Influence of media composition on the production of alkaline alfa-amylase from Bacillus subtilis CB-18
Treść / Zawartość
Background. Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving a-l,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior altemative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. Material and methods. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell-free culture supernatant after centrifugation at 2515 x g for 15 minutes. Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Results. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with a-amylase activity of 680 U/mL after 48 h. Metal ions including Ca2+, Mn2+ and Mg2+ stimulated enzyme production while Hg2+ and Ag+ repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. Conclusion. This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.
- Aegeter P., Dunlap C., 1980. Culture of five commonly used acid-producing bacteria on banana pulp. Appl. Environ. Microbiol. 39, 937-942.
- Arikan B., 2008. Highly thermostable, thermophilic, alkaline SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Biores Tech. 99 (8), 3071-3076.
- Babu V., Mital B.K., Graig S.K., 2002. Effect of tomato juice addition on the growth and activity of Lactobacillus acidophilus. Int. J. Food Microbiol. 17 (1), 67-70.
- Collins C.H., Lyne P.M., 1970. Microbiological methods. Butherworths, London.
- De Azeredo L.A.I., De Lima M.B., Coelho R.R.R., Freire D.M., 2006. A low cost fermentation medium for thermophilic protease production by Streptomyces sp. 594 using feather meal and com steep liquor. Curr. Microb. 53, 335-339.
- Fischer E.H., Stein E.A., 1960. The enzymes. Vol. 4. Academic Press, New York.
- Fogarty W.M., Griffin P.J., 1975. Punfreation and properties of (β-amylase produced by Bacillus polymyxa. J. Appl. Chem. Biotech. 25, 229-238.
- Gessesse A., 1997. The use of nug meal as low cost substrate for the production of alkaline protease by the alkalophilic Bacillus sp. AR 009 and some properties of the enzyme. Biores. Tech. 62, 59-61.
- Gupta R., Gigras R, Mohapatra H., Goswami V.K., Chauhan B., 2003. Microbial α-amylases: a biotechnological perspective. Process Biochem. 381, 599-1616.
- Haki G.D., Anceno A.J., Rakshit S.K., 2008. Atypical Ca2+ - independent, raw-starch hydrolyzing α-amylase from Bacillus sp. GRE1: characterization and gene isolation. World J. Microb. Biotech. 24, 2517-2524.
- Hang Y.D., Woodams E.E., 1977. Baked-bean waste: a potential substrate for producing fungal amylases. Appl. Environ. Microbiol. 33, 1293-1294.
- Heinken F.G., O’Connor R.J., 1972. Continuous culture studies on the biosynthesis of alkaline protease, neutral protease and α-amylase by Bacillus sublilis NRRL-B 3411. J. Gen. Microb. 73, 35-44.
- Hemandez M., Rodriguez M., Guerra N., Roses R., 2006. Amylase production by Aspergillus niger in submerged cultivation on two wastes from food industries. J. Food Engr. 73, 93-100.
- Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T., Williams S.T., 1994. Bergey’s manuał of determinative bacteriology. Williams and Wilkins, Baltimore, USA.
- Horikoshi K., 1996. Alkaliphiles from an industrial point of view. FEMS Microbiol. Rev. 18, 259-270.
- Johnvesly B., Manjunath B.R., Naik G.R., 2002. Pigeon pea waste as a novel, inexpensive substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB - 99. Biores. Tech. 82, 61-64.
- Ito S., Kobayashi T., Ara K., Ozaki K., Kawai S., Hatada Y., 1998. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics and structures. Extremo- philes 2 (3), 185-190.
- Kelly C.T., Nash A.M., Fogarty W.M., 1984. Effect of manganese on alkaline phosphatase production in Bacillus sp. RK11. Appl. Microbiol. Biotech. 19, 61-66.
- Kim T.U., Gu B.G., Jeong J.Y., Byun S.M., Shin Y.C., 1996. Purification and characterization of a maltotetrose - forming alkaline α-amylase from an alkalophilic Bacillus strain GM8901. Appl. Environ. Microb. 61, 3105-3112.
- Lequerica J.L., Lafuente B., 1977. Citrus by - product utilization II. Semisolid fermentation of orange peels by Candida utilis. Rev. Agroquim. Tech. Aliment. 12,71-78.
- Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J., 1951. Protein measurement with folin-phenol reagent. J. Biochem. 193, 265-275.
- Lu Y.H., Chem G.Q., Snyder C.L., Sun J., Li Y., Wang J.L., Xaio J., 2010. High-level expression purification and characterization of a recombinant medium - temperature α-amylase from Bacillus subtilis. Biotech Lett. 32, 119-124.
- Malhotra R., Noorwez S.M., Satyanarayana T., 2000. Production and partial characterization of thermostable and calcium independent α-amylase of an extreme thermophile. Bacillus thermooleovorans NP 54. Lett. App. Microb. 31, 378-384.
- Miller G.L., 1959. Use of dinitrosalicic acid reagent for determination of reducing sugar. Anal. Chem. 31,426-428.
- Nagata Y., Suga S., Kado O., Maruo B., 1980. N-terminal amino acid sequence of α-amylase from Bacillus subtilis var amylosacchariticus: comparison with that of a liquefying type α-amylase. Agric. Biol. Chem. 44, 215-216.
- Nigarajan D.R., Rajagopalan G., Krishnan C., 2008. Purification and characterization of a maltooligosaccha- ride-forming α-amylase from a new Bacillus subtilis KCC103. Appl. Microb. Biotech. 73, 591-597.
- Poonam N., Dalel S., 1995. Enzyme and microbial systems involved in starch processing. Enz. Microb. Tech. 17, 770-778.
- Priest F.G., 1992. Exracellular enzymes. In: Encyclopedia of microbiology. Vol. 2. Ed. I. Lederberg. Academic Press, San Diego, 81-93.
- Saretty I.P, Saxena Y., Kapoor A., Sharma M., Sharma S.K., Gupta V., Gupta S., 2011. Alkaliphilic bacteria: applications in industrial biotechnology. J. Ind. Microbiol. Biotechnol. 38, 769-790.
- Sivaramakrishnan S., Gangadharan D., Nampoothiri K.M., Soccol C.R., Pandey A., 2006. Alpha amylases from microbial sources - an overview on recent developments. Food Tech. Biotech. 44, 173-184.
- Sun H., Zhao P, Ge X., Xia Y, Hao Z., Liu J., Peng M., 2010. Recent advances in microbial raw starch degrading enzymes. Appl. Biochem. Biotechnol. 160, 988-1003.
- Upton M.E., Fogarty W.M., 1977. Production and purification of thermostable amylase and protease of Thermomonospora viridis. Appl. Environ. Microbiol. 33 (1), 59-64.
- Van der Veen M.E., Van der Goot A.J., Boom R.M., 2004. Production of glucose syrups in highly concentrated Systems. Biotec. Prog. 21, 598-602.
- Vidal M.E.F., Vivas A.F., Gonzalez F., A. Arias J.M., (1995). Properties and significance of an α-amylase produced by Myxococcus coralloides. J. Appl. Bacteriol. 78, 14-19.
- Yetti M., Nazamid S., Zaiton FL, Son R., 2000. Raw starchdegrading enzyme from newly isolated strains of endophytic fungi. World J. Microbiol. Biotech. 16, 573-578.
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