Isolation of Aspergillus niger from soil and production of Amylase enzyme
The present study covers the isolation of fungi Aspergillus niger from garden soil of government science collage, K.K.Shastri campus, maninager, Ahmedabad. The fungi was identified using lactophenol cotton blue staining method and was tested in starch hydrolysis agar medium for its amylase enzyme production; a highly demanded industrial enzyme in various sectors such as food, pharmaceuticals, textiles, detergents,etc. It also includes fermentative production of Amylase enzyme in submerged aerobic fermentation process using the isolate. The effect of different carbon source, nitrogen compound and physico-chemical conditions like temperature, pH and incubation periods were studied for derivation of amylase enzyme.
Key words: Aspergillus niger, amylase enzyme, submerged aerobic fermentation
Y. this research project would not have been possible without the confidence. will be useful in different stages of our lives.M. Dalal and our Head of the Department Dr. We are sincerely thankful to our supervisor. Yagnik for their wholehearted support and cooperation in producing the subject matter. Miss. Miss. Apart from the subject of our research. Mr. B. Tallika Patel for her guidance. which we are sure.
Authers: Khushboo. endurance and support of our families. Finally. Deepmala Lawani. We wish to thank our parents whose love.N. My sincere gratitude also goes to all those who instructed and taught me through the year. it would have been difficult to keep up the constant high spirit of work. understanding and patience. Y. teachings and support have brought us this far. Chirag Shah. We are greatly indepted to express our gratitude to the Almighty for such strength through the journey on path that leads to success. Arpan Bhatt for many helpful comments. We are especially grateful to our colleagues for their assistance. criticisms and useful insights. Sheth
. Last but not the least thanks to all readers for their keen interest in our work. We also like to express our special thank to our lab assistant Mr. we learnt a lot from her. We are thankful to our principal Dr. Mr. Mr. Manan thakker and Mr. Our families have always been a source of inspiration and encouragement. We wish to thank our brothers and sisters for their affection. Mrs. Omkar Panchal. Without her valuable guidance and time spent behind us. Pratima sharma. Ramesh Thoda. help and motivation.ACKNOWLEDGEMENT
We take this opportunity with much pleasure to thank all the people who have helped us through the course of our journey towards producing this research paper. We would like to express gratitude to the other faculty members Miss. Chinki Soni. without whom support the study could not be completed fairly. We hope you would undoubtedly find the matter interesting and informative as well. We are thankful to all the other fellow students (our seniors) of Government Science College (khokhara) with whom we share tons of fond memories. We would like to acknowledge the support and encouragement of all our friends.
Amylases from plant and microbial sources have been employed for centuries as food additives. They are synthesized in cells by the normal machinery of protein synthesis. Nowadays the new potential of using microorganism as biotechnological sources of industrially relevant enzymes has stimulated renewed interest in the exploration of extra cellular enzymatic activity in several microorganisms. nucleotides. The enzyme then folds spontaneously into its active conformation. Amylases are starch-degrading enzymes that catalyze the hydrolysis of internal a-1. and vitamins which can be added to food to enhance its flavor or increase its nutritive value. Microbial production of primary metabolites contributes significantly to the quality of life. Bacillus licheniformis. Due to the increasing demand for these enzymes in various industries. It is produced by a variety of living organisms ranging from bacteria. microorganisms growing on inexpensive carbon sources can produce valuable products such as enzymes. 15]. microbial sources. low-cost medium is required for the production of a-amylase. Bacillus amyloliquifaciens and Aspergillus niger. Posttranslational modifications may be required to target an enzyme to its ultimate intracellular or extracellular location.55]. Vyas
Isolation of Aspergillus niger from garden soil for the production of amylase enzyme and effect of various parameters on enzyme production is the main aim of this study. Although many microorganisms produce this enzyme. Barley amylases have been used in the brewing industry. Enzymes are protein catalysts synthesized by living systems and are important in synthetic as well as degradative process.4-O-glycosidic bonds in polysaccharides such as starch in to simple sugar (glucose & maltose). whose DNA base sequence is transcribed into a messenger RNA. Both SSF and submerged fermentation (SmF) could be used for the
