Abstract
A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in Ağri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28th hour of incubation and the highest α-amylase activity was obtained at the 40th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn2+ (123%), whereas it was strongly inhibited 1.5 mM by Hg2+. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.
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Abbreviations
- BMA, BMB and BMC:
-
basal medium A, B and C, respectively
- SD:
-
standard deviation
- SDS:
-
sodium dodecyl sulphate
References
Apar K.D. & Özbek B. 2004. α-Amylase inactivation by temperature during starch hydrolysis. Process Biochem. 39: 1137–1144.
Asgher A.M., Asad J.A.M., Rahman B.S.U. & Legge R.L. 2007. A thermostable α-amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. J. Food. Eng. 79: 950–955.
Atanassova M., Derekova A., Mandeva R., Sjøholm C. & Kambourova M. 2008. Anoxybacillus bogrovensis sp. nov., a novel thermophilic bacterium isolated from a hot spring in Dolni Bogrov, Bulgaria. Int. J. Syst. Evol. Microbiol. 58: 2359–2362.
Aygan A., Arikan B., Korkmaz H., Dinçer S. & Çolak Ö. 2008. Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68. Braz. J. Microbiol. 39: 547–553.
Behal A., Singh J., Sharma M.K., Puri P. & Batra N. 2006. Characterization of alkaline α-amylase from Bacillus sp. AB 04. Int. J. Agric. Biol. 8: 80–83.
Belduz A.O., Dulger S. & Demirbag Z. 2003. Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore forming bacterium. Int. J. Syst. Evol. Microbiol. 53: 1315–1320.
Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I., Lipman D.J., Ostell J. & Sayers E.W. 2013. GenBank. Nucleic Acids Res. 41: D36–D42.
Bernfeld P. 1955. Amylases, α and β. Methods Enzymol. 1: 149–158.
Chai Y.Y., Abd Rahman R.N.Z.R., Illias R.M. & Goh K.M. 2012. Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose. J. Ind. Microbiol. Biotechnol. 39: 731–741.
De Clerck E., Rodriguez-Diaz M., Vanhoutte T., Heyrman J., Logan N.A. & De Vos P. 2004. Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated from contaminated gelatin batches. Int. J. Syst. Evol. Microbiol. 54: 941–946.
Derekova A., Sjøholm C., Mandeva R. & Kambourova M. 2007. Anoxybacillus rupiensis sp. nov., a novel thermophilic bacterium isolated from Rupi Basin (Bulgaria). Extremophiles 11: 577–583.
Dulger S., Demirbag Z. & Belduz A.O. 2004. Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov. Int. J. Syst. Evol. Microbiol. 54: 1499–1503.
Farahmand M., Mozafari N.A., Mehrabian S. & Khavarinejad R.A. 2009. Characterization of α-amylase produced by thermophilic bacteria isolated from Iranian hot spring waters. Pajouhesh Va Sazandegi 21: 161–167.
Gul-Guven R., Guven K., Poli A. & Nicolaus B. 2008. Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov., a thermophilic bacterium isolated from a hot spring in Batman. J. Gen. Appl. Microbiol. 54: 327–334.
Gupta R., Gigras P., Mohapatra H., Goswami V.K. & Chauhan B. 2003. Microbial α-amylase: a biotechnological perspective. Process Biochem. 38: 1599–1616.
Goh K.M., Kahar U.M., Chai Y.Y., Chong C.S., Chai K.P., Ranjani V., Illias R.M. & Chan K.G. 2013. Recent discoveries and applications of Anoxybacillus. Appl. Microbiol. Biotechnol. 97: 1475–1488.
Haki G.D. & Rakshit S.K. 2003. Developments in industrially important thermostable enzymes a review. Bioresource Technol. 89: 17–34.
Hmidet N., Bayoudh A., Berrin J.G., Kanoun S., Juge N. & Nasri M. 2008. Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1. Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli. Process Biochem. 43: 499–510.
Kikani B.A. & Singh S.P. 2012. The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1. Process Biochem. 47: 1791–1798.
Konsoula Z. & Liakopoulou-Kyriakides M. 2004. Hydrolysis of starches by the action of α-amylase from Bacillus subtilis. Process Biochem. 39: 1745–1749.
Konsula Z. & Liakopoulou-Kyriakides M. 2007. Co-production of α-amylase and β-galactosidase by Bacillus subtilis in complex organic substrates. Bioresource Technol. 98: 150–157.
Kumagai Y., Satoh T., Inoue A. & Ojima T. 2013. Enzymatic properties and primary structures of two α-amylase isozymes from the Pacific abalone Haliotis discus hannai. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 164: 80–88.
