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    2016-Isolation, Molecular Characterization and Screening of Extracellular Enzymes Produced by Vibrio Alginolyticus

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    Ramesh

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    Seaweed Res. Utiln, 38(1) : 67-74, 2016 Andaman Islands Isolation, molecular characterization and screening of extracellular enzymes produced by Vibrio alginolyticus from the seaweeds of KADA NARAYANA MURTHY*, R. MOHANRAJU, P. KARTHICK AND CH. RAMESH Department of Ocean Studies and Marine Biology, Pondicherry University Brookshabad campus, Port Blair, Andaman & Nicobar Islands, India “Corresponding author: murthymarino@graal.corm ABSTRACT ‘The present study is focussed onthe isolation, molecular characterization and screening of extracellular enzymes. produced by Vibrio alginalyticus obtained from the seaweeds of Andaman Islands. Totaly twenty isolates from 9 species of seaweeds were and identified as Vibrio alginolyticus by PCR based methods and confirmed by 168, RNA sequencing. All the isolates were screened for the production of extracellular enzymes such as Agerase, Alginase, Amylase, Chitinase, Gelatinase and Pectinase, Majority ofthe isolates were foundto be excellentalginase producers. This study is nat only significant on t ecological role of Vibrios in biogeochemical cycling in the marine environment, but also shows the potential ofVibrios for industrially important enzymes. Introduction Studies on the microorganisms associated with seaweeds have become prominent recently due to novel secondary metabolites produced by various marine microorganisms. These microorganisms have significant importance in pharmaceutical and in industria applications. Vibrios being one of the ubiquitously distributed heterotrophic bacteria in the marine environmentand their association with seaweeds have not been much studied, Vibrio alginolyticus known for biofilm formation is not reported much from seaweeds. Marine microorganisms play an important role in biogeochemical cycling by producing various extracellular secondary metabolites such as enzymes helping them to degrade the organic material in the marine environment. Competition among the microorganisms far ‘space and nutrients in the marine environment led to the evolution, which in return triggered the marine microorganisms to generate diverse enzyme systems to adaptto the complicated marine environments (Zhang and Kim, 2010). Understanding the role of microbial extracellular enzymes is very significant as the enzymatic, hydrolysis is the initial and key step in the entire remineralisation cascade (Arnosti, 2011). The marine microbial enzymes are significantly different from the homologous enzymes of terrestrial origin as the marine environment ranges from nutrient rich to nutrient sparse regions with properties such as high salt tolerance, hyperthermostabilty, barophilicity and cold adaptability (Zhang and kim, 2010). Marine environment is @ complex niche comprising of various organisms whose body is majorly comprised of various polysaccharides such as chitin in case of fungi, zooplankton and crustaceans; agar in agaronhytes; alginates in alginophytes and carrageenan in case of carrageenophytes, besides pectin and cellulose in case of phytoplankton and other plant components such as mangroves and seagrasses. Along with polysaccharides Received: 18-01-2016; Revised : 26-01-2016; Accepted : 30-01-2016 7 Kada Nerayana Murthy, R. Mohanraju, P Karthick and CH Ramesh components marine organisms are rich in various proteins and lipids. These organic biopolymers act as substrate for the microbial communities to produce various substrate specific extracellular enzymes helping in the remineralisation of organic matter and recyting of nutrients inthe marine environment. Apart trom the ecological and environmental significance, microbial enzymes of marine origin are gaining importance in various industrial processes for their novelty as biocatalysts. The enzyme