. Enzymes are catalysts of biological processes. less time and space required for production and ease of process modification and optimization . the ones most commonly used for their industrial production are Bacillus subtilis. organic acids. Through fermentation. consistency. P. Enzymes (including amylases) are the primary metabolites of microorganisms. amino acids. A. Patel Deval. there is enormous interest in developing enzymes with better properties such as raw starch degrading amylases suitable for industrial applications and their cost effective production techniques. To meet the demand of industries. and the mRNA us translated from its triplet code into the amino acid sequence of the desired protein by the ribosome and associated factors [54. Among mold species producing high levels of amylase. fungi to plants and humans [14. namely fungal and bacterial amylases. The structure of any given enzyme is encoded by a structural gene.Yesha. Aspergillus niger is used for commercial production of alpha amylase. This enzyme is an extra–cellular enzyme and therefore can be easily separated from the cell mass. In spite of the wide distribution of amylases. are used for the industrial production due to advantages such as cost effectiveness. Fermentation can be defined as aerobic or anaerobic oxidation of substrate to form the product of high value using microorganisms. Fungal amylases have been widely used for the preparation of oriental foods.
The reaction is also dependent on pH and a working range of pH 9 to 10.production of amylases. liquid medium was used by shake flask method carried out at laboratory scale. because of its low cost. For amylase production. the incubation time is very critical for a reproducible assay.5 is essential. Also. and available technology. high purity and ready availability. Folin Lowry’s method is sensitive down to about 10µg/mL and is probably the most widely used protein assay. Purification processes in downstream processing after fermentation strongly depend on the market. with low risk of contamination and lesser chances of media and material wastage. In this method.
. final quality. although traditionally these have been obtained from submerged cultures because of ease of handling and greater control of environmental factors such as temperature and pH. Shake flask method is best suited at laboratory scale as maximum product formation can be obtained at low cost and a large number of experimental values can be evaluated in minimum time. optimum conditions for maximum enzyme production can be set for production at industrial scale. processing cost. Most enzymes are purified by chromatographic techniques after crude isolation by precipitation with ammonium sulphate and membrane separations .
Protein estimation in any sample can be carried out by following methods: (1) Biuret method (2) Folin Lowry’s method Most protein estimation techniques use Bovine Serum Albumin (BSA) universally as a standard protein.
REVIEW OF LITERATURE
Aspergillus niger is used for commercial production of alpha amylase. ß-amylase (Beta-amylase) . α-amylase (alpha-amylase) . Few attempts have been made to elucidate the control mechanism involved in the formation and secretion of the extra cellular enzymes. thereby producing maltose iii. stability of the generated products. or transglycosylating enzymes.Breaks successive bonds from the non-reducing end of the straight chain.Breaks the glucose-glucose bonds down by removing two glucose units at a time. Amyloglucosidase (AMG) . 6]. to the production of cyclodextrins for the pharmaceutical industry.Amylases:
Although amylases are widespread in nature. Amylases are one of the most important industrial enzymes that have a wide variety of applications ranging from conversion of starch to sugar syrups. Among mold species producing high levels of amylase. therefore producing varied sized chains of glucose ii. lower energy requirements and elimination of neutralization steps. These enzymes account for about 30 % of the world’s enzyme production . Amylases are an extracellular enzyme and are classified based on how they break down starch molecules i. The enzymatic hydrolysis is preferred to acid hydrolysis in starch processing industry due to a number of advantages such as specificity of the reaction. The amylase family can roughly be divided into two groups: The starch hydrolyzing enzymes and the starch modifying. To prepare these extra cellular enzymes on a commercial scale. many attempts have been made to specify cultural conditions and to select superior strains of the fungus [5.Reduces the viscosity of starch by breaking down the bonds at random. microbes serve as a preferred source of these enzymes because of their rapid growth. producing glucose Many microbial amylases usually contain a mixture of these amylases.
