Purification and activity of amylases of Marine Halobacillus sp amylus HM454199

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1 Indian Journal of Geo-Marine Sciences Vol. 42(6), October 2013, pp Purification and activity of amylases of Marine Halobacillus sp amylus HM K Ashwini, L Karthik, Gaurav Kumar & K V Bhaskara Rao* Environmental Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu, India [ kokatibhaskar@yahoo.co.in] Received 14 March 2012; revised 3 November 2012 Present study was focused on purification and activity of amylases of marine eubacteria. Among 23 bacterial isolates from Nicobar Islands, Halobacillus sp amylus was selected and further examined. Enzyme activity was evaluated at different temperatures (25, 30, 35, 40 and 45ºC), at varying ph (6.0, 6.5, 7.0, 7.5 and 8.0) and by using various carbon sources (starch, glucose, sucrose, fructose, xylose and lactose). Different organic nitrogen sources (yeast extract, meat extract, beef extract, nutrient broth, urea and casein) and various concentration of NaCl were also used to evaluate enzyme activity. Maximum enzyme activity was recorded at 40 C, ph 8.0 and 1.0% of substrate concentration. Halobacillus sp amylus gave U of amylase per litre of culture broth, 3 fold higher than before optimization in submerged fermentation. Thus, these observations conclude that the enzyme obtained was relatively heat sensitive and moderately alkalophilic amylase which can be developed for extensive industrial applications. [Keywords: Nicobar Islands, Halobacillus sp amylus, Optimization, Amylase, Microorganisms, Culture ] Introduction Amylases are one of the most important industrial enzymes and have great significance in biotechnological studies. Amylases are characterized by their ability to hydrolyze glucosidic linkages in polysaccharides. α-amylase (endo1, 4-α-D-glucan glucohydrolase) is an extracellular enzyme that acts on starch compounds. This enzyme can be derived from various sources such as plants, animals and microbes but the major advantage of using microorganisms for the production of amylases is in economical bulk production capacity and microorganisms are easy to manipulate to obtain enzymes of desired characteristics 1. The use of the submerged culture is advantageous because of the ease of sterilization and process control such as temperature and ph etc. is easier 2. These uses have placed greater stress on increasing indigenous amylase production and search for more efficient processes. Recently there is a remarkable interest and demand for enzymes with novel properties. Currently microbes from marine sources are used for industrial production of enzymes, although the potential for synthesis of several novel enzymes by terrestrial microorganisms have been recognized. MC Tigue (1995) observed that each application of amylases *Corresponding author requires unique properties with respect to specificity, stability, temperature and ph dependence 3. Screening of marine microorganisms with higher amylase activity could therefore facilitate the discovery of novel amylases suitable for new biotechnological and industrial applications 4,5. Present study report the findings of novel alkaline amylase produced by newly isolated marine bacteria, Halobacillus sp amylus HM Materials and Methods Soil samples from marine sediments were collected and brought to the laboratory in aseptic conditions. One gram portion of soil was mixed in 10 ml water, serially diluted up to 10-4 dilution. Further, spread plated on Basal media viz. Nutrient agar, Zobell marine agar, SYEP agar for about hr. The bacterial colonies showing morphological variations were selected and further purified on respective media. Finally, the isolated colonies were streaked and incubated for growth at 37 C for 24 hr. Bacterial colonies were identified based on morphological and microscopic examinations by standard methods, respective bacterial culture was preserved at 4 C and glycerol stock was maintained simultaneously. Primary screening was carried out to evaluate amylase-producing bacteria using Starch Agar media. About 100 ml of the Starch Agar media was prepared in 250 ml of conical flask and the contents were

