Skip to main content
SpringerLink
Log in

Identification and characterization of a novel alkaline α-amylase Amy703 belonging to a new clade from Bacillus pseudofirmus

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Alkaline α-amylases are of great interest in desizing processes and detergent industries. Here, an alkaline α-amylase gene amy703 from an alkaliphilic Bacillus pseudofirmus strain was cloned and sequenced. Its encoding product, Amy703, might represent a new clade of α-amylase family, because it shared only 35 % highest identity with all amylases characterized up to date and was not clustered into any subfamilies with amylase activity in glycoside hydrolase family 13. Heterologous expression and characterization of Amy703 showed that it is a metalloenzyme with maximal activity at 40 °C and pH 9.0. Its activity was significantly enhanced by 2- and 2.48-fold at the presence of 10 mM Ca2+ and Mg2+, respectively, while Hg2+ was a strong inhibitor of Amy703. Amy703 has a higher affinity (K m  = 3.92 mg/ml) for soluble starch compared to many other alkaline amylases. The computer modeling of its structure indicated that Amy703 contains typical amylase domains and a loop region appearing to bind the substrates. Site-directed mutagenesis suggested that a conserved residue Glu550 was essential for the activity of Amy703, and proposed it working together with other two residues to constitute a catalytic triad (Asp521, Glu550, and Asp615).

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Ahlawat S, Dhiman SS, Battan B, Mandhan RP, Sharma J (2009) Pectinase production by Bacillus subtilis and its potential application in biopreparation of cotton and micropoly fabric. Proc Biochem 44(5):521–526. doi:10.1016/j.procbio.2009.01.003

    Article  CAS  Google Scholar 

  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25(17):3389–3402. doi:10.1093/nar/25.17.3389

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Arikan B (2008) Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3–15. Biores Technol 99(8):3071–3076. doi:10.1016/j.biortech.2007.06.019

    Article  CAS  Google Scholar 

  4. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22(2):195–201. doi:10.1093/bioinformatics/bti770

    Article  CAS  PubMed  Google Scholar 

  5. Behal A, Singh J, Sharma M, Puri P, Batra N (2006) Characterization of alkaline α-amylase from Bacillus sp. AB 04. Int J Agri Biol 8(1):80–83

    CAS  Google Scholar 

  6. Boel E, Brady L, Brzozowski A, Derewenda Z, Dodson G, Jensen V, Petersen S, Swift H, Thim L, Woldike H (1990) Calcium binding in. α-amylases: an X-ray diffraction study at 2.1- A resolution of two enzymes from Aspergillus. Biochemistry 29(26):6244–6249. doi:10.1021/bi00478a019

    Article  CAS  PubMed  Google Scholar 

  7. 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 72:248–254

    Article  CAS  PubMed  Google Scholar 

  8. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucl Acids Res 37(suppl 1):D233–D238. doi:10.1093/nar/gkn663

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Chai Y, Rahman R, Illias R, Goh K (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(5):731–741. doi:10.1007/s10295-011-1074-9

    Article  CAS  PubMed  Google Scholar 

  10. Deb P, Talukdar SA, Mohsina K, Sarker PK, Sayem SA (2013) Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001. Springer Plus 2(1):154. doi:10.1186/2193-1801-2-154

    Article  PubMed Central  PubMed  Google Scholar 

  11. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific LLC, Palo Alto, California, USA. http://www.pymol.org

  12. Dumbrepatil AB, Choi JH, Park JT, Kim MJ, Kim TJ, Woo EJ, Park KH (2010) Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity. Proteins 78(2):348–356. doi:10.1002/prot.22548

    Article  CAS  PubMed  Google Scholar 

  13. Eisenberg D, Lüthy R, Bowie JU (1997) VERIFY3D: assessment of protein models with 3D profiles. Meth Enzymol 277:396–404. doi:10.1016/S0076-6879(97)77022-8

    Article  CAS  PubMed  Google Scholar 

  14. Fuwa H (1954) A new method for microdetermination of amylase activity by the use of amylose as the substrate. J Biochem 41(5):583–603

    CAS  Google Scholar 

  15. Gouet P, Courcelle E, Stuart DI, Mtoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15(4):305–308. doi:10.1093/bioinformatics/15.4.305