. the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their various applications.
[A] REAGENTS: Enzyme production:(1) Starch agar medium (gm/100ml):-
Soluble starch Beef extract Agar Distilled water pH
: : : : :
1.7H2O Soluble starch pH
: : : : : : :
1.1 0.2 0.
.7H2O FeSO4.5 0. Dissolve separately soluble starch in about 25ml of warm distilled water.
(2) Fermentation medium (gm/liter):-
KH2PO4 NH4NO3 KCl MgSO4.01 20 6. Medium is used for detection of amylase production (starch hydrolysis) by organism from soil sample. Allow to cool down to room temperature. Autoclave for 15mins at 15 lbs pressure and at 121ºC.4 10 0.3 3. Mix the above solution and adjust the final volume to 100ml.0 100ml 7.5
Distribute in 100 ml of medium into 250 ml Erlenmeyer flasks and sterilize by autoclaving at 121ºC for 15 minutes.5
Dissolve by heat beef extract and agar in 50ml of distilled water.
Sonar Axiva). Ltd.2)
(2) Ammonium sulphate Enzyme assay:(1) Starch hydrolysis assay:Phosphate buffer (20mM. Erlenmeyer flasks (Ambrosil). BSA solution: add 0. Samples are diluted 10 times for protein estimation. Petridishes.01N HCl 0. Tips. Enzyme purification:(1) Phosphate buffer (50mM. b.5mg/mL concentration of BSA solution to be used as standard protein solution. pH 7. Micropipettes. Pipettes.01N I2 solution Purified enzyme sample (2) Protein estimation:a.25g of BSA powder in 10mL distilled water to get 2.1% starch 0.
lactophenol cotton blue staining method under microscope. Model CL110) Microwave Oven (IFB) Incubator (from EIE Instrument Pvt.) Orbital shaker Centrifuge Digital photocolorimeter
[C] Others: Whatman filter paper-1 (Qualitative Disk Filters. pH 7.
[B] INSTRUMENTS: (1) (2) (3) (4) (5) (6) (7) Autoclave (from Sedko Laboratory Equipments) pH meter (from Chemline. Test tubes. Folin ciocalteau reagent c. Spreader.(3) Culture of Aspergillus previously isolated from soil and identified by using standard method.0) 0.
(1)Isolation and identification:
Isolation of amylase producer from soil:The soil contains a rich deposit of both bacteria and fungi. college. Soil samples were serially diluted up to 10-3 dilutions by the serial dilution method  and spraded on starch agar plate in to which an antibiotic streptomycin is added after sterilization of medium to avoid bacterial contamination. which produce amylases. Soil samples were collected from the garden of govt. sci. The plates were incubated for 72h at room temperature. foods or could be purchased. Starch hydrolyzing fungi or bacteria could be isolated from the soil. Khokhra. The highest enzyme producer was identified by layering 1% of iodine solution on the agar plates and zone of clearance was observed for screening the fungi . Maninager (E). The soil samples were collected in a sterile container and brought to the laboratory for further processing. lactophenol cotton blue staining method under microscope. Identification of Aspergillus niger strain producing amylase:After incubation mix culture of amylase producer fungi were obtained which were identified using standard method of fungal identification. The identified fungal colony was again incubated in starch agar medium for 72h to get pure culture of Aspergillus niger.K Shastri campus.
. Scrape with a wire loop to loosen the spores.Preservation of culture:The obtained pure culture of Aspergillus niger was preserved in starch agar slants covered with parafilm at lower temperature.0 X 106 cells) was transferred from a stock culture in 250ml flask containing 100 ml of growth medium.
Inoculum preparation:Pour 10 ml of sterile distilled water on the slant containing fungal spores. A standardized inoculum size of conidia (each ml of cells suspension contained 2.
