onto each square. For this test LB (Luria-Bertani) agar medium without NaCl (5. 10. As the population of fungi was less compared to bacteria. The plates were kept in the incubator at 300C for two days.4 Identifications of true marine microorganisms After preparing the primary stock cultures the first phase of the work was to check whether the microorganisms were “true” marine or terrestrial microorganisms adapted to the marine environment. 1.0 g peptone.3 Primary stock cultures preparation After seven days all the plates were taken out and all the colonies grown in the plates were marked and numbered.

DNase. Results suggest that very close geographical location can yield varying numbers of “true” marine bacteria. 2001). 10. Tech.e.50% 31.00% 36. Amylase.Table No.00% 1st 2nd 3rd 4th Total Lothian Lothian Sajnakhali Sajnakhali 1. In the second stage of screening spectrophotometric enzyme assay technique was applied.0 g L-asparagine for L-asparaginase producers. thesis (Ghosh .0 g starch for amylase producers.0 g casein for protease producers. nitrilase and peroxidases. L-glutaminase and cellulase a few other commercially important biocatalysts had been targeted for screening and they were xylanase.0 g carboxymethyl cellulose (CMC) for cellulase producers in 1000 ml artificially prepared sea water) to grow in his M. The author has done a preliminary survey of these enzyme producers by observing the growth of microorganisms in solid media in Petri plates by providing the microorganisms a single carbon source (i. protease. 10. 07 shows the numbers of “true” marine bacteria isolated from the four field surveys. L-asparaginase.00% 60. 10. RNase. esterase. 10. Table No.00% 14. L-glutaminase and cellulase producers were searched from the collection of “true” marine microorganisms. The fourth field (Sajnakhali) produced the maximum ratio of “true” marines whereas the third field (Sajnakhali) produced the lowest ratio of “true” marine bacteria. Along with protease. 07 : Isolated numbers of true marine microorganisms in four field surveys Field Locations Total number of microbial colonies isolated 400 200 200 250 1050 True marine microorganisms 138 62 28 150 378 Percentage of true marine microorganisms 34. With the exception of . 10. L- asparaginase.0 g L-glutamine for L-glutaminase producers.3 Enzyme screening Five different enzyme producing marine microorganisms were screened from the true marine microorganisms isolated from the first field sediment and water samples.

) at λ 600 . so the screening work in the laboratory bench was never been an easy task.D. salt tolerance and activity in a wide range of pH.5 g . Since marine microorganisms behave differently than terrestrial microorganisms due to a growth environment. the amylase production medium (5.1 Amylase Considering the fact that presence of starch stimulates the amylase production. In the first phase the different production media as well as the assay procedures were standardized. Optical density (O. 0. high salt tolerance and wide range of pH activity.peroxidases producers all the enzyme producers were found successfully. Merck (Germany). Since the objective of this work was to search for the marine enzymes with some special features such as thermostability. So culture management was a very important job. 0.3. Retaining the enzyme production levels of the microorganisms of the environmental samples is not in the hand of the researchers. For best results the assays were performed in duplicate sets and special care was taken for the control samples to correlate them with the experimental samples. as an indicator of growth was recorded for each samples before every assay as only after a proper growth of microorganisms in fermentation broth can lead to a good enzyme production. 1.3.02 g FeCl2. so a general estimation of the enzyme production of the microorganisms were done primarily. India). The details of the primary screening work of all enzymes are described hereafter. 1. SD Fine Chem (India) and Sigma Aldrich (USA). All the chemicals and reagents were bought from SRL India (Mumbai. 5. Then the enzymes were assayed. Finally few of them were chosen for the presence of special properties such as thermostability. Next the entire process was repeated to confirm the results of the first phase assay.0 g peptone. The enzymes assayed in these cases were all extracellular enzymes.1.1 Assay techniques Enzymes from marine source are always a big challenge for the marine biotechnologists.0 g yeast extract. This was achieved in two consecutive phases.

] 2. Beckman Instrument. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium.0 g of Na-K-tartrate is dissolved in 30 ml of distilled water. The entire solution was cooked for 5 min at 1000C boiling water bath.4 ml 0.1 M sodium solution were prepared and mixed.. The reaction was carried out at 40°C for 30 min and finally stopped by adding 1 ml freshly prepared DNS reagent. 1% w/v starch in 0.f.57. pH 7. USA) 5. 1000 ml natural sea water q.0 g starch. pH 7.0) was designed.4). The solutions or phosphoric acid dihydrogen phosphate (NaH2PO4) exact pH was achieved by adding the either simultaneously in minute amounts. Colour intensity was measured after proper dilution in spectrophotometer at λ 540 .c.5 ml culture supernatant (1. Volume was made up by adding 0. .1 M phosphate buffer.s.5dinitrosalicylic acid (DNS) reagent at λ 540 in Spectrophotometer.1 M of disodium hydrogen phosphate (Na2HPO4) and 22. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Amylase activity was assayed in a reaction mixture (1 ml) of 0. Control: Media filtrate without the substrate was taken as blank. 0. [Preparation: 1. pH 6. Along with it the reducing sugar transforms into oxidized form. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml amylase production medium.6 ml of 0.0 g DNS was mixed in 20 ml of 2 N NaOH solution and 30. for 5 min at 40C to separate supernatant and cells) and 0. Spectrophotometer (DU-6. Reagents and Instruments: 1. 0. DNS reacts with reducing sugar and in turn red coloured 3-amino-5-nitrosalicylate is produced which is measured at λ 540 .1 M phosphate buffer.4 [Preparation: 77.1 M phosphate buffer [Preparation: 1.1M phosphate buffer only. Enzyme activity was assayed in terms of the color intensity of 3. 5.003 g (NH4)2SO4.0 g soluble starch was dissolved in the above mentioned buffer and used as substrate. DNS solution.] 3.MgSO4.2H2O.5 ml culture centrifuged at 10 × 103 r. Both the solutions were mixed and the volume was adjusted to 100 ml] 4.5 ml substrate (1% w/v starch in 0.

Centrifuge (Eppendorf Centrifuge 5415 D). After a part of azocasein was dissolved it was kept in refrigerator at 40C for 30 min.1.] 2.Precaution: During heating in water bath care was taken to avoid the entry of water drops or vapor into the reaction tubes. 5. Reagents and Instruments: 1. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium.2 Protease Assay of protease could be done in many ways. which may cause the dilution of the reaction mixture and give faulty readings. 10% tri-chloro acetic acid (TCA) solution [Preparation: 10. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the production medium.3. which might lead to degradation of azocasein] 3.1 M Tris-HCl (pH 8. Enzyme assay: Reaction mixture was prepared with 100 µl culture supernatant (1. 0. Spectrophotometer (Amersham Pharmacia Biotech.1 M Hydrochloric acid (HCl).0 g of TCA was dissolved in distilled water and kept at 40C before use.5) [Preparation: 0.5 ml . 1. Ultrospec 1100 pro). But azoprotein substrates would be the best choice for specific as well as accurate enzymatic reaction.1 M of Tris-HCl solution was prepared in water and the desired pH was adjusted as required with 0. It should be freshly prepared before use and should not be shaken vigorously during preparation. The assay protocol was followed here was quite same as done by Sarath (1989) but with a change in the azocasein concentration.] 4. Azocasein (250 µg/ml solution): Substrate [Preparation: In accurately weighed azocasein half of the required amount of water was mixed in a container and it was dissolved by very slow and gentle shaking. Next the solution was taken out and rest half of water was mixed and remaining undissolved azocasein was dissolved with slow shaking of the container and kept at 40C for few min before use.

Spectrophotometer (Amersham Pharmacia Biotech. 0. Ultrospec 1100 pro).1 M Tris-HCl and 225 µL distilled water. Finally the O. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Volume was made up by adding 0. Enzyme assay: 200 µL culture supernatant (1. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. 15% TCA solution [Preparation: 15.0 g of TCA was dissolved in distilled water and kept at 40C before use].1 M Na-Borate buffer (pH 8. for 5 min at 40C. decahydrate) in water is prepared and the pH was adjusted to desired value with 0. 6.5 ml culture centrifuged at 10 × 103 r. was measured with required dilution at λ 366 .f.1 M Tris-HCl buffer only. Nessler’s reagent [SRL India] 5.f. Centrifuge (Eppendorf Centrifuge 5415 D). TCA was precipitated by centrifugation at 10 × 103 r. 2. 0.1. 3.04 M L-asparagine solution (substrate) [Preparation: L-asparagine crystals were accurately weighed and dissolved in distilled water]. Reagents and Instruments: 1.3 L-asparaginase L-asparaginase activity was assayed in terms of ammonia formed as end product of enzymatic reaction.culture centrifuged at 10 × 103 r. American Water Works Association (AWWA) and Water Pollution Control Facilities (WPCF) 1992].3.D.1 M boric acid or 0. .c.1 M sodium hydroxide solution]. Precaution: Azocasein being a very delicate and light substrate should be shaken gently.65) [Preparation: 0.c. 4. 50 µL 0. Control: Media filtrate without the substrate was taken as blank. for 5 min at 40C to separate supernatant and cells).c. 125 µL azocasein solution.f. The entire mixture was cooked for 30 min at 370C and then the assay was terminated by 500 µL ice cold 10% TCA solution. 1.1 M solution of Borax (Sodium tetra borate. estimated spectrophotometrically by Nesslerization method [American Public Health Association (APHA).

estimated spectrophotometrically by Nesslerization method (APHA.04 M L-asparagine and 250 µL Na-Borate buffer were the ingredients of the reaction mixture.4 L-glutaminase Like L-asparaginase.1 M Na-Borate buffer only.3. 250 µL of 0. L-glutaminase activity was also assayed in terms of ammonia formed as end product of enzymatic reaction.04 M L-glutaminase solution (substrate) [Preparation: L-glutaminase powder was accurately weighed and dissolved in distilled water.c.for 5 min at 40C to separate supernatant and cells).1 M Na-Borate buffer (pH 8.65) [Preparation: mentioned earlier in page: 58] L- . To avoid this proper dilution of samples was required as well as the diluted samples were measured in the spectrophotometer before any flocculation occurs. Finally the O. Precaution: O. was measured at λ 450 . The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Reagents and Instruments: 1.f. WPCF 1992) except the substrate was glutaminase instead of L-asparaginase. AWWA. So in this assay method. A second control was prepared in which 0. for 5 min at 40C. Control: Culture filtrate without the substrate was taken as blank. The entire mixture was incubated for 10 min at 370C and then the cooking was terminated by 250 µ L of ice cold 15% TCA solution.1.D. If the TCA treated supernatant is directly added to Nessler’s reagent or even the diluted samples are left for a long time the tendency of flocculation increases.04 M L-asparagine solution was taken and it was added to medium (autoclaved medium before fermentation). each sample had its own control. Volume was made up by adding 0. 0. 0.] 2. TCA was precipitated by centrifugation at 10 × 103 r. 1. 10 µ L supernatant was transferred to another microcentrifuge tube containing 965 µ L of distilled water and 25 µ L Nessler’s reagent. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium.D. measurements should be performed before flocculation occurs.

So in this assay method. Nessler’s reagent [ SRL grade] 5. The CMC is stained red to deep pink. If the TCA treated supernatant is directly added to Nessler’s reagent or even the diluted samples are left for a long time the tendency of flocculation increases. 15% TCA solution [Preparation: mentioned earlier in page: 58] 4. The entire mixture was incubated for 10 min at 370C and then the assay was terminated by 250 µ L of ice cold 15% TCA solution.04 M L-glutaminase and 250 µL Na-Borate buffer. 250 µ L of 0. Spectrophotometer (Amersham Pharmacia Biotech. Unstained area indicates where the CMC has been broken down to β −1. a benzidine derivative that can react with cellulose. each sample had its own control. measurements should be performed before flocculation occurs. Control: Culture filtrate without the substrate was taken as blank.D.5 Cellulase Cellulase producing microorganisms were primarily screened from the microorganisms by the activity zone technique with Congo Red dye. destaining is done. 10 µ L supernatant was transferred to another microcentrifuge tube containing 965 µ L of distilled water and 25 µ L Nessler’s reagent.c.4 glucans that contained fewer glucose residues. 6. Enzyme assay: The reaction mixture was comprised of 200 µL culture supernatant (1. 1.D. To avoid this proper dilution of samples was required as well as the diluted samples were measured in the spectrophotometer before any flocculation occurs. for 5 min at 40C.5 ml culture centrifuged at 10 × 103 r.3.Congo Red is a metachromatic dye. A second control was prepared in which 0.c. TCA was precipitated by centrifugation at 10 × 103 r. Precaution: O. It was a cheap and quite . After the treatment of CMC with cellulase. Finally the O.3.1 M Na-Borate buffer only. Ultrospec 1100 pro). It is a linea r molecule allowing the azo and amine groups of the dye to form hydrogen bonds with similarly placed hydroxyl radicals. Centrifuge (Eppendorf Centrifuge 5415 D). was measured at λ 450 .f.1.04 M L-glutamine solution was taken and it was added to medium (autoclaved medium before fermentation). Volume was made up by adding 0.f. for 5 min at 40C to separate supernatant and cells).

1.08: Composition of different flasks during cellulase assay Flask No. One Petri plate was used for one microorganism.0 g peptone. The microorganisms producing zone of activity were selected as cellulase producers for further treatments.0).5 g NaCl. 1000 ml SDW q. 1000 ml SDW q.s. The process was repeated to select the microorganisms for further treatment.1% Congo Red solution and kept for 4 h.5 g NaCl.0 g beef extract..s. 24..0 g peptone. Four different types of fermentation flasks were prepared each in duplicate sets for every microorganism and numbered properly. Culture preparation: Selected microorganisms were collected from respective working culture plates and inoculated into the fermentation medium (Nutrient broth containing 3. 48. 5.0 g beef extract. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 96 h in 250 ml Erlenmeyer flasks containing 25 ml nutrient broth medium and samples were collected at 0.0 g CMC. Since the productions of cellulase were not sufficient enough to label the microorganisms as potent cellulose producers. 0. The amount of CMC added was 01% w/v. this technique had been adapted for the primary screening. Activity zone technique: Each true marine strain was picked onto Petri plates with solid cellulase production medium (3.2). Table No. A special set of experiments were designed before conducting the spectrophotometric assay which is also described here. After 4 h excess dye was washed gently with tap water and the plates were checked for presence of any zone of activity. 1 2 3 4 5 6 7 Nutrient broth √ √ √ √ √ √ √ CMC √ √ × × √ √ × Microorganism √ √ √ √ × × × Designation Test Test Control Control Control Control Control . After 7 days the colonies were flooded with 0. The plates were kept in the incubator for 7 days at 270C. 72 and 96 h and assayed spectrophotometrically with the help of DNS reagent. Below is given the detail of the flasks for one microorganism in tabular form. pH 7. pH 7. 0. 5.accurate test.

0 g oat . Colour intensity was measured after proper dilution in spectrophotometer at λ 540 and dissolved in water.f. pH was adjusted with acetic acid and . 3.1.5 ml culture centrifuged at 10 × 103 r. for 5 min at 40C to separate supernatant and cells). pH 4.1% w/v CMC dissolved in Na-acetate buffer. Ultrospec 1100 pro) 5. The entire solution was cooked for 5 min at 1000C boiling water bath.5 M Na-acetate buffer (pH 4.8 √ Control × × Reagents and Instruments: 1.] 2. which may cause the dilution of the final reaction mixture and give faulty readings at the spectrophotometer. 1.0 g beef extract. DNS solution [Preparation: mentioned earlier in page: 56] 4. 0.8).The reaction was carried out at 50°C for 60 min and finally stopped by adding 700 µ L freshly prepared DNS reagent. (Amersham Pharmacia Biotech. 400 µ L Na-acetate buffer and 200 µ L substrate (0.0 g peptone.c. 5.5 g NaCl. Activity zone technique: Each true marine strain was picked onto Petri plates with solid xylanase production medium (3. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Cellulase activity was assayed in a reaction mixture of 100 µ L culture supernatant (1. Precaution: During heating in water bath care was taken to avoid the entry of water drops or vapor into the reaction tubes. Here also a primary screening had been done using Congo Red before conducting the secondary screening as described earlier in case of cellulase producing microorganisms.3. 0.6 Xylanase Xylanase producing microorganisms were also screened from the collection of marine microorganisms by the same technique as described for cellulase.1% w/v CMC in 0.5 M Na-acetate buffer.8) [Preparation: Na-acetate was accurately weighed NaOH. 0. 1. Spectrophotometer.