2 782 INDIAN J. MAR. SCI., VOL. 42, NO. 6, OCTOBER 2013 poured into the sterile petriplates after sterilization. A loop full of pure bacterial culture was inoculated into Starch Agar media and incubated at 37 C for 24 hours. After the incubation period the plates were flooded with iodine solution and checked for the presence of zone of hydrolysis around the bacterial colonies. Amylase-producing bacteria were selected and used for further study. Fifty ml of fermentation medium containing composition g -1 starch, 10; peptone, 10; yeast extract, 20; KH 2 PO 4, 0.05; MnCl 2.4H 2 O, 0.015; MgSO 4.7H 2 O, 0.25; CaCl 2.2H 2 O; 0.05; FeSO 4.7H 2 O; 0.01 was transferred to 250 ml Erlenmeyer flasks and sterilized in autoclave for 15 min. After cooling to room temperature, a loopful of bacterial culture was aseptically transferred to each flask. Flasks were kept in a rotary shaker incubator (Sanyo gallen kamp, UK) at 200 rpm for 24 hr. After incubation, the fermented broth was centrifuged at 7000 rpm for 15 min and the supernatant was used for the estimation of amylase activity. All the experiments were done in triplicate. Amylase activity was assayed using a reaction mixture comprising of 1 ml of crude enzyme, 1 ml of 1% (w/v) soluble starch solution. The reaction mixture was incubated at 25 C for 10 min. and the reaction was terminated by adding 2 ml of DNS to the reaction tube and then immersing the tube in the boiling water bath(100 C) for 5 minutes. Reducing sugar (maltose) liberated was estimated by the 3,5-Dinitrosalicylic acid (DNS) method. Absorbance was measured at 246 nm with spectrophotometer. One unit of amylase activity was defined as the amount of enzyme causing the release of 1µ mole of reducing sugar in one min., under the assay conditions. Protein content was assayed using a reaction mixture compromising of 1 ml of crude enzyme, 1ml of substrate solution and incubated for 10 min. Reaction was stopped by 0.1 ml phenol solution incubated for 30 min at room temperature. The absorbance was measured at 650 nm with UV-Visible spectrophotometer. One ml of crude enzyme was incubated at different temperature ranging from 25, 30, 35, 40 and 45 C for 24 hours. Likewise, 1 ml of crude enzyme was incubated with phosphate buffer of different ph ranged from 6.0, 6.5, 7.0, 7.5, and 8.0 at 37 C for 24 hours. Enzyme assay and protein assay was carried out as described earlier. Similarly, 1 ml of crude enzyme was incubated at different carbon source such as starch, glucose, sucrose, fructose and lactose at 40 C at ph 8 for 24 hours. Further, 1 ml of crude enzyme was incubated at different organic nitrogen sources such as yeast extract, meat extract, beef extract, nutrient broth, urea and casein at 40 C at ph 8 and dextrose as carbon source for 24 hours. Finally, to determine the effect of NaCl concentration on enzyme activity,1 ml of crude enzyme was incubated at different varied concentrations of NaCl (3.5, 4.5, 5.5, and 6.5% ) at ph 8 and at 40 C, dextrose as carbon source and yeast extract as nitrogen source for 24 hours. The enzyme assay and protein assay was carried out as described earlier. Hundred ml of cell free extract was saturated with ammonium sulphate for primary purification. Desired saturation of 80% was achieved by adding slowly ammonium sulphate. Contents were centrifuged at 5000 rpm and incubated over night. After incubation, the extract was centrifuged at 5000 rpm for 20 min and supernatant was collected. Supernatant was saturated at 90% again. Further, the contents were centrifuged at 5000 rpm for 20 min. After centrifugation, the supernatant was discarded and pellet was collected and used for further analysis. Secondary purification was done by placing the enzyme solution in a dialysis bag of selectively permeable and immersed in a large volume of phosphate buffer that is stirred by magnetic stirrer and maintained at about 4 C for 24 hrs. The potential isolate was screened based on the above traits and characterized based on 16S rrna sequencing. Primer (5 - ACG GCT ACC TTG TTA CGA CTT-3 ) and ABI3730XL capillary DNA sequencer (ABI Prism 310 Genetic Analyzer, Tokyo, Japan) was used for 16S rrna gene sequencing analysis. Phylogenetic tree was constructed and the genus and species were successfully identified. The restriction sites in the 16s rrna sequence of Halobacillus sp amylus were predicted using NEB cutter (version 2.0). Results and Discussion Five marine sediments were used in the present study to isolate marine eubacteria. Based on the colony morphology 23 different isolates were selected for further studies. Among the 23 isolates, four strains showed starch hydrolysis on starch nutrient agar. Finally, a isolate showing highest zone of hydrolysis was selected to study the amylase production and its activity. The production of enzyme, its protein content and bacterial growth was determined at different temperature ranging from 30 C to 50 C and optimum enzyme production was obtained at 40 C (Fig. 1). Above 40 C, the enzyme production was decreased