Liu W., Shi P., Chen Q., Yang P., Wang G., Wang Y., Luo H. & Yao B. 2010. Gene cloning, over-expression, and characterization of a xylanase from Penicillium sp. CGMCC 1699. Appl. Biochem. Biotechnol. 162: 1–12.
Liu X.D. & Xu Y. 2008. A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization. Bioresource Technol. 99: 4315–4320.
Lowry O.H., Rosebrough N.J., Farr A.L. & Randall R.J. 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193: 265–275.
Malhotra R., Noorwes S.M. & Satyanarayana T. 2000. Production and partial characterization of thermostable and calciumindependent α-amylase of an extreme thermophilic Bacillus thermooleovorans NP54. Lett. Appl. Microbiol. 31: 378–384.
Najafi M.F., Deobagkar D. & Deobagkar D. 2005. Purifcation and characterization of an extracellular α-amylase from Bacillus subtilis AX20. Protein Expr. Purif. 41: 349–354.
Nascimento W.C. & Martins M.L.L. 2003. Production and properties of an extracellular protease from thermophilic Bacillus sp. Braz. J. Microbiol. 35: 91–96.
Prakash B., Vidyasagar M., Madhukumar S., Muralikrishna G. & Sreeramulu K. 2009. Production, purification, characterization of two extremely halotolerant, thermostable, and alkalistable α-amylases from Chromohalobacter sp. TVSP 101. Process Biochem. 44: 210–215.
Pikuta E., Lysenko A., Chuvilskaya N., Mendrock U., Hippe H., Suzina N., Nikitin D., Osipov G. & Laurinavichius K. 2000. Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavitherms comb. nov. Int. J. Syst. Evol. Microbiol. 50: 2109–2117.
Poli A., Esposito E., Lama L., Orlando P., Nicolaus G., Appolonia F.D., Gambacorta A. & Nicolausa B. 2006. Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). Syst. Appl. Microbiol. 29: 300–307.
Qader S.A., Bano S., Aman A., Syed N. & Azhar A. 2006. Enhanced production and extracellular activity of commercially important amylolytic enzyme by a newly isolated strain of Bacillus sp. AS-1. Turk. J. Biochem. 31: 135–140.
Saxena R.K., Dutt K., Agarwal L. & Nayyar P. 2007. A highly thermostable and alkaline amylase from a Bacillus sp. PN5. Bioresource Technol. 98: 260–265.
Stamford T.L.M., Stamford N.P., Coelho L.C.B.B. & Araujo J.M. 2001. Production and characterization of a thermostable α-amylase from Nocardiopsis sp. endophyte of yam bean. Bioresource Technol. 76: 137–141.
Topal SÖ. 2000. Investigation of selected industrially important enzymatic activities of Turkish agricultural mycoflora — I: amylase, lipase, protease. Turk. J. Biol. 24: 79–93.
Ul-Haq I., Ashraf H., Iqbal J. & Qadeer M.A. 2003. Production of α-amylase by Bacillus licheniformis using an economical medium. Bioresource Technol. 87: 57–61.
Wind R.D., Buitelaar R.M., Eggink G., Huizing H.J. & Dijkhuizen L. 1994. Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable amylase-producing strain. Appl. Microbiol. Biotechnol. 41: 155–162.
Yang S.J., Lee H.S., Park C.S., Kim Y.R., Moon T.W. & Park K.H. 2004. Enzymatic analysis of an amylolytic enzyme from the hyper-thermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an α-amylase and a cyclodextrin-hydrolyzing enzyme. Appl. Environ. Microbiol. 70: 5988–5995.
Yang X., Zhang F., Ren W., Nie G., Li S., Ming H., Tang S., Zhang L. & Li W. 2012. A study on the best condition for thermostable α-amylase fermented by response surface methodology by strain Anoxybacillus sp.YIM 342. J. Yunnan University 4: 490–496.
Yumoto I., Hirota K., Kawahara T., Nodasaka Y., Okuyama H., Matsuyama H., Yokota Y., Nakajima K. & Hoshino T. 2004. Anoxybacillus voinovskiensis sp. nov., a moderately thermophilic bacterium from a hot spring in Kamchatka. Int. J. Syst. Evol. Microbiol. 54: 1239–1242.
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Matpan Bekler, F., Güven, K. Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey. Biologia 69, 419–427 (2014). https://doi.org/10.2478/s11756-014-0343-2
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DOI: https://doi.org/10.2478/s11756-014-0343-2