Four different types of fermentation flasks were prepared each in duplicate sets for every microorganism and numbered properly. 72 and 96 h and assayed spectrophotometrically with the help of DNS reagent. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 96 h in 250 ml Erlenmeyer flasks containing 25 ml Nutrient broth medium and samples were collected at 0.09: Composition of different flasks during xylanase assay Flask No. 0. 1000 ml SDW q.1% w/v oat spelt xylan dissolved in Na-acetate buffer 3.5) [Preparation: Na-acetate was accurately weighed and dissolved in water. After 7 days the colonies were flooded with 0. 48.5 M Na-acetate buffer (pH 5. One Petri plate was used for one microorganism. Ultrospec 1100 pro) . Spectrophotometer.. Table No. Below is given the detail of the flasks for one microorganism in tabular form. 0. pH 7.spelt xylan. The microorganisms responsible for producing activity zone were selected as xylanase producers for further treatments. DNS solution [Preparation: mentioned earlier in page: 56] 4.0). The amount of xylan added was 01% w/v. 24. After 4 h excess dye was washed gently with tap water and the plates were checked for presence of any activity zone. The plates were kept in the incubator for 7 days at 27 0C.s. (Amersham Pharmacia Biotech.] 2. pH was adjusted with acetic acid and NaOH.1% Congo Red solution and kept for 4 h. Culture preparation: Selected microorganisms were collected from respective working culture plates and inoculated into the fermentation medium (Nutrient broth). 1 2 3 4 5 6 7 8 Nutrient broth √ √ √ √ √ √ √ √ Xylan √ √ × × √ √ × × Microorganism √ √ √ √ × × × × Designation Test Test Control Control Control Control Control Control Reagents and Instruments: 1.

The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. A unit causes an increase in absorbance at λ of 0.5).001 min-1. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Xylanase activity was assayed in a reaction mixture of 100 µ L culture supernatant (1.f. pH 5. pH 5.25 mM MgSO4 dissolved in double distilled water (ddH2O) 4.0 under the specified conditions.0 [Preparation: CH3COONa was accurately weighed and dissolved in water. 6. DNase I. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the subculture medium.1.c. The entire solution was cooked for 5 min at 1000C boiling water bath.1% w/v xylan in 0. preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The assay method was developed by Kunitz (1950) based upon the increased absorbance at λ 260 observed during the depolymerization of DNA by 260 DNase. splitting phosphodiester linkages.7 DNase (Deoxyribonuclease) Deoxyribonuclease from beef pancreas.5.5 M Na-acetate buffer. was an endonuclease. 1. first crystallized by Kunitz (1950). DNA (Substrate) [Preparation: 0. Nuclease free double distilled water.3. 3.The reaction was carried out at 50°C for 60 min and the reaction was terminated by adding 700 µ L freshly prepared DNS reagent. 400 µ L Na-acetate buffer and 200 µ L substrate (0. Colour intensity was measured after proper dilution in spectrophotometer at λ 540 . which may cause the dilution of the final reaction mixture and give faulty readings at the spectrophotometer. for 5 min at 40C to separate supernatant and cells).] 2.ml-1 when acting upon highly polymerized DNA at 250C and pH 5. Precaution: During heating in water bath care was taken to avoid the entry of water drops or vapor into the reaction tubes. 1.0 M acetate buffer.5 ml culture centrifuged at 10 × 103 r. pH was adjusted with acetic acid or NaOH.01 g DNA was added to 200 ml MgSO4 solution and . Reagents and Instruments: 1.

10 : Specifications of the controls used in case of DNase assay Blank No. Ultrospec 1100 pro) 7. The substrate was kept at 40C for best use for 3–4 weeks. Control: Five different blanks were set for this assay method which is given below in the following table (Table No. for 5 min at 40C to separate supernatant and cells). The first four being the universal control. they were performed only once and the fifth one being the substrate blank. 50 µ L substrate and 750 µL double distilled water. The entire mixture was incubated for 10 min in the spectrophotometer at λ 260 and change in optical density was measured at every single minute interval (from 0th min to 10th min. Spectrophotometer (Amersham Pharmacia Biotech. The culture supernatant along with the DNA and double distilled water mixed in the same ratio (4:1:15) as done for the sample run.10) Table No. Substrate control prepared in a same way as substrate expect the addition of DNA 6. it was repeated along with every test sample run.kept overnight. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: The reaction mixture was comprised of 200 µL culture supernatant (1.f.] 5. Precaution: All the microtips and the microcentrifuge tubes were doubly autoclaved in order to avoid the presence of nucleases and the water used was also nuclease free double distilled water.5 ml culture centrifuged at 10 × 103 r. 25 ml of acetate buffer was added to the solution and the volume was made upto 250 ml with the addition of ddH2O.c. 1 2 3 4 5 Blank designation Substrate (contains DNA) Substrate without DNA Medium Supernatant + Substrate (contains DNA) Medium Supernatant + Substrate without DNA Culture supernatant + Substrate without DNA Blank specification Universal blank 1 Universal blank 2 Universal blank 3 Universal blank 4 Substrate blank The changes in optical density were monitored in cases of all the five. total 11 readings for each run). The entire process was handled by wearing latex gloves to prevent .

5 ml culture centrifuged at 10 × 103 r.10 M sodium acetate buffer.0 is determined by measuring the amount of acid soluble oligonucleotide released under defined conditions. (1959) has been used here. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. 60% perchloric acid 4.] 2. The method of Kalnitsky et al. Reagents and Instruments: 1.1. One unit causes an increase in absorbance of 1.c. Since the entire assay was done inside the spectrophotometer in UV mode. 5. Zimmerman and Sanderson (1965) described a sensitive assay using polycytidylic acid. Spectrophotometer (Amersham Pharmacia Biotech. pH 5.the contamination by nuclease from hand. 3. 1% RNA dissolved in 0.0 at λ 260 at 370C and pH 5. (1960) have published an assay using a synthetic substrate. Nuclease free double distilled water.0 under the specified conditions. 3'-phosphate. the quartz cuvette had an important role as for each cuvette a certain volume of solution (generally ≥ 800 µL for 1000 µL cuvette) was required for optical density measurement.10 M Sodium acetate buffer. 0.8 RNase (Ribonuclease) Standardization of ribonuclease activity has been difficult due to varying rates at which reactions occur as well as to the significant differences in nucleotide patterns in RNA isolated from biological sources. for 5 min at 40C to separate .0 [Preparation: CH3COONa was accurately weighed and dissolved in water. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. 1. The rate of hydrolysis of RNA at pH 5. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: RNase activity was assayed in a reaction mixture of 250 µl culture supernatant (1. Ultrospec 1100 pro) 6. Hence the cuvette volume had to be considered before designing the assay method.f. pH was adjusted with acetic acid or NaOH.3. Crook et al. cytidine 2'.

Apparently most cells. pH 5.10 M sodium acetate buffer. yeasts cells. It was also assayed in the same manner and finally diluted (1:20) before taking the reading in spectrophotometer. Precaution: All the microtips and the microcentrifuge tubes were doubly autoclaved in order to avoid the presence of nucleases and the water used was also nuclease free ddH2O. 1.supernatant and cells).1 . can hydrolyze FDA or carboxy fluorescein diacetate (cFDA) into the more polar fluorescent products by intracellular esterases.c. The entire process was handled by wearing latex gloves to prevent the contamination by nuclease from hand. however.10 M sodium acetate buffer. generally. p-nitrophenyl acetate or naphthyl acetate was used as esterase substrates. whether mammalian cells.f. for 5 min at 40C and 500 µL of nuclease free double distilled water was added. Instead 200 µL 0.0.10 M sodium acetate buffer was added to make up the volume. Control: Culture filtrate without the substrate was taken as blank. Tubes were centrifuged at 10 × 103 r.20) [Preparation: 173. 200 µL substrate (1% RNA in 0. Apart from this. Reagents and Instruments: 1.9 ml of 0. pH 5. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium.1. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Samples were diluted (1:400) to measure in spectrophotometer at λ 260 . The reaction was carried out at 370C for 30 min and finally stopped by adding 250 µL ice cold 60% perchloric acid after putting the reaction tubes into ice bath.2 M Na2HPO4 and 26. McIlvaine’s buffer (pH 7. So for each sample an individual blank was prepared. equilibrated at 370C prior to assay) and 200 µL 0. or Gram-positive or Gram-negative bacteria. medium before fermentation was so treated as a separate sample for assay as universal control.3.9 Esterase The assay technique applied here was done earlier by Breeuwer et al (1995) where the substrate used was fluorescein diacetate (FDA).0.

Spectrophotometer (Amersham Pharmacia Biotech.f. different from cyanogenesis: (i) nitrilase catalyzes the direct hydrolysis of nitriles to the corresponding carboxylic acids and ammonia (Eq.] 3.3.2 and the final pH adjustment was done by addition of either of the two solutions simultaneously. RCN + 2H2O → RCOOH + NH3 RCN + H2O → RCONH2 RCONH2 + H2O → RCOOH + NH3 (Eq. Control: Medium filtrate (autoclaved medium before fermentation) instead of culture supernatant had been considered as blank. 1997). was measured with required dilution at λ 490 .] 2.1 M citric acid were mixed to prepare the buffer of pH 7.2) (Eq.1) (Eq. and then to the acids and ammonia by amidase (Eq.c. for 5 min at 40C to separate supernatant and cells) and 200 µL McIlvaine’s buffer was preincubated at 400C for a brief period of time of 3 min.5 ml microcentrifuge tube and dissolved in dimethyl sulfoxide. and (ii) nitriles are catabolized in two stages-they are first converted to the corresponding amides by nitrile hydratase (Eq. for 5 min at 40C and the O.5 ml culture centrifuged at 10 × 103 r.D. Nitrilase activity was assayed according to the methods described by Kobayashi et al (1990) the substrate used was acetonitrile instead of crotonitrile. Ultrospec 1100 pro) 4.10 Nitrilase The microbial degradation of nitriles can occur via two enzymatic pathways. 3) (Shimizu et al. 1. The assay was terminated with 500 µL ice cold McIlvaine’s buffer. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Reaction mixture of 250 µL culture supernatant (1. 1).0 mM FDA solution.1. Then entire mixture was cooked for 30 min at 400C with the addition of 25 µL of 5. The final reaction mixture was centrifuged at 10 × 103 r.f.ml of 0.3) In this microbial enzyme screening process the enzyme nitrilase was targeted and it was achieved through the conversion of nitrile into carboxylic acid and ammonia. 2).c.0 mM FDA solution [Preparation: FDA powder was weighed accurately in a 1. The enzyme activity . 5.

was measured in terms of ammonia formed as end product of enzymatic reaction, estimated spectrophotometrically by Nesslerization method (APHA, AWWA, WPCF 1992). Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Reagents and Instruments: 1. 200 µ M acetonitrile (substrate) [Preparation: 200 mM stock solution of acetonitrile was serially diluted with distilled water to prepare the substrate of desired concentration.] 2. 20 µ M potassium phosphate buffer (pH 8.0) [Preparation: 93.6 ml of 20 µM Na2HPO4 and 6.4 ml of 20 µM NaH2PO4 were added and the final pH was achieved by mixing either of the two solutions simultaneously.] 3. 1 M HCl acid [Preparation: Desired amount of concentrated HCl was diluted with distilled water.] 4. 1 µ M Dithiothreitol (DTT) [Preparation: DTT was weighed accurately and dissolved in distilled water and the stock solution was serially diluted to achieve the desired concentration.] 5. Nessler’s reagent [SRL grade] 6. Spectrophotometer (Amersham Pharmacia Biotech; Ultrospec 1100 pro) 7. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: A reaction mixture of 250 µL culture supernatant (1.5 ml culture centrifuged at 10 × 103 r.c.f. for 5 min at 40C to separate supernatant and cells), 200 µ L potassium phosphate buffer, 50 µ L DTT and 50 µL acetonitrile was incubated for 30 minutes at 250C and then the assay was terminated by 200 µ L of 1 M HCl. Then after a brief centrifugation at 10 × 103 r.c.f. for 5 min at 40C,10 µ L reaction mixture supernatant was transferred to another microcentrifuge tube containing 965 µ L of distilled water and 25 µ L Nessler’s reagent. Finally the O.D. was measured at λ
450

.

Control: Culture filtrate without the substrate was taken as blank. Volume was made up

by adding 20 µ M potassium phosphate buffer only. So in this assay method, each sample had its own control. A second control was prepared in which 200 µ M acetonitrile solution was taken and it was added to medium filtrate (autoclaved medium before fermentation). Precaution: O.D. measurements should be performed before flocculation occurs. If the TCA treated supernatant is directly added to Nessler’s reagent or even the diluted samples are left for a long time the tendency of flocculation increases. To avoid this proper dilution of samples was required as well as the diluted samples were measured in the spectrophotometer before any flocculation occurs. 1.3.1.11 Peroxidase

The activity of oxidase enzymes generate H2O2 as a product. The kinetics of the enzyme with respect to H2O2 formation can be indirectly assayed by including peroxidase and a calorimetric indicator in the substrate solution. The peroxidase reduces the H2O2 to H2O and oxidizes the spectrophotometric indicator. Thus, the peroxidase acts as an indicator enzyme that mediates an indirect interaction (i.e., transfer of electrons) from the reaction product of the first enzyme and the spectrophotometric indicator. Examples of such spectrophotometric indicators that are useful for solid-phase enzyme screening include 4Chloro-1-naphthol, which generates a blue-purple precipitate or 3,3',5,5'-

Tetramethylbenzidine (TMB), which generates a blue product. Examples of peroxidases that can be used as indicator enzymes are horseradish peroxidase, soybean peroxidase, haloperoxidase (including both chloroperoxidase and bromoperoxidase),

myeloperoxidase, cytochrome c peroxidase, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, and peroxidase Novozyme. RTM. 502 (Coleman et al, 2002). Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Reagents and Instruments: 1. 1.0 M phosphate buffer (pH 7.0) [Preparation: 57.7 ml of 1.0 M Na2HPO4 and

42.3 ml of 1.0 M NaH2PO4 were added and the final pH was achieved by mixing either of the two solutions simultaneously.] 2. 0.02 g 4-chloro-1-naphthol [Preparation: 0.02 g of 4-chloro-1-naphthol was 17.6 mM H 2O2 : Substrate [Preparation: 120 µ L of H2O2 was dissolved in

dissolved in 100 ml of distilled water.] 3. 99.880 ml distilled water to make 100 ml 17.6 mM H2O2 solution.] 4. 5. Spectrophotometer (Amersham Pharmacia Biotech; Ultrospec 1100 pro) Centrifuge (Eppendorf Centrifuge 5415 D).

Enzyme assay: Reaction mixture was prepared with 500 µL culture supernatant (1.5 ml culture centrifuged at 10 × 103 r.c.f. for 5 min at 40C to separate supernatant and cells), 100 µL 1.0 M phosphate buffer (pH 7.0), 100 µL 0.02 g 4-chloro-1-naphthol and 100 µL 17.6 mM H2O2. The entire mixture was incubated for 30 min at 250C and then taken for centrifugation at 10 × 103 r.c.f. for 5 min at 40C. Finally the optical density was measured with required dilution at λ
575

.

Control: Medium filtrate (autoclaved medium before fermentation) without the substrate was taken as blank. Volume was made up by adding 1.0 M phosphate buffer only.

1.3.2

Results

The following table shows the results of the primary and secondary screening results of the enzyme producing marine bacteria. Table No. 11: Results of primary and secondary screening of different enzymes

Table No.08: Composition of different flasks during cellulase assay

Flask No. Designation 1 2 3

Nutrient broth

CMC

Microorganism

√ √ √

√ √ ×

√ √ √

Test Test Control

Spectrophotometer. DNS solution [Preparation: mentioned earlier in page: 56] 4.8) [Preparation: Na-acetate was accurately weighed NaOH. Precaution: During heating in water bath care was taken to avoid the entry of water drops or vapor into the reaction tubes.5 M Na-acetate buffer.1% w/v CMC in 0. pH was adjusted with acetic acid and .f. 1. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Cellulase activity was assayed in a reaction mixture of 100 µ L culture supernatant (1. pH 4.The reaction was carried out at 50°C for 60 min and finally stopped by adding 700 µ L freshly prepared DNS reagent. Here also a primary screening had been done using Congo Red before conducting the secondary screening as described earlier in case of cellulase producing microorganisms.1.] 2.6 Xylanase Xylanase producing microorganisms were also screened from the collection of marine microorganisms by the same technique as described for cellulase. for 5 min at 40C to separate supernatant and cells). 0. 3.c.4 5 6 7 8 √ √ √ √ √ × √ √ × × √ × × × × Control Control Control Control Control Reagents and Instruments: 1. The entire solution was cooked for 5 min at 1000C boiling water bath. . 400 µ L Na-acetate buffer and 200 µ L substrate (0. (Amersham Pharmacia Biotech. Ultrospec 1100 pro) 5.1% w/v CMC dissolved in Na-acetate buffer.5 M Na-acetate buffer (pH 4.8). 0.3. Colour intensity was measured after proper dilution in spectrophotometer at λ 540 and dissolved in water. which may cause the dilution of the final reaction mixture and give faulty readings at the spectrophotometer.5 ml culture centrifuged at 10 × 103 r.