industry has a significant global market of around $1 ailion in 2010 and estimated to increase 6.6% compound annual growth rate (CAGR) to reach $1.5 billion by 2015. Almost 75% ofall industrial enzymes are hydrolytic enzymes Carbohydrases, proteases and lipases dominate the enzyme market accounting for more than 70% of al enzymesales (Liet. a.,2012). Vibrioalginolyticusis one ofthe fastest growing bacteria among the Vibrios with a short generation time of 12- 25 min (Ulitzur, 1974) and found widely distributed in majority of the marine surfaces, as ane ofthe key blo-fouler. The present study focusses in exploring the extracellular enzyme potential of Vibrio alginolyticustromthe seaweeds of Andaman Islands. Materials and Methods Isolation of Vibrio alginolyticus In the present study 9 species of seaweeds comprising 4 green algae (Ulva lactuca, Caulerpa sertularioides, Caulerpa racemosa and Dictyosphaeria cavernosa), 3 red algae (Acanthophora spicifera, Gracilaria corticataand Mastophora rosea}and 2 brown algae (Sargassum turbinaroides and Turbinaria ornata) were screened for Vibrio aiginolyticus like organisms. Seaweed samples were collected from the intertidal regions of Port Bia, South Andaman and Little Andaman during low tide as the macro algal distribution is prominent in South and Little Andaman when compared to the North Andaman islands (Karthick et. af., 2013 a, b) The samples were immediately transferred to a sterile polythene cover and transported to the laboratory. Small portion of these samples were taken in sterile petridish, washed thrice with a spray of sterile filtered seawater to remove the unattached bacteria and other epiphytes. A surface swab from a small area of each sample was taken by using sterile cotton swab and transferred to a labeled Vial containing 20 mt of Alkaline Peptone Water (APW-pH 8.5, Hi media) and incubated for 6 hat37°C for enrichment of Vibrios. After the incubation 100 pl of inoculum were plated onto a TCBS agar plate (Thiosulphate Citrate Bile- salts Sucrose, Merck) and incubated at 37°C for 24 h (Farmer and Hickman-Brenner, 2006). Sucrose positive (Yellow) and Sucrose negative (Green) colonies. were picked and streaked on toa fresh TCBS plate and incubated at 37°C for 24h, Each isolated colony was further purified on Marine Agar plates (MA, Himedia, India) incubated and pure strains were obtained. All these isolates were then stabbed in Nutrient agar with 1% NaCl and 0.8% agar, sealed and stored at room temperature. Molecular Characterization of Epiphytic Vibrios DNA Extraction The genomic DNA was isolated by following standard protocol (Pang et. al., 2006). 1.5mlof overnight broth culture of the isolates was centrifuged at 8000 rpm for 5 min and the cell pellet obtained was washed twice with Phosphate buffered saline (PBS), centrifuged again to obtain pellets which were dissolved in 200 yl of TE ouffer (pH 8.0), boiled for 10 minutes, transferred to -20°C for 5 ‘minutes, for the release of DNA. This was then centrifuged at 8000 rpm for 5 min and the supernatant obtained was transferredtoa separate vialand stored at-20°C, PCR detection of Vibrio alginolyticus and 16S rRNA sequencing All the isolates obtained were subjected to PCR assay targeting collagenase gene by following the methodology described by Pinto et. al. (2005) to identity themto V. alginolyticus. The PCR products were resolved by electrophoresis in 2% agarose gel. An amplicon size of 737 bpis postive for Vibrioalginolyticus. Further among the 20 isolates 4 isolates viz. HAS4, PBKSW1A, HCSS and CCSW4b were subjected to 165 rRNA sequencing to confirm theiridentity. PCR amplification of 16S rRNA gene was carried out following the methodology described by Lane (1991) The PCR products were purified using Shrimpex’s GeNoRime PCR purification kitand sequenced by ABI 3500 DNA Sequencer (Applied Biosystems, Foster City, USA) at Shrimpex Biotech Services Pvt. Ltd, Chennai, India. The sequences were submitted to GenBank and accession numbers were assigned as KP271509, kP271511, KP271516 and