    Article  CAS  PubMed  Google Scholar 

  16. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Proc Biochem 38(11):1599–1616. doi:10.1016/S0032-9592(03)00053-0

    Article  CAS  Google Scholar 

  17. Hagihara H, Igarashi K, Hayashi Y, Endo K, Ikawa-Kitayama K, Ozaki K, Kawai S, Ito S (2001) Novel α-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus Isolate KSM-K38. Appl Environ Microbiol 67(4):1744–1750. doi:10.1128/aem.67.4.1744-1750.2001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Hashim SO, Delgado OD, Martínez MA, Kaul R-H, Mulaa FJ, Mattiasson B (2005) Alkaline active maltohexaose-forming α-amylase from B. halodurans LBK 34. Enzyme Microbiol Technol 36(1):139–146. doi:10.1016/j.enzmictec.2004.07.017

    Article  CAS  Google Scholar 

  19. Horinouchi S, Fukusumi S, Ohshima T, Beppu T (1988) Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents. Eur J Biochem 176(2):243–253. doi:10.1111/j.1432-1033.1988.tb14275.x

    Article  CAS  PubMed  Google Scholar 

  20. Howland J (1998) Extremophiles––Microbial life in extreme environments. In: Horikoshi K, Grant WD Wiley-Liss, New York pp 322. ISBN 0-471-02618-2. Biochem Edu 26 (4): 331–331 doi: 10.1016/S0307-4412(98)00171-X

  21. Janeček Š (2002) How many conserved sequence regions are there in the α-amylase Family. Biologia 57(Suppl 11):29–41

    Google Scholar 

  22. Janecek S, Svensson B, Henrissat B (1997) Domain evolution in the α-amylase family. J Mol Evol 45(3):322–331. doi:10.1007/PL00006236

    Article  CAS  PubMed  Google Scholar 

  23. Janeček Š, Svensson B, MacGregor EA (2013) α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci 1–22. doi:10.1007/s00018-013-1388-z

  24. Kim TU, Gu BG, Jeong JY, Byun SM, Shin YC (1995) Purification and characterization of a maltotetraose-forming alkaline (α)-amylase from an alkalophilic Bacillus strain, GM8901. Appl Environ Microbiol 61(8):3105–3112

    CAS  PubMed Central  PubMed  Google Scholar 

  25. Kumari A, Singh VK, Fitter J, Polen T, Kayastha AM (2010) α-Amylase from germinating soybean (Glycine max) seeds––purification, characterization and sequential similarity of conserved and catalytic amino acid residues. Phytochemistry 71(14–15):1657–1666. doi:10.1016/j.phytochem.2010.06.012

    Article  CAS  PubMed  Google Scholar 

  26. Lo H-F, Lin L-L, Chen H-L, Hsu W-H, Chang C-T (2001) Enzymic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant E. coli. Proc Biochem 36(8–9):743–750. doi:10.1016/S0032-9592(00)00273-9

    Article  CAS  Google Scholar 

  27. Lu ZH, JH H, Zhang GM (2013) Isolation of alkaline amylase producing bacteria and analysis of the enzymatic characteristic. J Anhui Agri Sci 41(7):2857–2859

    CAS  Google Scholar 

  28. Majzlová K, Pukajová Z, Janeček Š (2012) Tracing the evolution of the α-amylase subfamily GH13_36 covering the amylolytic enzymes intermediate between oligo-1, 6-glucosidases and neopullulanases. Carbohydr Res 367(15):48–57. doi:10.1016/j.carres.2012.11.022

    PubMed  Google Scholar 

  29. Matsuura Y, Kusunoki M, Harada W, Kakudo M (1984) Structure and possible catalytic residues of Taka-amylase A. J Biochem 95(3):697–702

    CAS  PubMed  Google Scholar 

  30. Najafi MF, Deobagkar D, Deobagkar D (2005) Purification and characterization of an extracellular α-amylase from B. subtilis AX20. Protein Expr Purif 41(2):349–354. doi:10.1016/j.pep.2005.02.015

    Article  CAS  PubMed  Google Scholar 

  31. Puspasari F, Radjasa OK, Noer AS, Nurachman Z, Syah YM, Maarel M, Dijkhuizen L, Janeček Š, Natalia D (2013) Raw starch––degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme. J Appl Microbiol 114(1):108–120. doi:10.1111/jam.12025