0. pH 7. The microorganisms responsible for producing activity zone were selected as xylanase producers for further treatments.] 2. pH was adjusted with acetic acid and NaOH. After 7 days the colonies were flooded with 0.0 g oat spelt xylan. One Petri plate was used for one microorganism.0 g beef extract. Four different types of fermentation flasks were prepared each in duplicate sets for every microorganism and numbered properly.1% w/v oat spelt xylan dissolved in Na-acetate buffer . 48. Table No. 1000 ml SDW q. The plates were kept in the incubator for 7 days at 27 0C. 0. 24.. Culture preparation: Selected microorganisms were collected from respective working culture plates and inoculated into the fermentation medium (Nutrient broth). 1. 72 and 96 h and assayed spectrophotometrically with the help of DNS reagent.5 M Na-acetate buffer (pH 5.0 g peptone. 5. The amount of xylan added was 01% w/v. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 96 h in 250 ml Erlenmeyer flasks containing 25 ml Nutrient broth medium and samples were collected at 0.5 g NaCl.09: Composition of different flasks during xylanase assay Flask No.Activity zone technique: Each true marine strain was picked onto Petri plates with solid xylanase production medium (3.1% Congo Red solution and kept for 4 h. 0. 1 2 3 4 5 6 7 8 Nutrient broth √ √ √ √ √ √ √ √ Xylan √ √ × × √ √ × × Microorganism √ √ √ √ × × × × Designation Test Test Control Control Control Control Control Control Reagents and Instruments: 1.5) [Preparation: Na-acetate was accurately weighed and dissolved in water.s. Below is given the detail of the flasks for one microorganism in tabular form.0). After 4 h excess dye was washed gently with tap water and the plates were checked for presence of any activity zone.

Ultrospec 1100 pro) 5.] 2. 400 µ L Na-acetate buffer and 200 µ L substrate (0.5 ml culture centrifuged at 10 × 103 r.c. DNase I. Colour intensity was measured after proper dilution in spectrophotometer at λ 540 .5 M Na-acetate buffer. Spectrophotometer. for 5 min at 40C to separate supernatant and cells). preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'.1. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the subculture medium. pH was adjusted with acetic acid or NaOH. A unit causes an increase in absorbance at λ of 0.5).1% w/v xylan in 0. The entire solution was cooked for 5 min at 1000C boiling water bath.f. (Amersham Pharmacia Biotech.3. pH 5. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium.0 under the specified conditions.ml-1 when acting upon highly polymerized DNA at 250C and pH 5. which may cause the dilution of the final reaction mixture and give faulty readings at the spectrophotometer. pH 5. 1.7 DNase (Deoxyribonuclease) Deoxyribonuclease from beef pancreas.0 M acetate buffer.001 min-1. Nuclease free double distilled water. DNS solution [Preparation: mentioned earlier in page: 56] 4.3.0 [Preparation: CH3COONa was accurately weighed and dissolved in water. Precaution: During heating in water bath care was taken to avoid the entry of water drops or vapor into the reaction tubes. The assay method was developed by Kunitz (1950) based upon the increased absorbance at λ 260 observed during the depolymerization of DNA by 260 DNase. Reagents and Instruments: 1. 1. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Xylanase activity was assayed in a reaction mixture of 100 µ L culture supernatant (1. was an endonuclease.The reaction was carried out at 50°C for 60 min and the reaction was terminated by adding 700 µ L freshly prepared DNS reagent. splitting phosphodiester linkages. . first crystallized by Kunitz (1950).

they were performed only once and the fifth one being the substrate blank. The culture supernatant along with the DNA and double distilled water mixed in the same ratio (4:1:15) as done for the sample run. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: The reaction mixture was comprised of 200 µL culture supernatant (1. Ultrospec 1100 pro) 7. Precaution: All the microtips and the microcentrifuge tubes were doubly autoclaved in .c. it was repeated along with every test sample run. DNA (Substrate) [Preparation: 0. 6. 1 2 3 4 5 Blank designation Substrate (contains DNA) Substrate without DNA Medium Supernatant + Substrate (contains DNA) Medium Supernatant + Substrate without DNA Culture supernatant + Substrate without DNA Blank specification Universal blank 1 Universal blank 2 Universal blank 3 Universal blank 4 Substrate blank The changes in optical density were monitored in cases of all the five.3. Control: Five different blanks were set for this assay method which is given below in the following table (Table No.] 5. for 5 min at 40C to separate supernatant and cells).25 mM MgSO4 dissolved in double distilled water (ddH2O) 4. Substrate control prepared in a same way as substrate expect the addition of DNA 6. The substrate was kept at 40C for best use for 3–4 weeks. The first four being the universal control. 25 ml of acetate buffer was added to the solution and the volume was made upto 250 ml with the addition of ddH2O. total 11 readings for each run).f. 50 µ L substrate and 750 µL double distilled water.01 g DNA was added to 200 ml MgSO4 solution and kept overnight.10 : Specifications of the controls used in case of DNase assay Blank No. The entire mixture was incubated for 10 min in the spectrophotometer at λ 260 and change in optical density was measured at every single minute interval (from 0th min to 10th min.10) Table No. Spectrophotometer (Amersham Pharmacia Biotech.5 ml culture centrifuged at 10 × 103 r.

Since the entire assay was done inside the spectrophotometer in UV mode. 5. pH was adjusted with acetic acid or NaOH. pH 5. (1960) have published an assay using a synthetic substrate.0 at λ 260 at 370C and pH 5.10 M sodium acetate buffer.3.order to avoid the presence of nucleases and the water used was also nuclease free double distilled water. 0. 1% RNA dissolved in 0.0 is determined by measuring the amount of acid soluble oligonucleotide released under defined conditions.1. (1959) has been used here. Spectrophotometer (Amersham Pharmacia Biotech. cytidine 2'.0 under the specified conditions. Nuclease free double distilled water.] 2. The rate of hydrolysis of RNA at pH 5. 3. Hence the cuvette volume had to be considered before designing the assay method. the quartz cuvette had an important role as for each cuvette a certain volume of solution (generally ≥ 800 µL for 1000 µL cuvette) was required for optical density measurement. The entire process was handled by wearing latex gloves to prevent the contamination by nuclease from hand. 1.10 M Sodium acetate buffer. Crook et al. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. Zimmerman and Sanderson (1965) described a sensitive assay using polycytidylic acid. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. Ultrospec 1100 pro) 6. 60% perchloric acid 4. One unit causes an increase in absorbance of 1. Reagents and Instruments: 1.8 RNase (Ribonuclease) Standardization of ribonuclease activity has been difficult due to varying rates at which reactions occur as well as to the significant differences in nucleotide patterns in RNA isolated from biological sources. Centrifuge (Eppendorf Centrifuge 5415 D) . 3'-phosphate. The method of Kalnitsky et al.0 [Preparation: CH3COONa was accurately weighed and dissolved in water.

can hydrolyze FDA or carboxy fluorescein diacetate (cFDA) into the more polar fluorescent products by intracellular esterases.10 M sodium acetate buffer. Control: Culture filtrate without the substrate was taken as blank. It was also assayed in the same manner and finally diluted (1:20) before taking the reading in spectrophotometer. The reaction was carried out at 370C for 30 min and finally stopped by adding 250 µL ice cold 60% perchloric acid after putting the reaction tubes into ice bath. Instead 200 µL 0. p-nitrophenyl acetate or naphthyl acetate was used as esterase substrates.10 M sodium acetate buffer. medium before fermentation was so treated as a separate sample for assay as universal control. Samples were diluted (1:400) to measure in spectrophotometer at λ 260 . Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. 200 µL substrate (1% RNA in 0. whether mammalian cells. The entire process was handled by wearing latex gloves to prevent the contamination by nuclease from hand. generally. So for each sample an individual blank was prepared. Precaution: All the microtips and the microcentrifuge tubes were doubly autoclaved in order to avoid the presence of nucleases and the water used was also nuclease free ddH2O.f. or Gram-positive or Gram-negative bacteria.0.3. .c.10 M sodium acetate buffer was added to make up the volume.9 Esterase The assay technique applied here was done earlier by Breeuwer et al (1995) where the substrate used was fluorescein diacetate (FDA).Enzyme assay: RNase activity was assayed in a reaction mixture of 250 µl culture supernatant (1. yeasts cells. Apart from this.f.5 ml culture centrifuged at 10 × 103 r. pH 5. Apparently most cells.1. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium. however.c. 1. for 5 min at 40C and 500 µL of nuclease free double distilled water was added. pH 5. for 5 min at 40C to separate supernatant and cells).0. Tubes were centrifuged at 10 × 103 r. equilibrated at 370C prior to assay) and 200 µL 0.

20) [Preparation: 173. and then to the acids and ammonia by amidase (Eq. 1.0 mM FDA solution. Ultrospec 1100 pro) 4. 3) (Shimizu et al.1 M citric acid were mixed to prepare the buffer of pH 7. different from cyanogenesis: (i) nitrilase catalyzes the direct hydrolysis of nitriles to the corresponding carboxylic acids and ammonia (Eq.10 Nitrilase The microbial degradation of nitriles can occur via two enzymatic pathways.1) (Eq.3. Then entire mixture was cooked for 30 min at 400C with the addition of 25 µL of 5.0 mM FDA solution [Preparation: FDA powder was weighed accurately in a 1. 5. The assay was terminated with 500 µL ice cold McIlvaine’s buffer. RCN + 2H2O → RCOOH + NH3 RCN + H2O → RCONH2 RCONH2 + H2O → RCOOH + NH3 (Eq.2) (Eq. Control: Medium filtrate (autoclaved medium before fermentation) instead of culture supernatant had been considered as blank.9 ml of 0. 2).f.] 3. for 5 min at 40C and the O. .c. and (ii) nitriles are catabolized in two stages-they are first converted to the corresponding amides by nitrile hydratase (Eq.2 M Na2HPO4 and 26. 1).1 ml of 0. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: Reaction mixture of 250 µL culture supernatant (1.1. The final reaction mixture was centrifuged at 10 × 103 r. for 5 min at 40C to separate supernatant and cells) and 200 µL McIlvaine’s buffer was preincubated at 400C for a brief period of time of 3 min. 1997). was measured with required dilution at λ 490 .5 ml microcentrifuge tube and dissolved in dimethyl sulfoxide. Spectrophotometer (Amersham Pharmacia Biotech.Reagents and Instruments: 1.2 and the final pH adjustment was done by addition of either of the two solutions simultaneously.f.5 ml culture centrifuged at 10 × 103 r.D. McIlvaine’s buffer (pH 7.c.] 2.3) In this microbial enzyme screening process the enzyme nitrilase was targeted and it was achieved through the conversion of nitrile into carboxylic acid and ammonia.

Reagents and Instruments: 1.] 2. 200 µ M acetonitrile (substrate) [Preparation: 200 mM stock solution of acetonitrile was serially diluted with distilled water to prepare the substrate of desired concentration. 1 µ M Dithiothreitol (DTT) [Preparation: DTT was weighed accurately and dissolved in distilled water and the stock solution was serially diluted to achieve the desired concentration. 200 µ L potassium phosphate buffer.6 ml of 20 µM Na2HPO4 and 6.f. for 5 min at 40C. WPCF 1992).Nitrilase activity was assayed according to the methods described by Kobayashi et al (1990) the substrate used was acetonitrile instead of crotonitrile.c.] 4. Nessler’s reagent [SRL grade] 6.f. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium.] 5.4 ml of 20 µM NaH2PO4 were added and the final pH was achieved by mixing either of the two solutions simultaneously. Centrifuge (Eppendorf Centrifuge 5415 D) Enzyme assay: A reaction mixture of 250 µL culture supernatant (1. estimated spectrophotometrically by Nesslerization method (APHA. AWWA. Ultrospec 1100 pro) 7. Spectrophotometer (Amersham Pharmacia Biotech. Then after a brief centrifugation at 10 × 103 r. 1 M HCl acid [Preparation: Desired amount of concentrated HCl was diluted with distilled water.10 µ L reaction mixture supernatant was transferred to another microcentrifuge tube containing 965 µ L of .5 ml culture centrifuged at 10 × 103 r. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium.c.0) [Preparation: 93. 20 µ M potassium phosphate buffer (pH 8. for 5 min at 40C to separate supernatant and cells).] 3. 50 µ L DTT and 50 µL acetonitrile was incubated for 30 minutes at 250C and then the assay was terminated by 200 µ L of 1 M HCl. The enzyme activity was measured in terms of ammonia formed as end product of enzymatic reaction.

502 (Coleman et al.5. myeloperoxidase. which generates a blue product. If the TCA treated supernatant is directly added to Nessler’s reagent or even the diluted samples are left for a long time the tendency of flocculation increases.D. A second control was prepared in which 200 µ M acetonitrile solution was taken and it was added to medium filtrate (autoclaved medium before fermentation). . lignin peroxidase. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 25 ml Marine Difco 2216 medium.D. The peroxidase reduces the H2O2 to H2O and oxidizes the spectrophotometric indicator.3'. carrot peroxidase. 2002). peanut peroxidase. the peroxidase acts as an indicator enzyme that mediates an indirect interaction (i. Volume was made up by adding 20 µ M potassium phosphate buffer only. Control: Culture filtrate without the substrate was taken as blank. To avoid this proper dilution of samples was required as well as the diluted samples were measured in the spectrophotometer before any flocculation occurs. measurements should be performed before flocculation occurs. RTM.. soybean peroxidase. Examples of such spectrophotometric indicators that are useful for solid-phase enzyme screening include 4Chloro-1-naphthol. each sample had its own control. Examples of peroxidases that can be used as indicator enzymes are horseradish peroxidase. Precaution: O. The kinetics of the enzyme with respect to H2O2 formation can be indirectly assayed by including peroxidase and a calorimetric indicator in the substrate solution.e. So in this assay method. 1. Finally the O. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. transfer of electrons) from the reaction product of the first enzyme and the spectrophotometric indicator. Thus.3. cytochrome c peroxidase. which generates a blue-purple precipitate or 3. haloperoxidase (including both chloroperoxidase and bromoperoxidase).11 Peroxidase The activity of oxidase enzymes generate H2O2 as a product. was measured at λ 450 . tulip peroxidase.5'- Tetramethylbenzidine (TMB). and peroxidase Novozyme.1.distilled water and 25 µ L Nessler’s reagent.

Ultrospec 1100 pro) Centrifuge (Eppendorf Centrifuge 5415 D). 100 µL 0. The entire mixture was incubated for 30 min at 250C and then taken for centrifugation at 10 × 103 r.3 ml of 1.c.6 mM H2O2 solution.880 ml distilled water to make 100 ml 17. Finally the optical density was measured with required dilution at λ 575 . 5. for 5 min at 40C.2 Results The following table shows the results of the primary and secondary screening results of the enzyme producing marine bacteria.6 mM H 2O2 : Substrate [Preparation: 120 µ L of H2O2 was dissolved in Enzyme assay: Reaction mixture was prepared with 500 µL culture supernatant (1. 1. Volume was made up by adding 1. 17.0 M phosphate buffer (pH 7.f.7 ml of 1.] 3.0 M phosphate buffer (pH 7. 1. .Reagents and Instruments: 1. Control: Medium filtrate (autoclaved medium before fermentation) without the substrate was taken as blank.0 M Na2HPO4 and 42.5 ml culture centrifuged at 10 × 103 r.] 4.02 g of 4-chloro-1-naphthol was dissolved in 100 ml of distilled water.f. 100 µL 1. 99.0 M NaH2PO4 were added and the final pH was achieved by mixing either of the two solutions simultaneously.c.] 2.02 g 4-chloro-1-naphthol and 100 µL 17.3. 0.0). Spectrophotometer (Amersham Pharmacia Biotech.02 g 4-chloro-1-naphthol [Preparation: 0.6 mM H2O2.0 M phosphate buffer only.0) [Preparation: 57. for 5 min at 40C to separate supernatant and cells).

the culture supernatant of the various enzyme producers were put through different extreme conditions to check whether they maintained their enzymes producing capabilities in desirable amounts. so it is . At the same time the importance of commercial enzymes active through a wide range of pH goes without saying. It was assumed that it would be easier to purify the enzymes if they successfully pass through these parameters. The above mentioned parameters were selected since commercially important bioactive catalysts with novel properties were targeted in this project work. The enzymes were assayed in the same way as done earlier but with a few alterations. such as pre-incubation temperatures upto 800C.1. then it would be very easy to choose solvents for chromatography. Cost is a very important factor which is counterbalanced with quality in case of commercialization of bioactive compounds. As the cultures used for testing are mesophilic and marine derived. Attaining low temperature is an important criterion for enzyme purification but since the enzyme producers selected through these procedures would be thermostable. If the enzymes are active through a wide range of pH.4 Screening for special enzyme properties In this part of the research work. pH in extreme acidic to basic ranges and salinity upto 5% w/v. so loss of target protein during purification would also be minimized.