KP319175. The partial 165 rRNA gene sequences were compared with those available in the public databases like National Contre for Biotechnology Information (NCBI) and European Molecular Biology Laboratory (EMBL). Sequence alignment and comparison were performed using the 68 © Seaweed Research and Utilisation Association Isolation, molecular characterization and screening of extracellular enzymes produced by Vibro alginolyticus multiple sequence alignment programme ClustalW (Higgins et. a/., 1994). Sequences were edited manually to remove the gaps. Neighbour-joining method was ‘employed to construct the Phylogenetictree using MEGAG software (Tamura et. al., 2018). Extracellular enzyme production Al the 20 isolates were subjected to various substrates such as Agar, Sodium alginate, Starch, Chitin, Gelatin and Pectin to know the potential of these isolates to produce extracellular enzymes such as Agarase, Alginase, Amylase, Chitinase, Gelatinase and Pectinase respectively (Baumann et, al., 1971; Huet. al, 2009; Tropeano et. a, 2012). Allthese assays were conducted on solid media by providing the respective substrate at 0.5 to 1% wiv for each enzyme, The results were recorded as grades viz. average, good, very good, excellent and negative based on their appearance and zone of hydrolysis of the substrate surrounding the bacteria on solid media. In the present study 49 Vibrio like isolates were obtained. Among the 49 isolates, 20 isolates were Fig.1. PCR assay largeting collagenase gene of Vibrio alginolyticus Va: Vibrio alginolyticus CRIM 516 T (Positive control) M-1 kb ladder Fig. 2. Neighbour-joining tree confirmed to Vibrio alginolyticus by PCR assay targeting collagenase gene (Fig.1). Four isolates that were subjected to 16S rRNA sequencing confirmed their identity as V alginolyticus with more than 98% homology with Genbank. Phylogenetic analysis had shown that all the four isolates showed closely related to Vibrio alginolyticus CECT 436 (FM204868) (Fig.2) Furthermore, all the 20 isolates were screened for 6 extracellular enzymes such as agarase, alginase, amylase, chitinase, gelatinase and pectinase (Table-1). Graphical representation ofthe results is presented in Fig.3 showing the number of isolates having potential for vatious extracellular enzymes. Among the 20 isolates, majority of the isolates produced amylase followed by gelatinase, alginase, agarase and pectinase at different grades of hydrolysis. Totally 7 isolates showed al the six enzymes; 7 isolates showed 5 enzymes each; 5 isolates showed 4 enzymes each and 1 isolate showed @ minimum of 3 enzymes each (Table-1) Neighbour-joining tree (Fig. 2) showing the phylogenetic positions of 4 Vibrio alginolyticus isolates obtained from the seaweeds of Andaman Islands and closest species based on 16S rRNA gene sequence data, Bootstrap values (1000 replications) greater than 50% are indicated on the branching nodes. Bar, 0.08 substitutions per nucleotide position. Staphylococcus aureus ATCC 43300 (AM980864) was usedas out-group, Discussion More than three quarters of the earth's surface is covered by sea providing abundant resources to the mankind. Enzymes have historical applications in dally life, although it was not known earlier and with the development of science, enzymes became inevitable for a range of industrial processes especially in “green” industrial processes as they are renewable, biodegradable, eco-friendly and non-hazardous (Sana, ‘Lb L de Nene Fig. 3. Extracellular enzyme potential of vibrios of Andaman Islands Research and Utilisation, Vol. 98(1), 2016, eal Kada Nerayana Murthy, R. Mohanraju, P Karthick and CH Ramesh Table-t. Extracellular enzyme potential of V.alginolyticus isolates of Andaman Islands S.No Strain ID ‘Agarase Aiginase Amylase Chitinase Gelatinase Pectinase Enzymes, produced 1 HBULS 6 2 HGs3 4 3. Hos4 4 4. HGs6 3 5. PBKSWIA * + 6 6. coswab + 5 7. coswaa * + 6 8. HAS 5 9. HASS 5 10. HGC4 . 4 11. HGCS . 