    Article  CAS  PubMed  Google Scholar 

  32. Ramasubbu N, Paloth V, Luo Y, Brayer G, Levine M (1996) Structure of human salivary-amylase at 1.6 A resolution: implications for its role in the oral cavity. Acta Crystallogr D Biol Crystallogr 52(3):435–446. doi:10.1107/S0907444995014119

    Article  CAS  PubMed  Google Scholar 

  33. Sambrook J, Russell DW, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York

  34. Sarethy I, Saxena Y, Kapoor A, Sharma M, Sharma S, Gupta V, Gupta S (2011) Alkaliphilic bacteria: applications in industrial biotechnology. J Ind Microbiol Biotechnol 38(7):769–790. doi:10.1007/s10295-011-0968-x

    Article  CAS  PubMed  Google Scholar 

  35. Shanmughapriya S, Kiran GS, Selvin J, Gandhimathi R, Baskar TB, Manilal A, Sujith S (2009) Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047. Biotechnol Bioproc Eng 14(1):67–75. doi:10.1007/s12257-008-0060-1

    Article  CAS  Google Scholar 

  36. Sharma A, Satyanarayana T (2012) Cloning and expression of acid stable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 16(3):515–522. doi:10.1007/s00792-012-0451-2

    Article  CAS  PubMed  Google Scholar 

  37. Souza PM, Magalhães PO (2010) Application of microbial α-amylase in industry––a review. Braz J Microbiol 41:850–861. doi:10.1590/S1517-83822010000400004

    PubMed Central  PubMed  Google Scholar 

  38. Takata H, Kuriki T, Okada S, Takesada Y, Iizuka M, Minamiura N, Imanaka T (1992) Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at α-(1–4)-and α-(1–6)-glucosidic linkages. J Biol Chem 267(26):18447–18452

    CAS  PubMed  Google Scholar 

  39. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi:10.1093/molbev/msr121

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25(24):4876–4882. doi:10.1093/nar/25.24.4876

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Tigue M, Kelly CT, Doyle EM, Fogarty WM (1995) The alkaline amylase of the alkalophilic Bacillus sp. IMD 370. Enzyme Microbiol Technol 17(6):570–573. doi:10.1016/0141-0229(94)00098-C

    Article  Google Scholar 

  42. Vallee BL, Stein EA, Sumerwell WN, Fischer EH (1959) Metal content of α-amylases of various origins. J Biol Chem 234(11):2901–2905

    CAS  PubMed  Google Scholar 

  43. Wx H, Hm C, Gy F, Me Z (2000) Study on enzyme in dexin soils polluted by mercury, chromium and arsenic. Acta Scientiae Circumstantiae 20(3):338–343

    Google Scholar 

  44. Yang H, Liu L, Li J, Du G, Chen J (2011) Heterologous expression, biochemical characterization, and overproduction of alkaline a-amylase from B. alcalophilus in B. subtilis. Microb Cell Fact 10:77. doi:10.1186/1475-2859-10-77

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Yang H, Liu L, Wang M, Li J, Wang NS, Du G, Chen J (2012) Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from Alkalimonas amylolytica for improved oxidative stability. Appl Environ Microbiol 78(21):7519–7526. doi:10.1128/AEM.01307-12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Zhang GM, Huang J, Huang GR, Ma LX, Zhang XE (2007) Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Appl Microbiol Biotechnol 74(2):339–346. doi:10.1007/s00253-006-0648-3

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by the Natural Science Foundation of Hubei Province (2011CDA00302), China 863 Program (2012AA022203) and TianjinZhuanxiang 13ZCDZSY05000.

Author information

Authors and Affiliations

  1. Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan, 430062, China

    Zhenghui Lu & Guimin Zhang

  2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Chaoguang Tian & Yanhe Ma

  3. The College of Life Sciences, Central China Normal University, Wuhan, 430079, China

    Aiying Li

Authors
  1. Zhenghui Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chaoguang Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aiying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guimin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanhe Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guimin Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, Z., Tian, C., Li, A. et al. Identification and characterization of a novel alkaline α-amylase Amy703 belonging to a new clade from Bacillus pseudofirmus . J Ind Microbiol Biotechnol 41, 783–793 (2014). https://doi.org/10.1007/s10295-014-1420-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-014-1420-9

Keywords

Search

Navigation