1. The preincubation temperatures was raised upto 800C for 30 min followed by the enzyme assay at 370C. So the buffers were prepared according to requirements (borate buffer for pH 8. Such work has not been done so far and is therefore an objective of this doctoral thesis.0 and pH 8. All the production media designed for the enzyme producers were either marine bacterial production medium (Marine Difco 2216) or specially designed marine production media (cellulase and xylanase production medium). The function of the reaction buffer was to maintain a stable reaction pH.4. The experiments were performed after pre-incubation over night at two different saline conditions. For testing the activity under saline conditions all the reagents and buffers were . Not all the screened enzyme producers were tested. Asp – 02.0). viz. 0% and 5% w/v NaCl. That these microorganisms would be producing thermostable and active enzymes in a wide range of pH needs to be experimentally verified.0 and acetate buffer for pH 4. Asp – 04 and Asp – 05) were assayed according to the protocol with some changes.4. they were not considered for this special property testing.1. The assays were done in two pH conditions. pH 4.0.1 Reaction conditions The selected five microorganisms (Asp – 01. Asp – 03. Since no potent amylase and peroxidase producers were found during primary screening operations. These were done by keeping it in mind that only those enzyme producers will pass through this test which was really robust to withstand high saline conditions as well as to check whether the NSW boosts up the enzyme productions. A minimum of five microorganisms for each different enzyme were selected.1 L-asparaginase 1.expected that they would be producing active enzymes under saline conditions. The assays were performed with the cultures prepared in the media made of single distilled water (SDW) as well as natural sea water (NSW).

0 4.0 800C 800C 800C 800C 0% 5% 0% 5% 0% 5% 0% 5% The figures (05 to 14) show L-asparaginase activity of the marine isolates (Asp-01 – Asp-05) based on the reaction conditions outlined in Table No.0 4.0 8. From those plots it was observed that maximum L-asparaginase activity was obtained with alkaline assay condition for both the media.0 NSW based medium pH Preincubation temp.113 – 0.2 4. The isolates had attained ample growth [O. 06).1 Asp-01 (L-asparaginase producer) Figure 05: Asp-01 in 100% SDW Marine Marine Difco medium Figure 06: Asp-01 in 100% NSW Difco medium The produced L-asparaginase by the microorganism Asp-01 was represented graphically (Figure 05.D. in the range of 0.611 (1:20 dilution)] before the assay was performed. 12: Different assay conditions for testing special enzyme properties for Lasparaginase SDW based medium Set pH Preincubation temp. Salinity 800C 800C 800C 800C Results 0% 5% 0% 5% 0% 5% 0% 5% Set A2 B2 C2 D2 E2 F2 G2 H2 Salinity A1 4. Table No. 4.0 4.0 8.0.1.0 B1 C1 D1 E1 F1 G1 H1 1. 1. Keeping the assay pH constant. the salinity and temperature were changed . 12.0 4.1.0 8.0 8.0 8.4. Below are given their details.2.0 4.4.0 8.0 8.0 8.prepared in 5% NaCl solution (5 g NaCl in 100 ml SDW). at 8. So 16 sets of reaction mixtures were prepared.

From those plots it was observed that maximum asparaginase activity was obtained with alkaline assay condition for both the media. 08). 1.0 the activity decreases significantly up to 6070% and at this acidic pH the effect of temperature as well as salinity do not affect the production in both media.0 pH the enzymatic activity remains almost same in all saline conditions (0% and 5% salinity).2. When the pH was maintained at 4.4. But in stressed conditions the situation becomes opposite. This microorganism showed good enzyme activity in stressed conditions when pH was maintained at 8.0. Lowering the pH at 4. . Though the activity lowers down significantly up to 6080% in 100% NSW Marine Difco but in case of 100% SDW Marine Difco medium the production almost equals the normal production.1. The produced L-asparaginase was represented L- graphically (Figure 07.the production reduces to nearly 40-50% which also can be considered as good activity.0 the activity does not vary significantly except at 800C temperature incubation and 5% salinity the enzyme production almost equals to the normal production rate. At 8. 370C incubation and 5% salinity a noteworthy production was achieved with the fermentation broth of 100% NSW Marine Difco medium.0 pH. Production of L-asparaginase in 100% NSW Marine Difco medium is higher than in 100% SDW Marine Difco medium.2 Asp-02 (L-asparaginase producer) Figure 07: Asp-02 in 100% SDW Marine Marine Difco medium Figure 08: Asp-02 in 100% NSW Difco medium From the fermentation broths of Asp-02 the produced enzymatic activity was measured under various stressed conditions. At 80 0C temperature and 8. By observing the two above mentioned figures it can be concluded that the production rate is higher when the microorganism was fermented in 100% SDW Marine Difco than 100% NSW Marine Difco.

10). 370C incubation the production was greater in case of 100% NSW Marine Difco medium than 100% SDW Marine Difco medium. At pH 4.0 at 370C incubation the production was higher in both the media at 5% salinity and at pH 4.0 this microorganism gave noteworthy enzyme production under stressed conditions and at 8.2.2.0.4.4 Asp-04 (L-asparaginase producer) Figure 11: Asp-04 in 100% SDW Marine Marine Figure 12: Asp-04 in 100% NSW .0 pH.1.1. 370C incubation and at 0% salinity). The activity decreased only up to 30-50% in other conditions. At pH 8. 1. At 800C the enzyme activity remained almost same for both the media and they were reduced by 50-55% than the alkaline condition (pH 8. From the plots it was observed that maximum L-asparaginase activity was obtained with alkaline assay condition for both the media.3 Asp-03 (L-asparaginase producer) Figure 09: Asp-03 in 100% SDW Marine Marine Difco medium Figure 10: Asp-03 in 100% NSW Difco medium The produced L-asparaginase by the microorganism Asp-03 in various stressed conditions was represented graphically (Figure 09.4.When overall production was estimated it was seen that this microorganism also has good enzymatic activity in 100% SDW Marine Difco medium and in stressed conditions the production was higher at basic pH. 1. Overall assessment of this microorganism is that it showed good activity in 100% SDW Marine Difco medium and activity at stressed condition was better at alkaline pH.0. 370C incubation and 5% salinity the activity was almost same with the normal condition.

1 Reaction conditions The selected two microorganisms (Glut-01.5 Asp-05 (L-asparaginase producer) Figure 13: Asp-05 in 100% SDW Marine Marine Difco medium Figure 14: Asp-05 in 100% NSW Difco medium The produced enzymatic activity from the fermentation broths of Asp-05 was measured under various stressed conditions and the produced L-asparaginase was represented L- graphically (Figure 13.0 and pH 8.2. 14). 1.Difco medium Difco medium From the figures 11 and 12 it was observed that maximum L-asparaginase activity by microorganism Asp-04 was obtained with alkaline assay condition for both the media. The assays were done in two pH conditions. The preincubation temperatures was raised upto 800C for 30 min followed by the enzyme assay at 370C. .1.0. For this Microorganism the enzyme activity lowered to 50-55% in all stressed conditions and the activity was better in case of 100% SDW Marine Difco medium production. 1. Glut-02) were assayed according to the protocol with same changes as done in case of L-asparaginase. pH 4.2 L-glutaminase 1.4. For this Microorganism the enzyme activity lowered to 50-55% in all stressed conditions and the activity was better in case of 100% SDW Marine Difco medium production. From those plots it was observed that maximum asparaginase activity was obtained with alkaline assay condition for both the media.4.4.2.

0 B1 C1 D1 E1 F1 G1 H1 4. The function of the reaction buffer was to maintain a stable reaction pH. 13: Different assay conditions for testing special enzyme properties for Lglutaminase SDW based medium Set pH Preincubation temp.178 – 0. in the range of 0. Table No.0 4. 1.0 800C 80 C 0 Salinity A1 4.0 8.0 8.0 4. 13. 0% and 5% w/v NaCl. Below are given their details. viz.D. The isolates had attained ample growth [O.The experiments were performed after pre-incubation over night at two different saline conditions.2.0 4.1 Glut-01 (L-glutaminase producer) Figure 15: Glut-01 in 100% SDW Marine Marine Difco medium Figure 16: Glut-01in 100% NSW Difco medium . Salinity 800C 80 C 0 NSW based medium Set A2 B2 C2 D2 E2 F2 G2 H2 pH Preincubation temp.0 8. 4.2 Results The figures (15 to 18) show L-glutaminase activity of the marine isolates (Glut-01 and Glut-02) based on the reaction conditions outlined in Table No.0 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% 800C 80 C 0 800C 80 C 0 1.4.2.0 8.0 8.2.4. For testing the activity under saline conditions all the reagents and buffers were prepared in 5% NaCl solution (5 g NaCl in 100 ml SDW).0).0 8.0 4. So the buffers were prepared according to requirements (borate buffer for pH 8.0 8. So 16 sets of reaction mixtures were prepared.0 4.500 (1:20 dilution)] before the assay was performed.0 and acetate buffer for pH 4.0 8.

At pH 8. From those graphs it was observed that maximum production was achieved at alkaline condition. From the graphs it was observed that maximum production was achieved at alkaline condition.2. At alkaline pH the production was reduced to 40-45%. 1. 16) in terms of liberated NH3 from NH4NO3 salt.4. Production was better in 100% SDW based Marine Difco medium.2.The produced L-glutaminase was represented graphically (Figure 15. At 370C temperature and 5% salinity in 100% NSW based Marine Difco medium the activity almost equaled the normal production rate and in stressed conditions the broth of 100% NSW based Marine Difco medium gave better activity. At acidic pH the activity reduced to 50-55% of the normal activity while the production in 100% SDW based Marine Difco medium was better this time. At acidic pH the activity falls down to 60-70% but the salinity and the temperature did not have much effect. And production was better in 100% NSW based Marine Difco medium. Overall better production was achieved in 100% SDW based Marine Difco medium broth.0 the activity reduced to 50% of its normal activity in 100% SDW based Marine Difco medium at different stressed conditions but the activity was almost equaled at 5% salinity and 370C temperature and the activity was reduced to 60% at higher temperature. 1.3 Protease .2 Glut-02 (L-glutaminase producer) Figure 18: Glut-02 in 100% Figure 17: Glut-02 in 100% SDW Marine NSW Marine Difco medium Difco medium The produced L-glutaminase was represented graphically (Figure 17.4. 18) in terms of liberated NH3 from NH4NO3 salt.

4. Prot-104 and Prot-105. in the range of 0.2 Results The figures (19 to 33) show protease activity of the marine isolates (Prot-101 – Prot-105) based on the reaction conditions outlined in Table No.4.3.1. Prot103.5. The isolates had attained ample growth [O.2. Tris buffer was prepared for pH 8. 1989).371 (1:20 dilution)] before the assay was performed.5 and the other pHs were obtained with phosphate buffer.3.D.0 (Sarath.0 and 8. 14.158 – 0. 7. Three pH conditions were chosen 6. Pre-incubation at 800C for 30 min at two different saline conditions of 0% and 5% w/v salinity were set to execute this experiment. The selected five microorganisms were Prot-101.1 Prot-101 (protease producer) Figure 19: Prot -101 in 100% SDW Marine Marine Difco medium Figure 20: Prot -101 in 100% NSW Difco medium The maximum production was achieved in neutral condition with the supernatant of 100% SDW based Marine Difco medium. The substrate azocasein had a limiting solubility below pH 6. 14: Different assay conditions for testing special enzyme properties for protease 1. By keeping the pH constant when temperature was increased the production decreased enormously but at 370C the microorganism showed very small change in production rate in 5% salinity in case of 100% SDW based .3. The following table expresses the details.0.1 Reaction conditions Two different types of Marine Difco 2216 media were prepared (100% SDW and100% NSW) for fermentation. Hence the acidic pH was chosen as 6. 1.4.0. Table No. Prot-102.

But in acidic pH in different stressed conditions the activity reduced to 20% of the activity found in 370C incubation and 0% salinity in case of 100% SDW based Marine Difco medium and with 100% NSW based Marine Difco medium supernatant the protease production was less than 100% SDW based Marine Difco medium. 1.4. In case of the enzyme produced in 100% NSW based Marine Difco medium at neutral .3. At pH 7. But with 100% NSW based Marine Difco medium the activity remained more or less same in all conditions (different salinity and incubation temperature) and the reduction of 65% activity was observed in neutral condition when the microorganism was treated with 100% NSW based Marine Difco medium instead of 100% SDW based Marine Difco medium.2.Marine Difco medium. In acidic pH the highest enzymatic activity was observed with 37 0C incubation and 0% salinity and it was almost 50% of the activity of the measured amount in neutral condition.2 Prot-102 (protease producer) Figure 22: Prot -102 in 100% NSW Figure 21: Prot -102 in 100% SDW Marine Marine Difco medium Difco medium The microorganism Prot-102 also gave maximum activity at neutral condition and in both fermentation broths the production was almost same. 20).0 salinity did not play any pivotal role in enzyme stability in case of the protease produced in 100% SDW based Marine Difco medium but at 800C temperature the production decreased by 75%. Overall the microorganism Prot-101 always showed better result when fermentation was carried out in 100% SDW based Marine Difco medium and it can be concluded that the produced enzyme can endure the stress of temperature and salinity at alkaline pH (Figure: 19. In alkaline pH the production of enzyme was found to be 25-30% of the maximum achieved activity. but it remained almost steady in every different stressed conditions with 100% SDW based Marine Difco medium but the activity was greater with 100% NSW based Marine Difco medium supernatant.

The stability of enzyme in 100% NSW based Marine Difco medium was maximum at alkaline pH with 0% salinity and 370C incubation temperature but in all . Nearly 45% reduction in enzyme production occurred at alkaline pH in 100% SDW based Marine Difco medium and temperature and salinity did not affect the stability of the enzyme at that pH. At alkaline pH the better production was observed in stressed conditions but the production was higher in 100% NSW based Marine Difco medium at 0% salinity and at 5% salinity the production was higher in 100% SDW based Marine Difco medium. At neutral pH salinity did not play any important role in enzyme stability but increased temperature reduced the stability of the enzyme remarkably. At pH 6. At pH 6. 22).pH in different salinity and temperature the production decreased up to 50% from the maximum production.3.2.0 better production was achieved in the 100% NSW based Marine Difco medium and temperature and salinity did not alter the enzyme stability so much. Overall Prot-102 gave better production in 100% NSW based Marine Difco medium and the produced enzyme is thermostable and salt tolerant in a pH range of 6.0 to 8.5 if cultivated in 100% NSW based Marine Difco medium (Figure: 21. 1.3 Prot-103 (protease producer) Figure 23: Prot -103 in 100% SDW Marine Marine Difco medium Figure 24: Prot -103 in 100% NSW Difco medium This microorganism gave maximum enzyme production in 100% SDW based Marine Difco medium at neutral condition.4.0 the temperature did not affect the stability of the enzyme but stability reduced as higher saline conditions were maintained for 100% SDW based Marine Difco medium supernatant.

2. Though at alkaline pH the produced enzyme in 100% NSW based Marine Difco medium was more stable in stressed conditions but overall enzyme stability was found more in 100% SDW based Marine Difco medium (Figure: 25. 24).4 Prot-104 (protease producer) Figure 25: Prot -104 in 100% SDW Marine Marine Difco medium Figure 26: Prot -104 in 100% NSW Difco medium The microorganism Prot-104 gave maximum activity in 100% SDW based Marine Difco medium at neutral condition. 1. This nature of the enzyme was also true for the production in 100% NSW based Marine Difco medium but the overall production was almost reduced to 35% of the production in 100% SDW based Marine Difco medium. 26).4. Overall the thermostability decreased as pH was increased and the salt tolerance was increased with the increase of pH for the protease produced by Prot-104. Here the maximum stability of the enzyme was found in 100% SDW based Marine Difco medium and the enzyme was found to be thermostable rather than salt tolerant.4. As the pH was increased the stability was found more in 100% NSW based Marine Difco medium than that of 100% SDW based Marine Difco medium and here also it was found that the enzyme was rather salt tolerant than thermostable.3. In 100% SDW based Marine Difco medium broth the produced enzyme was stable in 5% salinity but the stability lost remarkably with the increase of temperature.3.other cases the enzyme stability decreased by 75% and the overall assessment about the microorganism is that the produced enzyme is more thermostable and salt tolerant at higher pH if cultivated in 100% SDW based Marine Difco medium (Figure: 23. 1.5 Prot-105 (protease producer) .2. The situation was almost opposite when the pH was lowered down to acidic range.