4 12, HGCe ‘ 5 13, HBU2 5 14. HEU 5 15. HBUA 4 16. B4tI2a " 5 17. BBtt2a ” 1 6 18. Datta i" 6 18. Det128 i" 6 20._D6t12b : 6 trAverage; +++Good; ++++VeryGood; +++4+ Excelent; Negative 2013). Although many important enzymes have been isolated from plants and animals, microbial enzymes are found to be an excellent alternative due to their diverse biochemical properties, plasticity for easy genetic manipulation and large-scale production. Marine microbial enzymes are uniquein nature when compared to the enzymes of terrestrial origin due to their adaptability of marine bacteria towards an array of diverse environmental parameters like high salt concentration, extreme temperatures, acidic and alkaline pH, extreme barometric pressure and low nutrient availablity (Zhang and Kim, 2010). Further, marine environment is @ source of complex organic materials of high molecular weight and polymeric structure, mainly proteins, starch, lipids, pectin, Cellulose, chitin, nucleic acids, or lignin (Poremba, 1995; Arnosti et. al., 1998) which support the life through their contribution as live, dead and decaying marine flora and fauna. Microbial enzymes derived from such environment help to convert these complex organic materials into nutrients by degradation serving the purpose of nutrient regeneration supporting millions of floraand fauna living in itto build a complete trophic system. Every enzyme that is derived from marine microorganisms represents their ability to growin such environments rich in substrates they knowhowto process, Agar is a polysaccharide which exists as a primary coll-wall component of red algae (Rhodophyceae) such as Gelidium and Gracilaria species (Fu and Kim, 2010). Several Vibrio derived agarases were reported from the marine environment showing an excellent agarolytic activity like seawater (Aoki et. a/., 1990; Macian et. al., 2001) and sediments (Araki ot. al.,1998; Dong ot. 4a/., 2008, 2007; Liao et. al., 2011). Sugano et. al. (1993) isolated Vibrio sp. strain JTO107, an agar degrading bacteria that decomposes the cell-walls of some seaweeds including a Laminaria sp. and Undaria pinnatifida in Japan. In the present study, one isolate obtained from seaweed Mastophora rosea (HBU4) showed an excellent agarase activity and furthermore majority ofthe isolates showed agarase activity ranging from very good to average activity Alginate is a linear polysaccharide in which B-D- mannuronate (M) and c-L~ uluronate (6) are covalent'y (1-4)- linked in different sequences (Wong et. a, 2000). Alginates are quite abundant in nature especially in brown algae such as Sargassum sp. and Laminaria sp. where 40% of thelr dry weight is comprised of it (Albrecht and Schiller, 2005). Reports on Vibrio derived alginate lyases 79 © Seaweed Research and Utilisation Association Isolation, molecular characterization and screening of extracellular enzymes produced by Vibro alginolyticus were known since early 90's. Reichelt and Baumann (1973) reported that luminous bacteria belonging to V. harveyi possess alginases. Ramaiah and Chandramohan (1992) reported alginate lyase activity from V. harveyiand V, fischeri isolated from the seaweeds of Goa and Lakshadweep coral reef lagoons of India. Sawabe et. (1998) isolated Vibrio halioticoli an alginolytic marine bacteria from the gut of the abalone Haliotis discushannai, Li et. al. (2003) isolated novel alginate lyase from Vibrio sp. QY102 obtained from surface of Sargassum species. Wang et. al. (2006) isolated Vibrio sp. YWA from rotted part of the kelp which showed excellent alginolytic activity. A Na/K: dependent alginate lyase obtained from Vibrio sp. YKW-34 isolated from the gut contents of turban shell was purified and characterized by Fuet. al, (2007). Wang et. al. (2013) isolated a Vibrio ‘sp. QY105 was isolated from sea mud of Qingdao, China ‘showing alginate lyase activity. Kim ef. a/. (2013) isolated Vibrio hemicentrotifrom the gut microflora of sea urchin (Hemicentrotus puleherrimus) producing alginate lyase. In the present study, Vibrio alginolyticus was obtained from seaweeds Ulva Jactuca (HBULS), Caulerpa racemosa (KSW1a), Dictyosphaeria cavernosa (CCSW4d), Acanthophora spicifera (HAS4), Gracilaria corticata (HGC4, HGCS, HGC6), Mastophora rosea (HBU2, HBUS), Sargassum turbinaroides (84112a, 861122), Turbinaria ornata (D4112a, 06112a). Majority of the isolates showed alginase activity displaying their role in degradation and nutrient cycling in the marine environment Amylases are enzymes that hydrolyze starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units. These enzymes are of great significance in the present day biotechnology with applications in food, fermentation, textile and paper industries. Ratcliffe et. a/. (1982) observed extracellular amylase activity in Vibrio gazogenes. Joseph et. al. (1982) observed amylase activity in V. parahaemolyticus and other halophilic Vibrios. Yang ef. al. (1983) observed amylase activity in Vibrio orientalis isolated from the seawater off the coast of China, Mohandass and Raghukumar (2005) observed positive amylase activity in V. alginolyticus isolate No, NIO/DI/32 obtained from marine sediments of Zuari estuary, Goa. Abdallah ef. a/. (2009) observed amylase activity in V. parahaemolyticus and V. alginolyticus strains incubated in seawater for 8 months to evaluate their adaptative responses to starvation. Subin and Bhat (2011) observed amylase activity in the vibrios viz. neries, V. furnissii, V. ordali, V. harveyi, V. parahaemolyticus, V. alginolyticus, V. splendidus, ¥ metschnikovii, V. natriegens and V. proteolyticus ‘obtained from the marine sediments of eastand west coast of India. In the present study, majority of the V alginolyticusisolates showed good amylase activity. Chitin is widely distributed in nature as @ biopolymer with non-toxic properties and is the most common polysaccharide found in nature after cellulose which is the major structural component of most fungi cell-walls and also quite abundant in the crust of insects and crustaceans (Zhang and Kim, 2010). Annual generation ofchitinis about 1.0*10"t (Marguerite, 2006). Most of the marine zooplankton regularly undergo shedding of their exoskeleton leaving large amount of abandoned chitin which actsasarrich source of carban and energy for growth and reproduction of chitin degrading microorganisms. The total production of chitin in the whole marinebiocycleisatleast2.3 million metrictons per year (Jeuniaux and Voss-Foucart, 1991). Chitin utilization pathway is one of the conserved activities among the species of Vibrionaceae family (Hunt ef. al., 2008). Osama and Koga (1995) reported chitinase activity trom 4 species of vibtios viz. V. fluvialis, V. parahaemolyticus, V. mimicus and V. alginolyticus. Suginta et. al. (2000) reported chitinase from V. carchariae (=harveyi), V. alginolyticusand V. campbell. Inthe present study one isolate obtained from seaweed Ulva lactuca (HBULS) showed very good chitinase activity. Further, 7 isolates showed average activity which can be optimized for ‘enhanced enzyme production. Gelatin is a polypeptide derived by hydrolytic degradation of collagen, the principal component of animal connective tissue which is traditionally been extracted from the skin and bone collagens of certain mammalian species primarily cows and pigs and gelatin from marine sources especially from the fish waste (c.0. skin, bones, scales and fins) from the seafood processing industry has been looked upon as a possible alternative to bovine and porcine gelatin (Venkateshwarlu, 2013). Majority of the vibrios are known to produce gelatinase enzyme anditis one ofthe key biochemicals or phenotypic, characteristics to differentiate between species of vibrios (Noguerola and Blanch, 2008). Gelatinase production is recognized as a virulence factor in humans beings and marine animals (Zhang and Austin, 2000; Vergis ef. al. 2002) and itis found to be regulated by Quorum sensing Seawoed Research and Utilisation, Vol, 38(1), 2016 n Kada Nerayana Murthy, R. Mohanraju, P. Karthick and CH. Ramesh phenomenon in vibrios (Natrah ef. al., 2011). Manilal et. al, (2010) observed gelatinase activity in the vibrios isolated from diseased black