3 ASHWINI et al.: PURIFICATION AND ACTIVITY OF AMYLASES OF MARINE HALOBACILLUS SP AMYLUS 783 Fig. 1 Effect of temperature on amylase production by Fig. 3 Effect of sugar sources on amylase production by Fig. 2 Effect of ph on amylase production by Hallobacillus sp amylus dramatically. Shanmugapriya et al. (2009) obtained similar results for the production of amylase by sponge associated marine bacterium Halobacterium salinarum MMD This organism did not produce amylase at 27 C although it grew well at this temperature. Likewise Chandra et al. (1980) also reported that Bacillus licheniformis CUM 305, where maximum growth was obtained at 30 C, but no enzyme production was reported 7. Interestingly, Saito and Yamamoto (1975) reported that B.licheniformis strain which produced amylase at temperature around 50 C and never produced the enzyme at temperature below 45 C 8. ph is one of the most important factors that determine the growth and morphology of microorganisms as they are sensitive to the concentration of hydrogen ions present in the medium. ph is known to effect the synthesis and secretion of amylase just like its stability was described by Fogarty (1983) 9. Result of present study reveals that there is a stimulation of enzyme synthesis Fig. 4 Effect of nitrogen sources on amylase production by at ph 7.0 and optimum production was obtained at 8.0 (Fig. 2). Increasing ph of the medium above ph 9.0 resulted in decrease of the amylase production. This showed that the bacterium required a slightly alkaline ph for the production of amylase. Chakraborty et al., (2009), reported similar finding for the production of α-amylase from marine Streptomyces sp. D1 10. Many Researchers extensively studied alpha amylase production using various substrates by submerged fermentation and also studied factors influencing enzyme activity. Bajpai et al. (1989). reported that different carbon sources can greatly influence the production of amylase 11. Moreover, the amylase yield was similar in all the carbon sources and this was not considered to reflect inducibility which was reviewed by Meers (1971) and Nyiri (1971) 12,13. In the present study, we observed that Amylase production and protein content was optimum in dextrose followed by lactose (Fig. 3). This

4 784 INDIAN J. MAR. SCI., VOL. 42, NO. 6, OCTOBER 2013 Purification steps Enzyme activity mg Protein activity U/mL/mg Table 1 Purification of amylase from Halobacterium sp amylus Specific activity U/mL/mg Purification fold Crude extract Ammonium precipitation Dialysis Yield % Fig. 5 Effect on NaCl on amylase production by Hallobacillus sp amylus Fig. 6 Phylogentic Tree of Hallobacillus amylus coincides with the results of Halobacillus sp. strain MA-2 obtained moderate halophile environment by Chakraborty et al. (2009) 10. Similarly, in the optimization process of the enzyme activity by using different nitrogen sources reveals that yeast extract was found to be the most promising nitrogen source for the production of amylase followed by nutrient broth (Fig. 4). Results obtained are similar with the results obtained by Deltori-campus et al., Nguyen et al., and Pederson and Nielson Salinity was found to be a significant factor in the production of amylase. In the absence of sodium chloride, no enzyme production and growth were Fig. 7 Restriction sites of observed. Since the strain was a marine isolate, it showed amylase production in a medium supplemented with NaCl. The effect of NaCl on amylase production was optimum at 4.5% and as the concentration was increased there was gradual decrease in enzyme production and growth of the organism. Likewise, at 6.5% concentration the production of enzyme was decreased and there is also decrease in the protein content (Fig. 5). After optimization, the culture supernatant was prepared and used for partial purification of the enzyme. Partial purification of amylase enzyme was done by bringing the filtrate of culture to 90% saturation