4. 1. They were designated as S1.Figure 27: Prot -105 in 100% SDW Marine Marine Difco medium Figure 28: Prot -105 in 100% NSW Difco medium This microorganism Prot-105 of 100% SDW based Marine Difco medium gave high production in neutral condition and the enzyme produced in 100% NSW based Marine Difco medium was 50% less stable.4 Cellulase 1. analysis of cellulose production as well as reducing sugar. At alkaline pH the enzyme was found almost stable in all different stressed conditions for both 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium. overnight kept 25 ml nutrient broth . The run lasted for 10 h and 11 samples were collected. Each sample was treated for growth measurement. Agitation Aeration Inoculum 200 rpm 0. S11. The enzyme production in 100% NSW based Marine Difco medium restored its stability in different stressed conditions at this pH.4. the enzyme was found to be stable for both the media and can be designated as thermostable and salt tolerant (Figure: 27.1 Bioreactor specifications Cellulase was assayed from the fermentation supernatant of the microorganism 2/03 cultured in bioreactor as the sufficient production was not found in shake flask fermentation.4. At neutral pH the activity was not lost with increased salinity but 50% reduction was observed with increased temperature. 28). Overall. ………. S2. In acidic pH the result did not show much of difference but the stability at 0% salinity and 370C incubation almost equaled the maximum production by the microorganism.75 vvm Microorganism 2/03.

8.0) conditions.4. two saline . 0.451 (1:20 dilution)] before the assay was performed. O.0 g peptone.4.4.0 and 9. 5.D.3.1% Nutrient Broth with 0. 15.2 Reaction conditions The fermentation broth collected at 0th hour (S1) was taken as blank and the 9th hour broth (S10) was treated as sample for the tertiary screening of cellulase.medium (composition given in page: 61). The isolate had attained ample growth [O.4.8.1 % CMC (3.0 g CMC. 0.0 g beef extract.0) and Tris-HCl buffer (pH 9.5 g NaCl. 7.600 for inoculum 0.4.0). 1000 ml SDW q.402 at Inoculum volume Antifoam Medium 5 ml 0.3 Result The following figure (29) shows cellulase activity of the marine isolate (Cel-2/03) based on the reaction conditions outlined in Table No. 1. Overnight pre-incubation at saline conditions and 800C for 30 min was done. pH 7.2) 24 h [1:20 dilution] 1. 1.0 and 9. Three pH conditions 4.1 Cel-2/03 (Cellulase producer) Figure 29: Cel-2/03 in 100% SDW based nutrient broth with 0.1% CMC The special enzymatic properties were measured in three pH (4.D. 15: different assay conditions for testing special enzyme properties of cellulase 1.s. Table No.4.8 and pH 7.0 were achieved with citrate buffers (pH 4. 7..

O. 4. The maximum production was achieved in acidic reaction condition. They were designated as S1.4.2) 24 h [1:20 dilution] 200 rpm 0..0 and pH 9.8.1 Bioreactor specifications Xylanase was also assayed from the fermentation supernatant of the microorganism 2/47 cultured in bioreactor as the sufficient production was not found in shake flask fermentation.5.1% Nutrient Broth with 0.240 at Inoculum volume Antifoam Medium 5 ml 0.1% CMC (medium composition given at page: 90).D. 1000 ml SDW q. analysis of xylanase production as well as reducing sugar. The normal assay condition was 500C incubation.0 g oat spelt Xylan. pH 7.0 g beef extract.1 % Xylan (3.s. overnight kept 25 ml nutrient broth . 1.0 the enzyme behaved same as at pH 4.600 for inoculum 0. 0. In acidic pH the enzyme was found to be thermostable but not salt tolerant.4. S13.8 pH and 0% salinity and the fermentation medium was 100% SDW based nutrient broth with 0. S2. As the pH was increased the stability was decreased by 50% but at pH 7. 15). 1. Agitation Aeration Inoculum medium (composition given in page: 61). The run lasted for 12 h and 13 samples were collected.conditions (0% and 5%) and at two different temperatures (500C and 800C) (Table No. Each sample was treated for growth measurement.5 g NaCl.0 g peptone.5 Xylanase 1. 5. The amount of cellulase activity was measured in terms of D-glucose.50 vvm Microorganism 2/47. ……….

The isolate had attained ample growth [O. The maximum production was achieved in acidic reaction condition.3. 16).0).4.0 the enzyme behaved equally in different pH and temperatures.5. two saline conditions (0% and 5%) and at two different temperatures (500C and 800C) (Table No. 16: different assay conditions for testing special enzyme properties of xylanase 1.2 Reaction conditions The fermentation broth collected at 0th hour (S1) was taken as blank and the 10th hour broth (S11) was treated as sample for tertiary screening for xylanase.476 (1:20 dilution)] before the assay was performed. Three pH conditions 5.4.5.0 and pH 9. 7.1. In acidic pH the enzyme was found to be thermostable but not salt tolerant.0 and 9.3 Result The following figure (30) shows cellulase activity of the marine isolate (microorganism 2/47) based on the reaction conditions outlined in Table 16.1% xylan (medium composition given in page: 92).4.0 were achieved with citrate buffers (pH 5. 0. 1. 5. As the pH was increased the stability was decreased by 50% but at pH 7.4. The amount of xylanase activity was measured in terms of xylose. 7.1% xylan The special enzymatic properties were measured in three pH (5.6 DNase .5.5.1 Microorganism 2/47 (xylanase producer) Figure 30 :microorganism 2/47 in 100% SDW based nutrient broth with 0.D.5 and pH 7. 1.0) conditions. Overnight pre-incubation at saline conditions and 800C for 30 min was done. The normal assay condition was 500C incubation.5 pH and 0% salinity and the fermentation medium was 100% SDW based nutrient broth with 0.0) and Tris-HCl buffer (pH 9.5.0 and 9. Table No.

1 Reaction conditions Two microorganisms 2/01 and 2/22 were selected from primary screening for special property testing and they were cultured in both 100% SDW Difco and 100% NSW Difco. Like this four different sample sets were prepared. Table No.4. Fermentation supernatant of 2/01 and 2/22 were prepared. // 5% NaCl 5 = pH 8 // room temp. // 0% NaCl .2 Results Following are the graph legends for DNase producing microorganisms 1 = pH 5 // room temp. 48 different controls were run for this experiment. // 0% NaCl 2 = pH 5 // room temp. // 5% NaCl 3 = pH 5 // preincubation at 800C temp. Table No.6.1. Table No. Overnight incubation over saline conditionwas also done. 19: Different assay conditions for testing special enzyme properties of DNase 1.4. // 0% NaCl 4 = pH 5 // preincubation at 800C temp. As the assay is directly performed in spectrophotometer cuvette so control A 1 to C8 were tested at room temperature (RT) and control D1 to F8 were first pre-incubated upto 800C for 30 min before performing the assay.6.0 and pH 8. 17: Composition of various blanks for testing special properties of DNase. Below are given the details. The samples were also treated in the similar fashion during thermostability testing. 18: Different sample sets for the DNase producing microorganisms The following table exhibits all the combinations (16 types for each four set) with which the experiments had been performed. pH 5.0 and 0% and 5% salinity were considered to be other reaction criteria.

// 5% NaCl 7 = pH 8 // preincubation at 800C temp.109 to 0. // 5% NaCl The following figures (31 . As the pH was increased to 8.6. 1.34) shows DNase activity of the marine isolates (2/01 – 2/22) based on the reaction conditions outlined in above (1-8). 32). At acidic pH in 100% SDW based Marine Difco the enzyme activity did not change much in 5% salinity at RT and at 0% salinity in 800C but decreased enormously at 800C with 5% salinity.2 Microorganism 2/22 (DNase producer) Figure 33: Microorganism 2/22 2/22 in 100% SDW Marine Difco medium Figure 34: Microorganism in 100% NSW Marine Difco medium This microorganism also gave maximum production at acidic condition in 100% SDW . // 0% NaCl 8 = pH 8 // preincubation at 800C temp.6.4.4.317 (1:20 dilution)] before the assay was performed.6 = pH 8 // room temp.1 Microorganism 2/01 (DNase producer) Figure 32: Microorganism 2/01 in 100% NSW Marine Difco medium Figure 31: Microorganism 2/01 in 100% SDW Marine Difco medium This microorganism showed maximum enzyme activity at acidic assay condition in 100% SDW based Marine Difco and the stability decreased by 35% in 100% NSW based Marine Difco. At this pH the enzyme was found to be thermostable rather than salt tolerant (Figure: 31.2. In 100% NSW based Marine Difco medium the enzyme stability was affected with increased salinity. 0. The isolates had attained ample growth [O.2.D.0 the enzyme was found less stable for both the media and in all cases the stability decreased by 60% of the normal condition. 1.

4.based Marine Difco and in 100% NSW based Marine Difco medium the production decreased by 30%. .1 R-01 (RNase producer) Figure 35 : R-01 in 100 % SDW and 100% NSW Marine Difco medium At normal condition this microorganism produces maximum enzyme activity (Figure 35) and at 370C seawater reduced enzyme activity in all stressed conditions. The only change done in the assay protocol was the pre-incubation temperature of the reaction mixture.7. 1. But in all other stressed conditions the enzyme stability was very little. At 800C the stability of the enzyme was reduced to 50% but in all cases the enzyme showed impressive stability in higher temperature and salinity. The isolates had attained ample growth [O.7.717 (1:20 dilution)] before the assay was performed. 20.43) shows RNase activity of the marine isolates (R 01 – R 09) based on the reaction conditions outlined in Table No. Table No.7. 20: Different assay conditions for testing special enzyme properties of RNase 1.4.2 Results The following figures (35 .7 RNase 1. They were numbered as R-01 to R-09. In these tests the mixtures were pre-incubated for 30 min at 800C and with 5% w/v NaCl overnight.4. A set of eight different combinations as detailed in the below given table was designed for each microorganisms.2. 34).4.109 to 0.D. 1. In this assay method only thermostability and salt tolerance were tested as described earlier.1 Reaction conditions Nine microorganisms were selected as potent RNase producers. 0. So this microorganism can not be treated as thermostable and salt tolerant (Figure: 33.

7. At 80 0C the stability of the enzyme decreased up to 50% and effect of salinity could not be neglected.3 R-03 (RNase producer) Figure 37 : R-03 in 100 % SDW and 100% NSW Marine Difco medium In both medium the enzyme productions were always almost same but the 50% to 60% of the stability was lost in all other stressed conditions (Figure 37). At higher temperature 50% stability of the normal condition was lost in 0% salinity and increased salinity caused further loss of stability at 800C.5 R-05 (RNase producer) Figure 39 : R-05 in 100 % SDW and 100% NSW Marine Difco medium In both media the enzyme productions were always almost same for this microorganism but the enzyme lost 50% of its normal activity at higher temperature (Figure 39).2.4.4 R-04 (RNase producer) Figure 38 : R-04 in 100 % SDW and 100% NSW Marine Difco medium The production rate was higher in 100% SDW based Marine Difco than in 100% NSW based Marine Difco medium (Figure 38). This microorganism showed impressive stability at different saline conditions and increased salinity lowered the stability of the enzyme by 50% at 370C.2 R-02 (RNase producer) Figure 36 : R-02 in 100 % SDW and 100% NSW Marine Difco medium The enzyme production was greater in case of 100% SDW based Marine Difco medium than in case of 100% NSW based Marine Difco medium (Figure 36). 1.4.4.2. 1.7.1.2. At 370C salinity affected the stability in 100% SDW based Marine Difco medium but in 100% NSW based Marine Difco medium no such effect of salinity was observed. 1.7.4. .2.7.

8 R-08 (RNase producer) Figure 42 : R-08 in 100 % SDW and 100% NSW Marine Difco medium This microorganism only lost 40% of it’s RNase activity in higher salinity for both media at 370C temperature and a stable enzyme activity was obtained in higher temperature and salinity. 1.2. Production of enzyme was found to be better in 100% SDW based Marine Difco than in 100% NSW based Marine Difco medium (Figure 42).7.7.9 R-09 (RNase producer) Figure 43 : R-09 in 100 % SDW and 100% NSW Marine Difco medium All most equal production of RNase was achieved in both media at normal condition and not much effect was observed at higher saline condition at 370C.4.1.2. .4. Effect of salinity was almost 50% reduction in activity at 370C.7. Only 50% loss of stability was supported by different salinity and temperatures for both media.4. At higher temperature enzyme stability was impressive in different saline conditions for both the media (Figure 40).2.7.2.7 R-07 (RNase producer) Figure 41 : R-07 in 100 % SDW and 100% NSW Marine Difco medium This microorganism always showed good activity in 100% NSW based Marine Difco medium than in 100% SDW based Marine Difco medium in different temperature and salinity and it seemed that presence of seawater in production medium enhanced the enzyme production (Figure 41). At 800C the activity was decreased to 50% in 0% salinity and higher salinity caused further decrease in activity. 1. 1.6 R-06 (RNase producer) Figure 40: R-06 in 100 % SDW and 100% NSW Marine Difco medium The production was nearly double in case of 100% SDW based Marine Difco from 100% NSW based Marine Difco medium at normal condition.4.

1.4. Below are given the details of the combinations of the experimental samples. The substrate FDA does not work in alkaline pH.1 Es-01 (esterase producer) Figure 44: Es-01 in 100% SDW Marine Difco medium Figure 45: Es-01 in 100% NSW Marine Difco medium This microorganism produced almost same amount of enzyme in both types of media. 0. Es-03.53) shows esterase activity of the marine isolates (Es-01 to Es-05) based on the reaction conditions outlined in Table 21.D.8. The isolates had attained ample growth [O.Overall the microorganism maintained a stable activity profile in all stressed conditions (Figure 43).8. Table No.217 (1:20 dilution)] before the assay was performed. 1.2 and pH 7.1 Reaction conditions Thermostability and salt tolerance were both tested in five esterase producers (Es-01. so the experiment was limited within acidic to normal pH range. Es02.2 Results The following figures (44 .4.2) as well as in two different fermentation media (marine Difco in 100% SDW and 100% NSW).2 were prepared for this test.109 to 0.8 Esterase 1. At .8.4. Es-04 and Es-05) in two different pHs (pH 2.2.4. Preincubation at 800C for 30 min and saline condition (overnight) was done. 21: Different assay conditions for testing special enzyme properties of esterase 1.2 and pH 7. McIlvaine buffer of pH 2.

45).2 Es-02 (esterase producer) Figure 47: Es-02 in 100% NSW Difco medium Figure 46: Es-02 in 100% SDW Marine Marine Difco medium Good enzyme activity was observed in neutral condition in 100% SDW based Marine Difco medium for this microorganism and the activity was better than in 100% NSW based Marine Difco medium. 49).2. Only the productions were reduced by 40%.8.this pH effect of salinity and temperature was very little. 1. 1.4 Es-04 (esterase producer) .3 Es-03 (esterase producer) Figure 49: Es-03 in 100% NSW Difco medium Figure 48: Es-03 in 100% SDW Marine Marine Difco medium The production of enzyme at neutral condition was three times higher in 100% SDW based Marine Difco medium than from 100% NSW based Marine Difco medium. 1. In 100% NSW based Marine Difco medium the production was 60% of the normal condition. For both media at neutral pH at higher temperature the production did not reduce so much but reduction in activity was observed in case of higher salinity. The produced enzyme in both media did not show any effect with increased salinity but at higher temperature almost 50% of its normal activity was reduced.2. The most interesting feature of the enzyme was that it produced hardly activity at acidic pH in 100% SDW based Marine Difco medium. At acidic pH the productions were almost negligible with respect to normal production and at the same time no effect of salinity and temperature was observed (Figure: 48.4. At acidic pH the productions were very less with respect to normal production and at the same time no effect of salinity and temperature was observed (Figure: 46.8. but at acidic pH sea water increased the activity (Figure: 44.2.4. But it did not have any effect of salinity and temperature.4. 47).8. Production in 100% NSW based Marine Difco medium at acidic pH was slightly higher than in 100% SDW based Marine Difco medium.