Take the absorbance at 580nm using digital photocolorimeter and phosphate buffer (pH 7. The flasks were incubated for 72 hrs at 28°C ± 2°C on a rotatory shaker at 150 rpm.7H2O (0.L-1) and pH adjusted at 6. Pour the whole content of the flask containing the growing fungus through a funnel fitted with Whattman number 1 filter paper. Incubate all 3 flasks for 10mins at room temperature (25-30°C) in dark.0).
Purification (precipitation) of enzyme:The enzyme was precipitated from a clear supernatant at 4°C by adding solid ammonium sulphate to achieve 85% saturation.5. MgSO4.0) as blank (table: 2).
(3)Extraction and purification of enzyme:
Extraction of enzyme:It is very easy to remove the fungal mycelium from the enzyme production medium.1 gm. The specific activity was defined as number of units per gram protein.L-1). The reaction mixture contained the following in a total volume of 8ml: 5ml of phosphate buffer (pH 7. The enzyme activity was expressed in number of units. After incubation period. 2ml 0.5 gm. keeping the solution in ice and the protein was allowed to precipitate by keeping it overnight at 4°C.2) and used immediately for activity determination.
Enzyme activity was determined by DNS method described by using starch as the substrate.1% starch solution.01 gm. inactivate the enzyme activity by adding 2ml of 0. 1 unit of enzyme was defined as the amount of enzyme (protein) in milligram required for hydrolysis of starch to produce a millimolar of reducing sugar (glucose) in one hour under assay conditions.Submerged fermentation was carried out in the Ehlenmeyer flasks by taking 100 ml of amylase production medium . Add 4ml of 0. containing KH2PO4 (1.4 gm. 1ml of active enzyme in test flask and 1ml of inactive enzyme in both the controls (starch control and enzyme control). Soluble starch (20 gm. pH 7.01N I2 solution in each flask and makeup the volume up to 100ml using distilled water. FeSO4. The filtrate contains the crude amylase. The protein was separated by centrifugation at 2000 rpm for 30 minutes at 4°C.7H2O (0.
.L-1).L-1).01N HCl.L-1). dissolved in minimum volume of phosphate buffer (50mM. The ammonium sulphate was added slowly .L-1). KCl (0. NH4NO3 (10 gm.
Reagent Phosphate buffer 0. Protein estimation was carried out according to following assay system.0ml 4.0ml 4.0ml 1.0ml 2.0ml 2. Samples were diluted 10 times for estimation (table 1). Table: 1 2.D at 580nm Enzyme assay: To check the enzyme activity as unit per liter.
Test flask 5.0ml 1.0ml -
Incubate for 15mins at room temperature 0. In this method.01N I2 solution 2.0ml 4. standard graph was prepared by estimation using standard protein solution (BSA solution= 2.0ml
Folin Lowry’s method:The protein content of enzyme solutions extracted was estimated by Folin Lowry’s method.0ml
Enzyme control 5.0ml 2.01N HCl 0.0ml Make up the volume up to 100ml O.1%starch Active enzyme Inactive enzyme
Starch control 5.5mg/mL concentration).
Allow it to react at room temperature for 30 minutes
Table 1: Protein estimation by Folin Lowry’s method for different substrate concentration
Incubate at room temperature for 15 minutes
Aliquot protein solution (mL)
Distilled water (mL)
Alkaline copper solution(mL)
Folin ciocalteau reagent(mL)
Enzyme purified was tested in the pH range (pH 3 to 8). sucrose. fructose and sorbitol could be considered as moderate source. were found to give significant yields of alpha amylase at pH 5. Sucrose and dextrin and galactose were good carbon sources for amylase production. Effect of different nitrogen sources on the production of alpha amylase was studied.50. sorbitol. pH is known to affect the synthesis and secretion of alpha amylase just like its stability (49). niger (Figure 2). viz: glucose. The influence of these carbon sources were tested at different concentrations (0. (4) Different pH values:pH is one of the important factors that determine the growth and morphology of microorganisms as they are sensitive to the concentration of hydrogen ions present in the medium.5 to 2. (3) Incubation temperature:The influence of temperature on amylase production is related to the growth of the organism. started after 24 hours of inoculation and showed maximum production on 5th day of incubation period for cells of Aspergillus niger (Figure 4).1N HCl. Hence. However. The initial pH of medium was adjusted to variable pH range by adding the 0.