tiger shrimp Penaeus monodon. Vanmaele et. al. (2015) observed gelatinase activity in the Vibrio isolates belonging to harveyi clade isolated from a shrimp hatchery and concluded that itis ‘one of the virulence factors causing disease in shrimps. In the present study, majority of the isolates showed gelatinase activity but the production ranged between goodand average which can be enhanced by optimization As marine environment is a major source of fishes which contribute to marine derived gelatin, the occurrence of gelatin hydrolyzing microorganisms explains their role as, intermediate decomposers of dead and decaying marine organisms. Besides, they also act collectively with collagenases produced by same bacteria or other pathogens playing a major role fish pathogenicity. The ‘occurrence of gelatin hydrolyzing vibrios on the surface of ‘seaweeds explains that they are not the part of the true epiphytes. Pectin is a complex high molecular mass glycosidic macromolecule found in higher plants, mainly presentinthe primary cel allandis the major component of the middle lamellae, largely responsible for the structural integrity and cohesion of plant tissues. Pectin is widely distributed in marine environment among ‘seagrasses (Khotimchenko ef al., 2012) and frustules of diatoms (Castro and Huber, 2008). Pectinases are group of enzymes that catalyse the degradation of pectic substance through depolymerization and deesterification reactions, widely used in the food industry for juice and wine production and occupies about 10% of the overall manufacturing of enzyme preparations (Pedrolli et.al. 2009). Pectinases from marine microorganisms are not much reported. Fenice ef. a/, (2007) studied extracellular enzyme activities by bacterla isolated trom marine samples of Tyrrhenian Sea and found isolates with tremendous pectinase activity. Loperena et. a/. (2012) studied extracellular pectinase produced by microorganisms. isolated from maritime Antarctica Vibrios are also studied for pectinase activity. Obiv and Umezurike (1981) reported pectin degrading Vibrio sp. from rotted onions (Allium cepa). Ramaiah and Chandramohan (1992) reported pectinase activity from luminous V. harveyi and ¥. fischeri isolated from the seaweeds of Goa and Lakshadweep coral ree lagoon of India, Subin and Bhat (2011) reported pectinases from V neries and V. furnissii. Hugouvieux-Cotte-Pattat ef. al. (2014) reviewed bacterial pectate lyases and reported pectinases from vibrios. Besides these few reports theres, not much study on pectinases from vibrios. in the present study, 9 isolates showed very good pectinase activity indicating their potential in processing the dead and decaying plant materials inthe marine environment. The present study emphasizes the role of vibrios in the marine environment. Several studies were conducted earlier on screening of extracellular enzymes from marine bacteria and stated that marine bacteria are found to be excellent source for extracellular enzymes of commercial value. In this study Vibrio alginolyticus isolates obtained from various seaweed surfaces showed very good potential for extracellular enzymes signifying their role in biodegradation, nutrient cycling and pathogenicity in the marine environment, Further, there are not many studies on extracellular enzymes from seaweed associated bacteria especially trom vibrios. Y. alginolyticus being fastest growing bacteria with shortest doubling time, it would be an excellent arganism for exploring extracellular enzymes of commercial importance besides their role in nutrient regeneration and cycling inthe marine environment. Acknowledgement This research work is a part of UGC Major Research Project. The first two authors thank the University Grants Commission (UGC), Govt. of India, New Delhi for providing Major Research Project (Sanction Letter No. 42-463/ 2013 (SR), Dt. 22-03-2013) and the University authorities for providing facilities. References Abdallah, FB, H,Kallel and A, Baknrouf 2009. 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