5 Es-05 (esterase producer) Figure 52: Es-05 in 100% SDW Marine Marine Difco medium Figure 53: Es-05 in 100% NSW Difco medium The production of enzyme at neutral condition was almost five times higher in 100% SDW based Marine Difco medium than from 100% NSW based Marine Difco medium. The produced enzyme in both media did not show any effect with increased salinity but at higher temperature almost 50% of its normal activity was reduced.9 Nitrilase 1. At acidic pH the productions were almost negligible with respect to normal production and at the same time no effect of salinity and temperature was observed (Figure: 52. N-03. The produced enzyme in 100% SDW based Marine Difco medium did not show any effect with increased salinity but at higher temperature almost 50% of its normal activity was reduced.4.1 Reaction conditions Nitrilase was assayed in the same manner as done in cases of L-asparaginase and Lglutaminase. The enzyme activity was same for all stressed conditions.0 and .9.8. Five nitrilase producing microorganisms (N-01. 53). At acidic pH the productions were almost negligible with respect to normal production and at the same time no effect of salinity and temperature was observed (Figure: 50.Figure 50: Es-04 in 100% SDW Marine Marine Difco medium Figure 51: Es-04 in 100% NSW Difco medium The production of enzyme at neutral condition was little higher in 100% SDW based Marine Difco medium than from 100% NSW based Marine Difco medium. 1. N-04 and N05) were selected from primary screening. 1.4. N-02.2.4. In case of special property testing pH 4. 51).

D.0 4.0 800C 800C 800C 800C 0% 5% 0% 5% 0% 5% 0% 5% 1.4. Table No. But the effect of temperature was observed with . In 100% NSW based Marine Difco medium at higher salinity and temperature the activity was reduced by 60% at alkaline pH.0 8.22: Different assay conditions for testing special enzyme properties of nitrilase SDW based medium Set pH Preincubation temp. In 100% SDW based Marine Difco medium much effect of salinity was not seen in the activity of enzyme.0 8.1 N-01 ( nitrilase producer) Figure 55: N-01 in 100% NSW Difco medium Figure 54: N-01 in 100% SDW Marine Marine Difco medium The enzyme produced in 100% NSW based Marine Difco medium at alkaline condition was better than that of 100% SDW based Marine Difco medium. 0. Preincubation at high temperature and saline condition were done as described in case of other enzymes.0 NSW based medium pH Preincubation temp.0 4.9.156 to 0. Salinity 800C 800C 800C 800C 0% 5% 0% 5% 0% 5% 0% 5% Set A2 B2 C2 D2 E2 F2 G2 H2 Salinity A1 4. 22.0 B1 C1 D1 E1 F1 G1 H1 4.2 Results The following figures (54 .0 8.2.0 8.63) shows nitrilase activity of the marine isolates (N-01 – N05) based on the reaction conditions outlined in Table No.0 8. The isolates had attained ample growth [O.0 were selected as acidic and basic pH.4.9.0 8.pH 8.0 4.614 (1:20 dilution)] before the assay was performed.0 8. 4.0 was attained with citrate buffer and pH 8.0 8. pH 4.0 was attained with phosphate buffer. 1. The below given table shows the details of the each sample set.0 4.0 4.

1. In case of 100% NSW based Marine Difco medium the produced enzyme showed more or less equal reduction in different stressed conditions.2. 1.2.9.9. 59).4.2 N-02 ( nitrilase producer) Figure 57: N-02 in 100% NSW Difco medium Figure 56: N-02 in 100% SDW Marine Marine Difco medium The microorganism N-02 gave maximum production at alkaline condition in 100% SDW based Marine Difco medium and it was nearly double of the production in 100% NSW based Marine Difco medium.9.4.4. 1. At acidic pH a good production was found in 100% SDW based Marine Difco medium and at higher salinity the production remained almost unchanged but at higher temperature the production reduced by 50% (Figure: 56. A 60% reduced activity was observed at stressed conditions at alkaline pH but the activity was always found to be steady. 55). At acidic pH 100% SDW based Marine Difco medium broth gave better enzyme activity than 100% NSW based Marine Difco medium broth and in both media the enzyme was found more salt tolerant and less thermostable (Figure: 58.4 N-04 ( nitrilase producer) Figure 60: N-04 in 100% SDW Marine Figure 61: N-04 in 100% NSW .2.3 N-03 ( nitrilase producer) Figure 59: N-03 in 100% NSW Difco medium Figure 58: N-03 in 100% SDW Marine Marine Difco medium This microorganism gave better production at alkaline condition in 100% NSW based Marine Difco medium than that of 100% SDW based Marine Difco medium but the 100% SDW based Marine Difco medium produced enzyme which did not show much effect at higher salinity but at higher temperature the production was less. 57). At acidic pH the production was better in 100% SDW based Marine Difco medium and overall steady enzyme activity was observed in all other conditions (Figure: 54.reduced activity.

At acidic pH salt tolerant enzyme activity was found in 100% SDW based Marine Difco medium but activity was reduced to 35% at higher temperature and at 80 0C and 5% salinity the activity became nil in 100% SDW based Marine Difco medium (Figure: 60. 1997).5 N-05 ( nitrilase producer) Figure 63: N-05 in 100% NSW Difco medium Figure 62: N-05 in 100% SDW Marine Marine Difco medium N-05 gave maximum production at alkaline condition and almost double activity was achieved in 100% SDW based Marine Difco medium than that of 100% NSW based Marine Difco medium. In case of 100% NSW based Marine Difco medium broth no significant effect of salinity and temperature was observed (Figure: 62. At acidic pH the production was better in 100% SDW based Marine Difco medium and showed good salt tolerancy. are antagonistic to the growth or life of other microorganisms (O’Grady et al. 1. 61). 2 Bioprospecting for antibiotics Antibiotics have been defined as substances produced by microorganisms that. 63).2.Marine Difco medium Difco medium The 100% SDW based Marine Difco medium broth gave maximum production at alkaline condition and the enzyme showed good thermostability and salt tolerancy. In 100% NSW based Marine Difco medium the activity at stressed conditions was found to be 75% reduced than that of normal conditions. in high dilution.4. At alkaline pH in 100% SDW based Marine Difco medium the enzyme was found to be less salt tolerant and thermostable than that of 100% NSW based Marine Difco medium broth. At higher temperature the activity reduced to 30%. There is no doubt that the discovery of antibiotics ushered in a new age of .9.

are easy to maintain. and bacteremia. VRE and Multi-drug resistant Tuberculus bacillus were not used owing to the hazards involved in working with these organisms. It is resistant to the ‘last resort’ antibiotic. dissertation work (Ghosh. DG III and DG IV. an age when many predicted an end to diseases that have plagued us for centuries.1 Screening Four microorganisms were screened for antibacterial activity from the first field samples by the author in his M. While animals and plants of marine origin are possible sources of antibiotics along with marine microorganisms. vancomycin. The microorganisms were initially named as DG I. DG II. These four microorganisms were primarily screened against the test organism E. Staphylococcus aureus is a particularly virulent organism responsible for anything from pimples to pneumonia. Aspergillus niger. methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant Mycobacterium tuberculoris. Staphylococcus aureus. Three species of bacteria are actually of alarming concern: vancomycin-resistant Enterococus (VRE). Peerless Hospital and . Salmonella enterica and Klebsiella pneumoniae collected from a pathological unit of a local hospital. 2001). 2000). Although most antibiotics have been derived from terrestrial life. But the recent emergence of antibiotic-resistant bacteria threatens the idea that science can eradicate disease. MRSA was used in this project as indicator organism along with MSSA.medicine. it is the Earth’s last frontier–the ocean that may provide the pharmaceutical industry with the next generation of medicines to combat these recalcitrant microorganisms. and are relatively straightforward and easy to manipulate genetically. our research team has limited the exploration to bacteria by keeping the facts in mind that they reproduce quickly. VRE is a common intestinal bacterium and is one of the leading causes of hospital infections. Methicillin-resistant S. Tech. Candida albicans as well as against some pathogenic microorganisms. osteomyelitis. coli DH5α and later reconsidered for testing against Bacillus subtilis. endocartitis. Escherichia coli. If MRSA develops or acquires vancomycin resistance there will be nothing with which to fight it (Henry. 2. aureus is the most worrisome of all the multi-drug resistant bacteria because it is resistant to every presently available antibiotic except vancomycin.

Kolkata.05 g KCl.5.05 g K2HPO4. Cup-plate method: This method was similar to zones of inhibition for pure antibiotics measured in a disc agar diffusion assay using a test organism with the only difference was that here instead of using paper discs saturated with antibiotic samples. overlay assay technique (useful in case of fungus) etc. 1.7H2O. Only the fungal strains were fermented along with the sample microorganisms in fungal medium (0. niger.05 g MgSO4. Assay Techniques: Antimicrobial compounds were expected to be found in the late log phase and early stationary phase as they are secondary metabolites.c. 0. Among them cup plate method was found to be most sensitive as well as easy and trustworthy method.5 g sucrose. for 5 min at 40C to separate supernatant and cells and the supernatant were recentrifuged for another 5 min to carry on the experiments. 0. 0. Roy Research Centre. Sample cultures were centrifuged at 10 × 103 r. Culture preparation: Microorganisms were collected from respective working culture plates and inoculated into the fermentation medium. 0. Various microbiological techniques are available to assay the antibiotic lead compounds eg disc diffusion method.0 . Before filling the wells with sample supernatants the plates were swabbed with test organism cultures on the respective plates i.e. 0. from the culture broth. cup plate method.K. The hospital strains were also treated likewise but of course with extra special cautions since these strains had been identified as disease causing.5% agar) for fungal cultures (A.B. C. albicans).4 g yeast extract. E.5) kept in another incubator at 230C. aureus.f.2 g NaNO3. B. pH 5. distilled water 100 ml q. nutrient agar (Nutrient Broth with 1. Hospital strains were also swabbed like wise. The second time centrifugation showed good results for the sample isolates so it was standardized for this zone of inhibition . The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 50 ml marine Difco 2216 medium.s. sample supernatants were poured into the wells dug in the Petri plates of solid agar medium.5% agar) for bacterial test cultures (S. Bacteria cultured in nutrient broth were kept in incubator at 370C.001 g FeSO4. coli. Test culture preparation: Test organisms were cultured in the same fashion 24 hours after the samples had been inoculated. subtilis) and fungal agar medium (Fungal medium with 1.

Kolkata Resistance Pattern: Resistant : Ciprofloxacin. Clinical features of the hospital derived pathogenic bacteria: Five human isolates which were collected from the hospital are profiled below. ISOLATE: A Name of Clinical Isolates: MRSA I (Strain No. 23) the preliminary screening results are given. Cloxaicillin. 2001 Material: Throat Swab Place: Peerless Hospital & B.technique. Erythromycin. Ray Research Centre. 221005881 Date of Isolation: 26th August. Cotrimoxazole. Primary results: The bacterial plates used to show result after 12 to 20 h but the fungal plates used to give results after 30 to 40 h due to slow growth rate of fungi. 23411) Isolated from: Patient No. Vancomycin ISOLATE: B Name of Clinical Isolates: MRSA II (Strain No. Methicillin. Cefataxime. K. Ceftriaxone Sensitive : Augmentin. Nutrient broth and fungal broth supernatants were treated same like the samples and used as blanks. Norfloxacin. Ofloxacin Intermediate : Amikacin. 2001 . 23602) Isolated from: Patient No. 23: Result of primary screening of antibiotic producing microorganism against various test organisms. All the samples were used in duplicate sets to confirm the results. Doxycyclin. Table No. 221005880 Date of Isolation: 24th August. Bacterial plates were incubated at room temperature and the fungal plates were incubated at 230C. In the following table (Table No. None of them produced any zone of inhibition in any plates.

Gentamycin Sensitive : Netilmicin. Cefuroxime. Cephalexin. K. Piperacillin Intermediate : Ceftriaxone Sensitive : Augmentin. 721069794) Isolated from: Patient No.Material: Throat Swab Place: Peerless Hospital & B. 221005450 Date of Isolation: 11th August. Ceftriaxone. Norfloxacin Sensitive : Amikacin. Netilmicin. 721044438) Isolated from: Patient No. Ray Research Centre. Amikacin . Cefuroxime. Piperacillin. Place: Peerless Hospital & B. Kolkata Resistance Pattern: Resistant : Augmentin. Gentamycin. 2001 Material : Drain Tip. Doxycyclin. 221003615 Date of Isolation: 30th June. Erythromycin. Ciprofloxacin. K. Methicillin. Cefoperazome ISOLATE : D Name of Clinical Isolates: Salmonela enterica (Diagonistic Serial No. Vancomycin ISOLATE : C Name of Clinical Isolates: MSSA (Diagonistic Serial No. Kolkata Resistance Pattern: Resistant : Amikacin. Ciprofloxacin. Cloxaicillin. K. Methicillin. Ray Research Centre. Ciprofloxacin. Cefataxime. Kolkata Resistance Pattern: Resistant : Cefataxime. Cotrimoxazole. Cefoparazone. Ofloxacin. Ofloxacin Intermediate : Augmentin. Doxycyclin. Sparfloxacin Intermediate : Cefataxime. Ceftriaxone. Ofloxacin. 2001 Material: Urine Place: Peerless Hospital & B. Ray Research Centre.

2-7. Kolkata Resistance Pattern: Resistant to all common antibiotics in clinical use (natural and synthetic) After primary screening the isolate DG I was considered for further tests due to its broad spectrum activity. 0. 0. 2.4).2.0 g NaCl.5 g yeast extract.05 g yeast extract.s.4 g lactose. 1. 0. 0. 50 ml SDW q.01 g MgSO4.025 g NaCl.. pH 7. 0.3 g yeast extract. 2.0005 g ZnSO4. 0. 0. pH 7.0 g NaCl.ISOLATE : E Name of Clinical Isolates: K.0005 g KH2PO4. 100 ml SDW. aeration etc. pH 7.. 0. 0. 50 ml NSW.1 Media standardization and day wise study Four production media were designed for the following experiment. pH.4).01 g FeCl3. 1.2 Medium design for optimum antibiotic activity After detecting the antimicrobial activity of the antibiotic lead compound of the microorganism DG I. 100 ml SDW q.0 g peptone.0002 g FeSO4. Other production parameters such as dissolved oxygen consumption.6 g peptone.0 g lactose. 0. 0. 1.2 g soyabean meal.15 g beef extract. 1. 0.0005 g CuSO4. For these studies A.4).s.0005 g MnCl2. Behala. temperature. niger was considered as a test organism.0 g peptone. PMII (0. 2001 Material: Unknown Place: Das Research Laboratory. Design of the production medium and the requirement of antifoam were determined in shake flasks and the results taken up in the bioreactor. were best checked in bioreactor.27. 0. They were PM I (0.2-7. 0.0002 g . 0. PMIV (0.5 g yeast extract. the various production parameters were studied by altering the media compositions along with day wise study for maximum production as well as the effect of production with different antifoam concentrations.0005 g MnCl2. pneumoniae Isolated from: Unknown Date of Isolation: 5th September.1 g glucose. 0.0005 g K2HPO4. PMIII (0. 1.

2.2-7. Table No.4).ZnCl2. the effect of antifoam was observed and it was found that the presence of . 100 ml SDW q. This medium was considered to be best production medium for the microorganism DG I.03 g NaCl. Table No.2. 24: Production of antibiotic in different production media in different time intervals The result shows that the medium PM1 was found to give maximum antibiotic activity at 48 h. 36. pH 7. 0. The experiment was commenced in triplicate sets.4 Results for antifoam activity The below given table shows the diameters of zone of inhibitions in cm for different antifoam concentrations. This medium contains 1% lactose which was found to be a critical component of the medium. Since during bioreactor experiments the use of antifoam is compulsory for synthetic media.3 Antifoam activity Production medium PMI was considered for the experiment of the effect of antifoam (a vegetable oil) in production of antibiotic lead compound by DG I. 48. 2. 60 and 72 h. 12. considering the 0 h sample as blank. Samples were taken at 0. 2.s. 25: Effect of antifoam in the production of antibiotic from DG I in PM1. 24. The diameters of zone of inhibitions were measured in cm.2.. Antifoam was added to the PMI at various concentrations and zone diameter was measured for the samples collected after 48 h of fermentation. 2.2 Results for media standardization The following table shows the diameters of zone of inhibitions in cm for different production media.