(2) Various nitrogen sources:-
The availability of the nitrogen source is the major controlling factor in the final biomass yield. The production of alpha amylase was found to be the best at pH 5. xylose. the optimum temperature for enzyme production was 30°C for cells of A. starch. (5) Incubation period:The alpha amylase activity was determined after every 24 hours of incubation in order to determine the optimum incubation period for maximum production of extra cellular alpha amylase. Peptone supported maximum production of enzyme whereas urea produced considerable amount of alpha amylase from cells of Aspergillus niger. Xylose.0 in SmF (38. fructose. Fungi of Aspergillus sp. it was observed that casein and gelatin caused poor enzyme production. Here we used the mesophilic strain of Aspergillus niger from soil and therefore assay of enzyme production was carried out at various temperature ranges 20 to 50°C for 24 hrs.0 (figure 3).0–6.0%).
. Starch was recorded to be the best carbon source for production of alpha amylase from cells of Aspergillus niger (Table 1).51).03% (Figure 5). the optimum temperature depends on whether the culture is mesophilic or thermophilic. Starch. galactose and dextrin.Parameters controlling amylase production
(1) Various carbon sources:-
Flasks containing production media was supplemented with different carbon sources. It was found that cells of Aspergillus niger showed considerable amount of growth at 20°C but there was less enzyme production. The enzyme production however. The optimum concentration of peptone was 0.
Therefore. addition of these surfactants is used for the production of extracellular enzymes. Triton X-100 and SDS favored more amylase production in culture media. niger (Table 2).002% and 0. Tween-80 at 0.
.02% caused maximum enhancement where as Triton X-100 and SDS increased amylase activity at 0. The detergent Tween-80.(6) Use of surfactants:Surfactants in the fermentation medium are known to increase the secretion of proteins by increasing cell membrane permeability.0002% respectively for cells of A.
28/1. V(actuvuty) = 49. V(actuvuty) = [1.35*(1/15)*0.
Effect of temperature on extra cellular enzyme production
.D at 580nm 0.00 Starch control OD1 0. 1.28 A = 12.45-1. 1.35 (constant for amylase) T = time of incubation.33
Calculation for enzyme activity:-
V(actuvuty) = [E0-Et/Et]*A*(1/T)*(1/v) *1000 Where.50 Test OD3 1.21 Unit per liter.5*1000 So.05 = 1.45
Et = OD3-OD1. E0 = OD2-OD1.50-0.05 = 1.33-0.21 Unit per liter From the above table and calculation the result obtained for enzyme activity under normal lab condition is 49.05 Enzyme control OD2 1. 2ml So.28]*12.RESULT
Blank O. 15mins V = volume of starch.
5 1.85 0.5 1.5 30.0 2.002 59.6 23.6 27.1 SDS 0.0002 44.0 0.
Table 3.2 41.0 0.5 1.6 41.8 35.0 2.5 22.0002 55.0 2. Effect of various carbon sources of various Concentrations.0 2.66 0.0 2.2 0.5 1.69 Table 4.5 1.0 0.5 1.69 0.0 2.2 28.52 1.002 45.5 29.4 24.5 26.2 25.0
Enzyme activity unit/cell 44.0 2.0 0.0 0.2 50.4 31.