5 ASHWINI et al.: PURIFICATION AND ACTIVITY OF AMYLASES OF MARINE HALOBACILLUS SP AMYLUS 785 with solid ammonium sulfate, followed by dialysis for overnight. Partially purified enzyme from Halobacterium sp amylus exhibited specific activity of U/mL/mg which corresponds to 8.07 Purification fold and 74.02% Yield (Table 1). Potential amylolytic bacteria were identified as Halobacterium sp amylus (Fig. 6). It shows 93% similarity with Halobacterium trueperi. Similarly the restriction sites prediction of the 16S rrna gene of Halobacterium sp amylus showed the restriction sites for various commercial and NEB restriction enzymes such as AciI, EcoRI, MboII, EcoRV, BaeI etc. (Fig. 7). Microbes from terrestrial sources are presently in use for industrial production of enzymes, although the potential for production of several novel enzymes by marine microorganisms have been known. In the past two years, a few Indian researchers were focused on the detection of enzymes like phosphatases, asparaginase, glutaminase, amylase, proteases, lipase, cellulase, urease and lactamase produced by marine bacteria 17. Among these enzymes, amylases are having wide range of applications. Each function of amylases requires distinctive properties with respect to specificity, stability, temperature, and ph dependence 3. Screening of microorganisms with higher amylase activities could therefore facilitate the discovery of novel amylases suitable for new industrial applications 18,19. Hence in future, amylases from Halobacterium sp amylus can be suitable for new industrial applications. Conclusion Amylases are among the most important enzymes and are of great significance having approximately 25% of enzyme market. They find potential applications in food, pharmaceutical and fine chemical industries. Based on the present study it appears that dextrose can serve as most suitable for maximum production of amylase by marine Halobacillus sp amylus. Under optimal conditions of starch (1% w/v), temperature (40 C), ph 8.0, inducers yeast extract (1% w/v), Halobacillus sp amylus produced U of amylase per litre of culture broth, which is 3 fold higher than before optimization and this strain was also found to be moderately thermophilic with alkaline ph. Acknowledgements Authors wish to thank the Management and Staff of VIT University for providing necessary facilities to carryout this study. References 1 Aiyer P V, Amylases and their applications, Afr J Biotechnol, 4 (2005) Vidyalakshmi R, Paranthaman R & Indhumathi J, Amylase Production on Submerged Fermentation by Bacillus spp, World Journal of Chemistry, 4 (2009) 89-91, M C Tigue, M A Kelly, C T Doyle, E M & Fogarty W M, The alkaline amylase of alkalophilic Bacillus sp. IMD 370, Enzyme. Microbial. Technology, 17 (1995) Gupta R, Gigras P, Mohapatra H, Goswani V K & Chaudan B, Microbial α-amylase: a biotechnological perspective, Process Biochem, 38 (2003) Wanderley K J, Torres F A G, Moraes L M P & Ulhoa C J, Biochemical characterization of α-amylase from the yeast Cryptococccus flavus, FEMS Microbiol. Lett, 231 (2004) Shanmugapriya S, Seghal K G, Joseph S, Ganhimathhi R, Bastin B T, Aseer M & Sujith S, Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047. J. Biotech. Bioprocess. Eng, 14(2009) Chandra A K, Meeda S & Bhadra A K, Production of extracelluar thermostable α-amylase by Bacillus licheniformis, J Ferment Technol, 58 (1980) Saito N & Yamamto K, Regulatory factors affecting α- amylase production in Bacillus licheniformis, J Bacteriol, 121 (1975) Fogarty W, Microbial Enzymes and Biotechnology. Applied science publishers, (1983), London and New York. 10 Chakraborty S, Khopade A, Kokare C, Mahadik K & Chopade B, Isolation and characterization of novel α-amylase from marine Streptomyces sp. D1, J. Mol. Catal. B. Enzyme, 58 (2009) Bajpai P & Bajpai P K, High-temperature Alkaline α- amylase from Bacillus licheniformis TCRDC-B13, Biiotechnol Bioeng, 33(1989) Meers J L, The regulation of α-amylase production in Bacillus licheniformis, Antonie Van Leeuwenhoek, 38 (1972) Nyiri L, The preparation of enzyme by fermentation, Int.J.Chem.Eng, 11 (1971) Dettoii-Campus B G, Priest F G & Stark J R, Hydrolysis of starch granules by the amylase from Bacillus stearothermophillus NCA 26, Process Biochem, 27 (1992) Nguyen Q D, Rezessy-Szabo J M & Hoschke A., Optimization of composition of media for the production of amylolytic enzyme by Thermomyces lanuginosus.atcc 34626, Food Technol Biotechnol, 38 (2000) Pederson H & Nielson J, The influence of nitrogen sources on the α-amylase productivity of Asperrgillus oryzae in continuous cultures, Appl Microbiol Biotechnol, 53 (2000) Chandrasekaran M, Industrial enzymes from marine microorganisms: the Indian scenario, J Mar Biotechnol, 5 (1997) Gupta R, Gigras P, Mohapatra H, Goswami V K & Chauhan B, Microbial α-amylases: a biotechnological perspective. Process Biochem, 38 (2003) Wanderley K J, Torres F A G, Moraes L M P & Ulhoa C J, Biochemical characterization of α-amylase from the yeast Cryptococcus flavus, FEMS Microbiol. Lett, 231 (2004)

Optimization, production and partial purification of extracellular α- amylase from Bacillus sp. marini

Optimization, production and partial purification of extracellular α- amylase from Bacillus sp. marini Available online at www.scholarsresearchlibrary.com Scholars Research Library Archives of Applied Science Research, 2011, 3 (1): 33-42 (http://scholarsresearchlibrary.com/archive.html) ISSN 0975-508X CODEN