0 showed the best result. 2. The entire experiment was done in three sets for three different pH but here only results with pH 4. The solvent was chosen with trial and error method. and 9.0) of the media supernatant was achieved in order to find at which pH the extraction was maximum and pH 4. Ethyl acetate being lighter would constitute the upper layer and the media supernatant was in the lower phase. 100 ml supernatant was considered for extraction. 2. Before extraction three different pH (4. First ethyl acetate (CH3COOC2H5) was selected and if proper extraction was not achieved then higher acetates or other organic solvents would be considered.3 Extraction of antibiotic into organic solvent The secondary metabolites produced the microorganisms accumulate in the fermentation medium. Then the entire mixture was allowed to settle for another ten minutes to be separated into two layers.3.antifoam did not affect the antibiotic production.0. The two phases were collected separately . dissolved into media water was to be extracted into organic solvent.0 are shown.3.2 Extraction procedure Fermentation media were collected and centrifuged (Eppendorf Centrifuge 5415 D) to collect the media supernatant. The fermentation was carried out at 270C with orbital shaking at 120 rpm for 48 h in 250 ml Erlenmeyer flasks containing 50 ml PMI (composition given at page: 120). Three such flasks were prepared. 7. 2. The pH of the supernatant was adjusted accordingly and the entire volume was taken into a 500 ml separating flask.0. First 30 ml solvent was mixed and shaken gently (rolling arm movement. The antibiotic lead compound. 100 ml of ethyl acetate (organic solvent) was mixed with it into three parts.1 Culture preparation DG-I was collected from respective working culture plates and inoculated into the fermentation medium. 360 0) for ten minutes.

3.0). Next the ethyl acetate was filtered and concentrated into rotary vacuum evaporator (Eyela) to collect the antibiotic lead compound by evaporating the solvent. Table No 26: Diameters of zones of inhibition of antibiotics produced by DG I before and after extraction .coli. 2. Fresh MeOH of same pH was taken as blank. A. considered for this experiment were E. The collected media supernatant was considered as experimental sample with the idea that any zone of inhibition produced by it would result unsatisfactory extraction. aureus.3 Test culture preparation Test organisms. The compound extracted from the solvent was then dissolved into MeOH (methanol) and the pH of the solution was measured (pH 6.3. 2.and the organic phase (CH3COOC2H5) was kept aside. With the collected media supernatant the same process was repeated twice with 30 ml and 40 ml CH3COOC2H5 respectively and the entire ethyl acetate extract was treated with 15% (w/v) Na2SO4 (to absorb the remaining water during extraction) for overnight. In the next table the entire results have been summed up and the zones of inhibition were measured in cm. 2. S. niger and E.3.coli (pathogenic hospital strain) and prepared in the same way as done before. To correlate with the extraction results crude media supernatant before extraction was also taken into account as samples.4 Cup-plate method The assay was done in the same fashion as done before.5 Results The results were quite satisfactory as the extracted samples exhibited bigger zones of inhibition than the crude ones and at the same time did not produce any zone for the collected media supernatant to confirm the complete extraction of the antibiotic lead compound into organic solvent (CH3COOC2H5).

3 Bioprocessing Bioprocessing. 3.50 L fermentor. as the name suggests. The aeration rate and stirrer speed were maintained at different levels in order to optimize the best condition . 1 3. This doctoral thesis deals with the upstream conversions of some enzymes and an antibiotic lead compound which had been screened previously.1. which is followed by downstream processing. Figure 66: Eyela MBF Bioreactor The working volume was 2. The culture pH and temperature was measured using inbuilt probes.0 L. Below are given the experimental details and results for each run.1 Bioreactor Run No.1 Bioprocessing of enzymes The enzyme producing microorganisms were bioprocessed in the Eyela MBF Bioreactor (Tokyo Rikakikai. The main objective was to study whether the production of enzymes and antibiotic lead compound improved through the bioreactor. 3.1.The media were sterilized ex-situ and after sterilization they were allowed to cool by circulating cold water in the jacket.1 Operating conditions Organism Agitation Microorganism 2/47 (Cellulase producing marine bacterium) 200 rpm .1. so that they would be easily cultivable commercially. The upstream conversion is the scale-up process by which the desired bioproduct is produced from the living source in large volume in a bioreactor and then the bioproduct is purified by means of downstream processing. Japan) which is a bench top submerged 2. The first one is upstream conversion. is actually a combined action of two steps. Tokyo.

pH 7.2 Offline analysis The run lasted for 32 h and 21 samples were collected.1% Nutrient Broth with 0.2) 5 ml 0. Reducing Sugar measurement: Since the cellulose was supposed to break the CMC into cellobiose and then finally into glucose (reducing sugar).1. 3. Single distilled water 1000 ml q.1. overnight kept 25 ml nutrient broth medium (composition given at page: 61).s. 0.0 g beef extract.D. Cellulase production: Each sample was assayed spectrophotometrically as done before during screening. analysis of cellulose production as well as reducing sugar. at λ 600 .5 g NaCl.3 Result The following figure (67) gives the graphical representation of cellulase production at . S21.600 for inoculum 0. 1. ………. The O. Growth measurement: Samples were properly diluted to measure O.. 5. Each sample was treated for growth measurement.Aeration Inoculum 0. It was done by mixing freshly prepared DNS reagent (preparation given at page: 56) with sample supernatant by 1:1 and cooked in a boiling water bath for 5 min.1 % CMC (3.1.0 g CMC. O.50 vvm Microorganism 2/47. They were designated as S1.281 at 24 h [1:20 dilution] Inoculum volume Antifoam Medium peptone. S2. hence the amount of glucose left in the sample can be easily assessed by measuring reducing sugar.D.1.D.0 g 3. was measured at λ 540 and concentrations of the glucose were determined with the help of a standard curve for glucose.

variation of pH.0 g CMC.75 vvm Microorganism 2/03. pH. pH 7. O.0 g beef extract.600 [1:20 dilution]. 2 3. 5.s.46 times higher than that produced in shake flask in 120 h (data not given). in terms of glucose).1. ……….1% Nutrient Broth with 0. 1000 ml SDW q. S11. 1.2. Production (g/100 ml.1 % CMC (3. S2. Each sample was treated for growth measurement.0 g 3. in terms of glucose) Figure 67: production of cellulase in Bioreactor by micoorganism 2/47 With in 36 h the maximum production (Figure 67) of cellulase was achieved which was found to be almost 2..D.600 for inoculum 0.2) 5 ml 0. pO2. pO2 and consumption of D-glucose Graph legend: Time in hour.different time intervals along with the growth of microorganism.1.2.402 at 24 h [1:20 dilution] Inoculum volume Antifoam Medium peptone.1. Growth O. Reducing sugar (g/100 ml.2 Bioreactor Run No. 0.5 g NaCl. analysis of cellulose .D. 3.2 Offline analysis The run lasted for 10 h and 11 samples were collected. They were designated as S1.1 Operating conditions Organism Agitation Aeration Inoculum Microorganism 2/03 (Cellulase producing marine bacterium) 200 rpm 0. overnight kept 25 ml nutrient broth medium (composition given at page: 61).

Reducing Sugar measurement: As done in case of Bioreactor run No. Production (g/100 ml.1. in terms of glucose) Figure 68: production of cellulase in Bioreactor by micoorganism 2/03 In this bioreactor run maximum production (Figure 68) of cellulase was achieved in 9 h and which was almost 3 times higher than that produced in shake flask after 120 h (data not shown).1.3 Results The following figure (68) gives the graphical representation of cellulase production at different time intervals along with the growth of microorganism. This bioreactor sample was treated for checking special enzymatic properties which have been discussed under section 1.1. pH.1 Operating conditions Organism Agitation Aeration Inoculum Microorganism 2/47 (Xylanase producing marine bacterium) 200 rpm 0.4.production as well as reducing sugar. pO2. Growth measurement: Samples were properly diluted to measure O. overnight kept 25 ml nutrient broth medium .2. at λ 600 .3.D. 1 (page: 129).3. 3. pO 2 and consumption of D-glucose Graph legend: Time in hour.600 [1:20 dilution].D. variation of pH.1 3. Growth O.3 Bioreactor Run No.4. in terms of glucose). Cellulase production: Each sample was assayed spectrophotometrically as done before during screening. 3 3. Reducing sugar (g/100 ml.50 vvm Microorganism 2/47.

hence the amount of xylose left in the sample can be easily assessed by measuring reducing sugar.3 Results The following figure (69) gives the graphical representation of xylanase production at different time intervals along with the growth of microorganism.240 at 24 h [1:20 dilution] Inoculum volume Antifoam Medium peptone.2 Offline analysis The run lasted for 12 h and 13 samples were collected.3. 1.D. Reducing Sugar measurement: Since the xylanase was supposed to break the xylan into xylobiose and then finally into xylose (reducing sugar). It was done by mixing freshly prepared DNS reagent (preparation given at page: 56) with sample supernatant by 1:1 and cooked in a boiling water bath for 5 min.5 g NaCl.3.0 g oat spelt xylan. SDW 1000 ml q. S13. was measured at λ 540 and concentrations of the xylose were determined with the help of a standard curve for xylose. O. pO2 and consumption of xylose.s.D.2) 5 ml 0. pH 7. 0. analysis of xylanase production as well as reducing sugar. Growth measurement: Samples were properly diluted to measure O. variation of pH. S2. 5. The O.1. at λ 600 .1 % xylan (3.0 g 3. .600 for inoculum 0. 3.. ……….1% Nutrient Broth with 0.(composition given at page: 61).1. Xylanase production: Each sample was assayed spectrophotometrically as done before during screening.D. They were designated as S1. Each sample was treated for growth measurement.0 g beef extract.

D. S2.600 [1:20 dilution].4.D.4.4. Reducing sugar (g/100 ml. S11.5 times higher than that produced in shake flask in 120 h (data not given).1% Marine Difco 2216 at 100% SDW (composition given at page: 51) Offline analysis The run lasted for 10 h and 11 samples were collected.3 Results The following figure (70) gives the graphical representation of DNase production at different time intervals along with the growth of microorganism. in terms of xylose) Figure 69: production of xylanase in Bioreactor by micoorganism 2/47 The maximum production (Figure 69) of xylanase was achieved within 10 h and it was found to be almost 3. in terms of xylose).50 vvm Microorganism 2/01. 4 Operating conditions Microorganism 2/01 (DNase producing marine microorganism) 200 rpm 0. overnight kept 25 ml nutrient broth medium (composition given at page: 61).1 3. 3.3. pO2. pH.4. variation of pH and . Growth measurement: Samples were properly diluted to measure O. DNase production: Each sample was assayed spectrophotometrically as done before during screening. O.1.1.1.5.1 Organism Agitation Aeration Inoculum h [1:20 Inoculum volume Antifoam Medium 3. at λ 600 .Graph legend: Time in hour. Growth O.D.204 at 24 dilution] 5 ml 0.2 Bioreactor Run No.4 3. This bioreactor sample was treated for checking special enzymatic properties which have been discussed under section 1. ………. Each sample was treated for growth measurement and analysis of DNase production. They were designated as S1. Production (g/100 ml.600 for inoculum 0.1.

600 [1:20 dilution].1 Operating conditions Organism Agitation Aeration Inoculum Microorganism R-01(RNase producing marine microorganism) 300 rpm 0. Graph legend: Time in hour. O. 3. Production (U.1% [1:20 dilution] Marine Difco 2216 at 100% SDW (composition given at page: 51) 3. S11. at λ 600 .D. S2.3 Results .221 at 24 h Inoculum volume Antifoam Medium 5 ml 0. RNase production: Each sample was assayed spectrophotometrically as done before during screening. overnight kept 25 ml nutrient broth medium (composition given at page: 61).5.D.ml-1) Figure 70: production of DNase in Bioreactor by micoorganism 2/01 3.5. Growth O.D.2 Offline analysis The run lasted for 10 h and 11 samples were collected.5. pH.50 vvm Microorganism R-01. pO2.1. They were designated as S1. 5 3.1.1.600 for inoculum 0.pO2.1. ………. Growth measurement: Samples were properly diluted to measure O. Each sample was treated for growth measurement and analysis of RNase production.5 Bioreactor Run No.

O. S12.D.600 for inoculum 0.6 Bioreactor Run No.1.1 Operating conditions Organism Agitation Aeration Inoculum medium Microorganism Prot-105 (protease producing marine bacterium) 300 rpm 0. pH.1.ml-1 which exhibited a 2. Each sample was treated for growth measurement and analysis of protease production. Growth O. Protease production: Each sample was assayed spectrophotometrically as done before during screening. variation of pH and pO2. pO2.6. Production (U.2 fold increase in activity than the fermentation done in shake flask 3. S2.204 at 24 h Inoculum volume Antifoam Medium 3.D. Growth measurement: Samples were properly diluted to measure O.6. Graph legend: Time in hour. ………. at λ 600 .The following figure (71) gives the graphical representation of RNase production at different time intervals along with the growth of microorganism.ml-1) Figure 71: production of RNase in Bioreactor by micoorganism R-01 At 9 h the microorganism achieved maximum production of 2147 U. overnight kept 25 ml nutrient broth (composition given at page: 61).D.75 vvm Microorganism Prot-105. They were designated as S1. .1% [1:20 dilution] Marine Difco 2216 at 100% SDW (composition given at page: 51) Offline analysis The run lasted for 11 h and 12 samples were collected. 6 3.2 5 ml 0.1.600 [1:20 dilution].

s.0 g NaCl. Growth O.1. variation of pH and pO2. pH.600 for inoculum 0. 10.D.ml-1) Figure 72: production of protease in Bioreactor by micoorganism Prot-105 3. Production (U. O.0% [1:20 dilution] 5.D. 10.1 Operating conditions Organism Agitation Aeration Inoculum medium Microorganism DG I 150 rpm 1.1.1 Bioreactor Run No. pH 7. pO2.135 at 24 h Inoculum volume Antifoam Medium 1000 ml 10 ml 1. SDW q.0 g yeast extract. Japan).. 10.3.4 .2 Bioprocessing of antibiotics The antibiotic lead compound DG I was bioprocessed in the same bioreactor (Tokyo Rikakikai.0 g peptone.3 Results The following figure (72) gives the graphical representation of protease production at different time intervals along with the growth of microorganism.0 g lactose.2-7.2. Tokyo. overnight kept 25 ml Marine Difco 2216 (composition given at page: 51). 1 3.0 vvm Microorganism DG I. Graph legend: Time in hour.6.600 [1:20 dilution]. 3.2.

1. pO2.3.1.1 for DG I 3.600 [1:20 dilution]. 2 3.D.f. microbial growth and lactose utilization at different time intervals. S10.2 Off line analysis The process was on for 27 h and 10 samples were collected as S1. 26 h rpm . niger). Assay of the lactose utilization: Freshly prepared DNS reagent was mixed with sample supernatant by 1:1 and kept in a boiling water bath for 5 min.D.1 Organism Agitation Aeration Inoculum medium Operating conditions Microorganism DG I 150 rpm (After 4 h rpm – 200. For each sample the cells were separated by centrifugation (10 x 103 r.2. overnight kept 25 ml Marine Difco 2216 (composition given at page: 51).3 Result The following figure (73) represents the production. Antibiotic assay: Samples were taken and then 150 µL of the supernatant was added to the wells in the nutrient agar plates which were previously swabbed with an overnight culture of the test organism (A. The O.300 & 27 h rpm – 350) 1.2. Time in hour Figure 73: Bioreactor Run No.2.2.2 Bioreactor Run No. 3.c. Production in mm.145 at 24 . After 30 h the Petri plates were observed for zone of inhibition and the diameter of zone of inhibition were measured. O.0 vvm Microorganism DG I. S2.D.. Graph Legend: Growth O. pH. pH and pO 2 variation. Lactose utilization (g/l). for 5 min). The supernatant were used for different off-line analyses.600 for inoculum 0.2.……. was measured at λ 540 and concentrations of the lactose were determined from the standard curve.

3 Bioreactor Run No.5 g beef extract. pH 3. Antibiotic assay: Samples were taken and then 150µL of the supernatant was added to the wells in the nutrient agar plates which were previously swabbed with an overnight culture of the test organism (A.c..h Inoculum volume Antifoam Medium glucose. After 30 h the Petri plates were observed for zone of inhibition and the diameter of zone of inhibition were measured.2 for DG I The maximum antibiotic production was achieved at 32 h during the late log phase of the microorganism and at that time medium pH was in neutral range and pO 2 value was 22.. pH.……. 3.2. The O.s.002 g FeSO4. pH and pO 2 variation.2-7. 1000 ml SDW q.1.4) [1:20 dilution] 10 ml 1.0. microbial growth and glucose utilization at different time intervals. 0. Glucose utilization (g/l).002 g ZnSO4. 3.005 7.0% (1. Production in mm.3 g NaCl.D. 10. 3. for 5 min).f. 0. S13.2. 0. pO2.2. niger).005 g KH2PO4. 0.2. Time in hour Figure 74: Bioreactor Run No.0 g 0. 6.2.1 g MgSO4. S2. Assay of the glucose utilization: Freshly prepared DNS reagent was mixed with sample supernatant by 1:1 and kept in a boiling water bath for 5 min.2 Off line analysis The process was on for 49 h and 13 samples were collected as S1. The production almost increased 1. Graph Legend: Growth O. was measured at λ 540 and concentrations of the glucose were determined from the standard curve. g MnCl2. 3 .3 Result The following figure (74) represents the production.0 g peptone. The supernatant were used for different off-line analyses.005 g K2HPO4.D.600 [1:20 dilution].5 fold in bioreactor than in shake flask and till that time 73% of the D-glucose was consumed by the microorganism. For each sample the cells were separated by centrifugation (10 x 103 r.0 g yeast extract. 0.