.5 1.88 42.88 0. Effect of various surfactants of various concentrations.2 42.8 10.4 34.6 37.002 45.44 7.3 TritonX-100 0.02 46.5
starch glucose dextrin fructose sorbitol xylose galactose
Concentration Enzyme of surfactant activity unit/cell Tween 80 0.8 0.6 33.0002 50.5 1.8 28.0 0.02 42.8 36.2 32.66 0.8 0.2 48.0 2.0 0.02 55.2 0.Carbon source sucrose
Effect of pH on extracellular enzyme production
Effect of incubation period on extracellular enzyme production
Burhan. Biotechnol. 6.C. thermophilic. 38 (2003) 1397–1403. isolate ANT-6. J. van der Veen.M. M. J. In: Enzyme Technology. Microbiol. A.). C. Asiatech Publishers Inc. alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. Soccol. India (2005) pp. Properties and applications of starch-converting enzymes of the a-amylase family. Assoc. Dijkhuizen. Gokhan. H. New Delhi. Ichishima E (1971). Pandey.M.E. Nisa. M. 3. Mahrtre NS (1965). C. J. Biochem. Ashabil. C.Effect of various nitrogen sources at 0. 7: 273-304. Rao. Adv. Appl. 5. Windish WW. van der Maarel. Advances in research on biochemistry of Takaamylase. G. T..
. 189–220.03% concentration
2. Leemhuis. Omer. Ferment. Enzymatic properties of a novel thermostable. C.U. 94 (2002) 137–155. L. U.L. Uitdehaag. A. Ezhilvannan: a-Amylases. A.J. Process. Larroche (Eds.R. 4. Osman. Microbial amylases. Webb. B. Satyanarayana. C.C. 29:107-142. J.
Singh. Applied Microbiology. Biotechnol. Process Biochem. A. Dar hell. Kabler PW. 20. Applied Science Publishers Ltd.. Z. process. Application of a statistical design to the optimization of culture medium for a-amylase production by Aspergillus niger ATCC 16404 grown on orange waste powder.Freufekder. Soccol. Heidelbergh Springer Betlin 2006. 2000 a. http://www. S. In: Microbial Enzymes and Biotechnology. Roche C (1976). 17.R. Appl. Mandels M. Measurement of Saccharifying cellulose. M. pp 205-218. 31: 135 – 152. Bioeng. Baltimore: Molecular Cell Biology. Fogarty: Microbial Amylases. Growth and a-amylase production by Aspergillus oryzae during continuous cultivations. Scientific American Books. M. Environ. C. Bernfed P. J. Nielsen. pH regulation of gene expression in fungi.A. Biotechnol. Enzymes of Starch degradation and synthesis Advance in enzymology 12: 379-481. Expanded bed affinity purification of bacterial a-amylase and cellulase on composite substrate analogue–cellulose matrices. 70. Biochem. Funga Gen. VT. Biol. Biochem. Nigamp V. Newyork1990. A. International Book Company.14. M. A. 51. Biochem 35: 1153 – 1169. 52 (1986) 1068–1073. Gheribi-Aoulmi. Meraihi. Enzyme associated aqueous extraction of Pea nut oil 79. 45 (1996) 81–93. Advances in microbial amylases. J. Bennamoun. 21. J. 1951. Advances in microbial amylases. Andreotti RE.T. H. J.
. 54. 15. Pandey. Lali. Lodish. Geldrich EF. Soccol. 73 (2005) 190–197. D. D and Mohan R. 18. 1: 5-53. W. Villadsen.org/JBPR 19. Production and characteristics of raw-starch-digesting a-amylase from a protease negative Aspergillus ficuum mutant. Amritkar. S. L. Singh. 39 (2004) 565–570. Food Eng.e3journals. 16. UK (1983) pp. 29: 61 –71. Kamat. 6: 21-33. Symp. 50.M. Teramoto. Carlsen. Z. N. D. 2006. Djekrif-Dakhmouche. Jones and Barlett. Denison SH (2000). D and Mohan. 55. 31 35-152. Boston 1987.). Pandey. J. Pandey. Molecular Biology. Clark HE. Nigamp. Y. 38.W. New developments in solid state fermentation... Hayashida. NewYork. D. Microbiol. Fogarty (Ed. 1–92. Huff CB (1988). 2000b. SoccoL. London.. CR and Mitchell. 49.