Extremophiles

, Volume 17, Issue 2, pp 339–348 | Cite as

Purification and biochemical characterization of an acidophilic amylase from a newly isolated Bacillus sp. DR90

  • Ahmad Asoodeh
  • Ashraf Alemi
  • Akbar Heydari
  • Jafar Akbari
Original Paper

Abstract

An acidophilic and Ca2+-independent amylase was purified from a newly isolated Bacillus sp. DR90 by ion-exchange chromatography, and exhibited a molecular weight of 68.9 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme were found to be 4.0 and 45 °C, respectively. The enzyme activity was increased by Ba2+, Fe2+ and Mg2+, and decreased by Hg2+ and Zn2+, while it was not affected by Na+, K+, phenylmethylsulfonyl fluoride and β-mercaptoethanol. Ca2+ and EDTA did not have significant effect on the enzyme activity and thermal stability. The values of Km and Vmax for starch as substrate were 4.5 ± 0.13 mg/ml and 307 ± 12 μM/min/mg, respectively. N,N-dialkylimidazolium-based ionic liquids such as 1-hexyl-3-methylimidazolium bromide [HMIM][Br] have inhibitory effect on the enzyme activity. Thin layer chromatography analyses displayed that maltose and glucose are the main products of the enzyme reaction on starch. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.

Keywords

Bacillus sp. DR90 Acidophilic amylase Starch industry Characterization Ionic liquids 

Supplementary material

792_2013_520_MOESM1_ESM.doc (139 kb)
Supplementary material 1 (DOC 139 kb)