Assay of the lactose utilization: Freshly prepared DNS reagent was mixed with sample supernatant by 1:1 and kept in a boiling water bath for 5 min.. 0.2.3.2-7.600 for inoculum 0.1 g FeCl3.3. pH 7. For each sample the cells were separated by centrifugation (10 x 103 r.25 g NaCl.0 vvm (after 24 h aeration 0. niger).0% (4. 0.2.005 g MnCl2.3 Result . After 30 h the Petri plates were observed for zone of inhibition and the diameter of zone of inhibition were measured.3.005 g SDW q.D. 3.f. 0. 1000 ml 10 ml 1.s.2 Off line analysis The process lasted for 32 h and 13 samples were collected as S 1. Antibiotic assay: Samples were taken and then 150µL of the supernatant was added to the wells in the nutrient agar plates which were previously swabbed with an overnight culture of the test organism (A.c.3. 0.0 g soyabean meal. ZnSO4.0 g lactose.1 Operating conditions Organism Agitation Aeration Inoculum medium Microorganism DG I 350 rpm (after 24 h agitation .0..4) [1:20 dilution] 3. S2.5 g yeast 0. O.D. S13. for 5 min). overnight kept 25 ml Marine Difco 2216 (composition given at page: 51). was measured at λ 540 and concentrations of the lactose were determined from the standard curve.180 at 24 h Inoculum volume Antifoam Medium extract.200) 1.2. The O.5 vvm) Microorganism DG I. The supernatant were used for different off-line analyses. 2.…….005 g CuSO4.

for 5 min).0 g NaCl. 4 3.1 Organism Agitation Aeration Inoculum medium Operating conditions Microorganism DG I 200 rpm 1.4 Bioreactor Run No. O.600 for inoculum 0.600 [1:20 dilution]. 10.f. pO2.2 [1:40 dilution] 10 ml 1. Graph Legend: Growth O. . microbial growth and lactose utilization at different time interval. 1000 ml NSW pH 7. S12. 3 for DG I The maximum antibiotic production was achieved at 29 h during the late log phase of the microorganism and at that time medium pH was in slightly basic range and pO2 value was in over range.2) Off line analysis The process lasted for 44 h and 12 samples were collected as S 1.c.. The production almost increased 1.s.D.1 fold in bioreactor than in shake flask and till that time 51% of the lactose was consumed by the microorganism. Time in hour Figure75: Bioreactor Run No. Production in mm. pH and pO 2 variation.2.D.4. Lactose utilization (g/l). 3. The supernatant were used for antibiotic assay.The following figure (75) represents the production.……..0% (5.2.0 g yeast extract. For each sample the cells were separated by centrifugation (10 x 103 r. pH. 3. overnight kept 25 ml Marine Difco 2216 (composition given at page: 51). S2. 10.0 g peptone.2.280 at 24 h Inoculum volume Antifoam Medium q.4.0 vvm Microorganism DG I. Antibiotic assay: Samples were taken and then 150 µL of the supernatant was added to the wells in the nutrient agar plates which were previously swabbed with an overnight .

pO2.2.culture of the test organism (A.2.4. Time in hour Figure 76: Bioreactor Run No. . 3. Production in mm.D.3 Result The following figure (76)gives the antibiotic production at different time intervals along with bacterial growth and pH and pO2 variation Graph Legend: Growth O. After 30 h the Petri plates were observed for zone of inhibition and the diameter of zone of inhibition were measured. pH. niger).600 [1:20 dilution]. 4 for DG I The maximum antibiotic production was achieved at 22 h during the mid log phase of the microorganism and at that time medium pH was in slightly basic range and pO 2 value was 0. The production almost increased 1.1 fold in bioreactor than in shake flask.

DISCUSSION .

1.1.2 1.1.2.1.1.1.2 1.1. 1 1.2.3 1.2.1.1 1.2.1 Bioprospecting in deltaic Sundarbans Marine enzymes Screening of marine enzymes Special enzyme properties L-asparaginase L-glutaminase Protease Cellulase Xylanase DNase RNase Esterase Nitrilase Overall assessment of marine enzymes Marine antibiotics Antibiotic profile of screened antibiotic lead compounds Standardization of optimum antibiotic production by microorganism DG I 1.2.1.9 1.1.2.1 1.2.2.1.1.7 1.5 1.1.2.3 1.4 1.8 1.2.The entire experimental work has been discussed under the below given headings.3 1.2.2 1.1.2 1.1.2 Extraction of antibiotic lead compound in organic solvent Bioreactor study of the antibiotic producer DG I Overall assessment on marine antibiotic lead compound .6 1.2.2.1 1.1 1.2.

2004).1. Since no published data of microbiological survey of this area was available. The entire work is now discussed in two headings. Isolating and bioprocessing the bioactive compounds from the marine microorganisms is an uphill task and a great deal of theoretical and practical knowledge was needed to accomplish the job. this project targeted to isolate bioactive compounds from marine microorganisms isolated from the sediment sea water of this area. The usefulness of terrestrial microorganisms as a source of bioactive compounds have already been established with numerous published journals and books and since the last 50 years the need to search for new bioactive compounds has led us to look into perhaps mankind’s last frontier in Earth – the water world.1 Marine enzymes In order to isolate marine enzymes the first job was to isolate marine microorganisms from soil and water samples and out of total 1050 microbial isolates a total of 378 true marine bacteria were isolated in four fields. one being marine enzyme and other being marine antibiotic. India.1 Screening of marine enzymes .1 Bioprospecting in deltaic Sundarbans This doctoral work exhibits the first ever bioprospecting work for commercially important marine enzymes and antibiotic lead compounds in the deltaic Sundarbans of Bay of Bengal. 1. The current work on marine enzymes will be published shortly as a review (Ghosh et al. 1.

The maximum activity achieved by the microorganism was Asp-05 in normal condition and that is 24.2. Dnase. the best producers were again screened under various stressed conditions in order to find any enzyme producers those produced high thermostable and high salt tolerant (5% w/v NaCl) enzyme and the found producers were recultivated in bioreactor. L-asparaginase.1.0) the enzyme activity were measured in two saline conditions (0% and 5% w/v NaCl) as well as in two different temperatures (370C and 800C). In two pH (4. nitrilase and peroxidase). All the assay techniques were standardized and each true marine bacteria were screened for enzymatic activity. In case of all the microorganisms (Asp-01. 370C incubation and 0% salinity.1 L-asparaginase The fermentations of microorganisms producing L-asparaginase were performed in both 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium. The assay condition during primary and secondary screening was 8. 1. Asp-04 and Asp-05). L-glutaminase. protease. RNase.0 and 8. xylanase. in which maximum enzyme activity was obtained. it was found that production was always better in 100% SDW Marine Difco medium. Asp-02. esterase.1.2 Special enzyme properties From each type of screened enzyme producers. cellulase.0 pH. 1. The average activity of the microorganisms in alkaline condition was nearly 20 .The isolated true marine bacteria were screened for 10 different commercially important enzymes (amylase. So it can be concluded that the effect of sea water was not so important for these five Lasparaginase producers. The enzyme activity was tested in various stressed conditions.6 U/ml but over all the activity of Asp-03 was found to be good in all stressed conditions. Apart from amylase and peroxidase all other enzyme producers were successfully screened. Asp-03.

The microorganism Prot-105 was found to be most impressive among the microorganisms tested and it gave the highest production in all conditions.1. pH 7. 1.U.ml-1 and in stressed conditions the average activity is nearly 10 U. which proves the thermostability.ml-1.0) the enzyme stability was measured in two saline conditions (0% and 5% w/v NaCl) as well as in two different temperatures (370C and 800C).2.0 pH.0.2. pH stability as well as salt tolerance of the enzyme. The enzyme activity was tested in various stressed conditions. Between the two microorganisms Glut-01 and Glut-02.5 pH. The enzyme activity was tested in various stressed conditions. 370C incubation and 0% salinity.5) the production of protease was measured in two saline conditions (0% and 5% w/v NaCl) as well as in two different temperatures (370C and 800C) (Table No. So it can be concluded that in stressed conditions the produced enzyme retained 50-55% of the activity.3 Protease The fermentations of microorganisms producing protease were performed in both 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium.:14). 1. the later exhibited maximum activity and both the microorganisms retained 50% of their normal activity in stressed conditions. In two pH (4.1.0. 370C incubation and 0% salinity. The assay condition for primary and secondary screening was 8.0 and 8.0 and 8. In three different pH (6.2 L-glutaminase The fermentations of microorganisms producing L-glutaminase were performed in both 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium. 7. The assay condition for screening was 8. All the five microorganisms (Prot-101 to Prot-105) showed good stability in different stressed conditions and it was observed that sea water did not have much effect in the production as well as the stability of the enzyme. So this microorganism was further studied in bioreactor and . 370C incubation temperature and 0% salinity was considered as neutral condition.

6 DNase The fermentation of DNase producing microorganisms 2/01 and 2/22 were both . It was found that the stability of the produced cellulase by microorganism Cel2/03 was satisfactory and the enzyme could be considered as thermostable and little a slightly salt tolerant (Figure: 29). The special enzyme properties were only checked with the bioreactor broth of the microorganism Cel-2/03 (Bioreactor Run No.0 (Figure: 30). 1. 3).5 Xylanase The enzyme xylanase was also initially screened with Congo Red like cellulase producers and later the selected ones were fermented in shake flasks and it was observed that the production was very poor in shake flasks. 2).1.the enzyme production was sucessfully scaled up (Figure 72). The special enzymatic properties were also checked with the bioreactor broth (Bioreactor Run No.2. Among them microorganism 2/47 was treated in bioreactor and checked the reproducibility of the enzyme. Among them two best cellulase producers.4 Cellulase The enzyme cellulase was initially screened with Congo Red and later the selected ones were fermented in shake flasks and it was observed that the production was very poor in shake flasks. 1.2.1. It was found that the stability of the produced xylanase by microorganism 2/47 was satisfactory and the enzyme could be considered as thermostable and salt tolerant at pH 7.1. 1.2.0 and 9. microorganism Cel-2/03 and microorganism 2/47 were treated in bioreactors and checked their reproducibility.

All the nine microorganisms exhibited an impressive activity profile at two different salinity and incubation temperature for both the media and maintained a noteworthy stability at higher temperature and salinity. microorganism R-01 produced maximum amount of enzyme at normal condition and the enzyme showed best stability at different stressed conditions in both media. Table No. The enzymes were treated in acidic and neutral pH with 0% and 5% salinity at 400C and at 800C. R-02. R-07. The microorganisms’ produced esterase were all active at neutral pH and . 1.1. Es-03. R-05. The assay condition for screening was pH 5. R08 and R-09) were selected for checking special enzymatic properties in both 100% SDW based Marine Difco medium and in 100% NSW based Marine Difco medium at pH 5 with 0% and 5% salinity and at 370C and 800C temperature.2 pH.2. R-06.0. which led all of their produced enzymes to be thermostable and salt tolerant. 0% salinity and room temperature (RT) incubation. 0% salinity and 7. 1.2. Es-02. Special enzymatic properties were tested at RT and 800C temperature and 0% and 5% salinity.7 RNase Nine RNase producing microorganisms (R-01.performed in shake flasks in 100% SDW based Marine Difco medium as well as in 100% NSW based Marine Difco medium. 20 gives a clear picture of different reaction conditions. Among these two microorganisms 2/01 and 2/22. 370C temperature incubation and 0% salinity.0. R-04. The reaction condition for the primary and secondary screening of esterase producers was 400C incubation. Es-04 and Es-05) were treated for checking special enzyme properties with the shake flask fermentation of two different types of media.8 Esterase Five esterase producing microorganisms (Es-01. microorganism 2/01 was found to be most impressive and it was cultivated in bioreactor for reproducibility. Among them.1. 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium. R-03. The assay condition for screening was pH 5. This microorganism was further cultivated successfully in bioreactor in order to reproduce the enzyme.

1.1. Marine enzymes producers found here enlightened us to proceed with further bioprospecting work in this part of the world as the microbial biodiversity of the entire area is not fully explored.9 Nitrilase The fermentations of microorganisms producing nitrilase were performed in both 100% SDW based Marine Difco medium and 100% NSW based Marine Difco medium. 250C incubation and 0% salinity in which maximum enzymatic activity was obtained and the activity was measured in terms of liberated NH 3 from NH4NO3.3 Overall assessment of marine enzymes The search of commercially important marine enzymes from microbial source in Sundarbans region of Bay of Bengal. The assay condition for primary and secondary screening was 8. The microorganism Es-03 produced maximum enzyme at neutral condition in 100% SDW based Marine Difco medium and seawater did not increase the enzyme production. India was done for the first time and this thesis work represents the entire screening work. The produced enzyme showed almost no activity at acidic pH. Overall the production of nitrilase in 100% SDW based Marine Difco medium was found to be better and no dominant effect of sea water was observed.0 and 8. Being active at .2.especially in 100% SDW based Marine Difco medium.0) the enzyme stability was measured in two saline conditions (0% and 5% w/v NaCl) as well as in two different temperatures (250C and 800C). Some of the enzymes showed unexpectedly good thermostability and salt tolerance and most of them are effective in a wide range of pH. Some of the microorganisms were bioprocessed in bioreactor and their enzyme productions were scaled up successfully. The enzyme activity was tested in various stressed conditions. In two pH (4. The microorganism N-04 was found to be best producer of nitrilase among the microorganisms tested and this microorganism gave a very good enzyme activity profile in all stressed conditions. 1.0 pH.1.

(Biotechnology) dissertation works (Ghosh.2 Marine antibiotics The search for new marine antibiotic lead compounds was initially done by the author in his M.2. 2001) and the activities were tested against E.2. The PMI was considered to be best production medium for this antibiotic producing DG I. coli DH5α .1. DG II. niger at an interval of every 12 h. DG I was found to have maximum anti bacterial and anti fungal activity and being a marine Pseudomonous sp. Since during bioreactor experiments the use of antifoam is compulsory for synthetic media the effect of antifoam was observed by adding the antifoam in various concentrations (from 0% to .1 Antibiotic profile of screened antibiotic lead compounds In order to find new bioactive compounds from marine source the microorganisms were treated with a number of bacteria and fungi as test organisms along with some recalcitrant pathogenic strains. Four different bacterial antibiotic production media were designed and the activity of the antibiotic was tested from the fermentation broth of those media in shake flasks against A. 1.1 Standardization of optimum antibiotic production by microorganism DG I Among the four screened microorganisms. Four microorganisms (DG I. DG III and DG IV) were found to be effective against most of the strains and they were summarized in Table No. Those microorganisms were further screened with various test organisms.high temperature and high saline conditions it is expected that they can be purified easily so that they will be cost effective during their commercial production. 23. 1. (GenBank Accession Number: AY546087) with anti fungal activity the further study was continued with this microorganism. 1.Tech.

the presence of lactose proved to be critical for antibiotic production and its maximum utilization occurred during log phases when the production was also found to be maximum. In bioreactor also.2. The extracted sample gave bigger zones of inhibition with respect to the fermented broth of PMI (Table No.2 Extraction of antibiotic lead compound in organic solvent The antibiotic was extracted with ethylacetate and the maximum extraction was achieved by lowering the pH of the fermented broth of PMI at 4.0.1.0 as it was seen that maximum extraction was achieved at this acidic pH instead of pH 7. 1.3 Bioreactor study of the antibiotic producer DG I The fermentation of DG I was again performed in bioreactor in order to scale up the antibiotic production. like shake flask fermentations.2 Overall assessment on marine antibiotic lead compound All in all the microorganism DG I can be considered as a very promising candidate for good antibiotic lead compound producer as the produced antibiotic can be easily extracted and a very simple synthetic medium is required for the optimum antibiotic production. 1.2.1 was found to be most promising and it showed the maximum scaled up production. . In this study the media was altered in each run and the rate of agitation and aeration were standardized.1.0 and 9. It was also observed that complete extraction was achieved with ethyl acetate and the antibiotic was dissolved in MeOH. 26).2. 1.1% v/v) in shake flask fermentation with PMI medium and it was observed that the presence of antifoam did not affect the antibiotic production. Among the 4 bioreactor runs Run No.

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