Skip to main content
Log in

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey

  • Section Cellular and Molecular Biology
  • Published:
Biologia Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

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.

    Article  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    PubMed  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bernfeld P. 1955. Amylases, α and β. Methods Enzymol. 1: 149–158.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Gupta R., Gigras P., Mohapatra H., Goswami V.K. & Chauhan B. 2003. Microbial α-amylase: a biotechnological perspective. Process Biochem. 38: 1599–1616.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Haki G.D. & Rakshit S.K. 2003. Developments in industrially important thermostable enzymes a review. Bioresource Technol. 89: 17–34.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Konsoula Z. & Liakopoulou-Kyriakides M. 2004. Hydrolysis of starches by the action of α-amylase from Bacillus subtilis. Process Biochem. 39: 1745–1749.

    Article  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Topal SÖ. 2000. Investigation of selected industrially important enzymatic activities of Turkish agricultural mycoflora — I: amylase, lipase, protease. Turk. J. Biol. 24: 79–93.

    CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Fatma Matpan Bekler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11756-014-0343-2

Key words

Navigation