References

  1. Aguilar G, Morlon-Guyot J, Trejo-Aguilar B, Guyot JP (2000) Purification and characterization of an extracellular α-amylase produced by Lactobacillus manihotivorans LMG 18010T, an amylolytic lactic acid bacterium. Enzyme Microb Tech 27:406–413CrossRefGoogle Scholar
  2. Alamri SA (2010) Isolation phylogeny and characterization of new α-amylase producing thermophilic Bacillus sp. from the Jazan Region, Saudi Arabia. Int J Biotechnol Biochem 6:537–547Google Scholar
  3. Al-ZaZaee MMA, Neelgund S, Gurumurthy DM, Rajeshwara AN (2011) Identification, characterization of novel halophilic Bacillus cereus Ms6: a source for extra cellular A-amylase. Adv Environ Biol 5:992–999Google Scholar
  4. Anupama A, Jayaraman G (2011) Detergent stable, halotolerant α-amylase from Bacillus aquimaris vitp4 exhibits reversible unfolding. IJABPT 2:366–376Google Scholar
  5. Aqeel B, Umar D (2008) Effect of alternative carbon and nitrogen sources on production of alpha-amylase by Bacillus megaterium. WASJ 8:85–90Google Scholar
  6. Arikan B (2008) Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3–15. Bioresour Technol 99:3071–3076PubMedCrossRefGoogle Scholar
  7. Asoodeh A, Ghanbari T (2013) Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806. J Mol Catal B Enzym 85–86:49–55CrossRefGoogle Scholar
  8. Asoodeh A, Lagzian M (2012) Purification and characterization of a new glucoamylopullulanase from thermotolerant alkaliphilic Bacillus subtilis DR8806 of a hot mineral spring. Process Biochem 47:806–815CrossRefGoogle Scholar
  9. Asoodeh A, Chamani J, Lagzian M (2010) A novel thermostable, acidophilic α-amylase from a new thermophilic “Bacillus sp. Ferdowsicous” isolated from Ferdows hot mineral spring in Iran: purification and biochemical characterization. Int J Biol Macromol 46:289–297PubMedCrossRefGoogle Scholar
  10. Bai Y, Huang H, Meng K, Shi P, Yang P, Luo H, Luo C, Feng Y, Zhang W, Yao B (2012) Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry. Food Chem 131:1473–1478CrossRefGoogle Scholar
  11. Bergey DH, Holt J (1994) Bergey’s manual of determinative bacteriology. Williams & Wilkins, BaltimoreGoogle Scholar
  12. Bernfeld P (1955) Amylases, α and β. Method Enzymol 1:149–158CrossRefGoogle Scholar
  13. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  14. Dabirmanesh B, Daneshjou S, Sepahi A, Ranjbar B, Khavari-Nejad R, Gill P, Heydari A, Khajeh K (2011) Effect of ionic liquids on the structure, stability and activity of two related α-amylases. Int J Biol Macromol 48:93–97PubMedCrossRefGoogle Scholar
  15. Gangadharan D, Nampoothiri K, Sivaramakrishnan S, Pandey A (2009) Biochemical characterization of raw-starch-digesting alpha amylase purified from Bacillus amyloliquefaciens. Appl Biochem Biotech 158:653–662CrossRefGoogle Scholar
  16. Goyal N, Gupta JK, Soni SK (2005) A novel raw starch digesting thermostable α-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microb Tech 37:723–734CrossRefGoogle Scholar
  17. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial alpha-amylases: a biotechnological perspective. Process Biochem 38:1599–1616CrossRefGoogle Scholar
  18. Hashida M, Bisgaard-Frantzen H (2000) Protein engineering of new industrial amylases. Trends Glycosci Glycotechnol 12:389–401CrossRefGoogle Scholar
  19. Hassan SA, Ali SA, Abbasi A, Kamal M (2011) Purification and biochemical characterization of a Ca2+-independent, thermostable and acidophilic α-amylase from Bacillus sp. RM16. Afr J Biotechnol 10:6082–6089Google Scholar
  20. Kolcuoğlu Y, Colak A, Faiz O, Belduz A (2010) Cloning, expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4. Process Biochem 45:821–828CrossRefGoogle Scholar
  21. Liu X, Xu Y (2008) A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization. Bioresour Technol 99:4315–4320PubMedCrossRefGoogle Scholar
  22. MacGregor EA, Janecek S, Svensson B (2001) Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta Protein Struct Mol Enzymol 1546:1–20CrossRefGoogle Scholar
  23. Metin K, Koç O, Ateşlier BB, Biyik HH (2010) Purification and characterization of α-amylase produced by penicillium citrinum HBF62. Afr J Biotechnol 9:7692–7701Google Scholar
  24. Mollania N, Khajeh K, Hosseinkhani S, Dabirmanesh B (2010) Purification and characterization of a thermostablephytate resistant α-amylase from Geobacillus sp. LH8. Int J Biol Macromol 46:27–36PubMedCrossRefGoogle Scholar
  25. Niehaus F, Bertoldo C, Kahler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51:711–729PubMedCrossRefGoogle Scholar
  26. Prakash O, Jaiswal N (2010) alpha-Amylase: an ideal representative of thermostable enzymes. Appl Biochem Biotechnol 160:2401–2414PubMedCrossRefGoogle Scholar
  27. Sajedi R, Naderi-Manesh H, Khajeh K, Ahmadvand R, Ranjbar B, Asoodeh A, Moradian F (2005) A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104. Enzyme Microb Tech 36:666–671CrossRefGoogle Scholar
  28. Shaw A, Bott R, Day A (1999) Protein engineering of α-amylase for low pH performance. Curr Opin Biotech 10:349–352PubMedCrossRefGoogle Scholar
  29. Simpson R (2004) Purifying proteins for proteomics: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  30. Suman S, Ramesh K (2010) Production of a thermostable extracellular amylase from thermophilic Bacillus species. J Pharm Sci Res 2:149–154Google Scholar
  31. Uyar F, Baysal Z, Dogru M (2003) Purification and some characterization of an extracellular α-amylase from a thermotolerant Bacillus subtilis. Ann Microbiol 53:315–322Google Scholar
  32. Vahidi H, Shafagi B, Mirzabeigi Z (2005) Culture medium optimization of α-amylase producing organism Mocur spp. using the variable size-simplex algorithm. Daru 13:20–22Google Scholar
  33. Valaparla VK (2010) Production of α-amylase by Acremonium sporosulcatum. J Pure Appl Microbiol 4:169–176Google Scholar
  34. Van Der Maarel MJEC, Van Der Veen B, Uitdehaag JCM, Leemhuis H, Dijkhuizen L (2002) Properties and applications of starch-converting enzymes of the alpha-amylase family. J Biotechnol 94:137–155PubMedCrossRefGoogle Scholar
  35. Vidyalakshmi R, Paranthaman R, Indhumathi J (2009) Amylase production on submerged fermentation by Bacillus spp. World J. Chem 4:89–91Google Scholar
  36. Zhang Z, Xie J, Zhang F, Linhardt RJ (2007) Thin-layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Anal Biochem 371:118–120PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Ahmad Asoodeh
    • 1
    • 2
  • Ashraf Alemi
    • 1
  • Akbar Heydari
    • 3
  • Jafar Akbari
    • 3
  1. 1.Department of Chemistry, Faculty of SciencesFerdowsi University of MashhadMashhadIran
  2. 2.Institute of BiotechnologyFerdowsi University of MashhadMashhadIran
  3. 3.Department of ChemistryTarbiat Modares UniversityTehranIran

Personalised recommendations