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Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose

  • Environmental Microbiology
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD–Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6–10. At 60°C in the absence of Ca2+, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca2+ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.

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Acknowledgments

This work was financially supported by the Malaysian Ministry of Higher Education, Fundamental Research Grant Scheme (FRGS) and Universiti Teknologi Malaysia internal grant. We thank Dr. Stefan Janecek and Prof. Bernard Henrissat for their helpful discussion.

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Authors and Affiliations

  1. Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia

    Yen Yen Chai, Rosli Md. Illias & Kian Mau Goh

  2. Enzyme and Microbial Technology Research, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia (UPM), 43400, Serdang, Selangor, Malaysia

    Raja Noor Zaliha Raja Abd Rahman

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  1. Yen Yen Chai
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Correspondence to Kian Mau Goh.

Electronic supplementary material

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10295_2011_1074_MOESM1_ESM.pdf

Amino acid sequence alignment between ASKA, ADTA, and known α-amylases. Enzyme sources are abbreviated as follows: ASKA, strain SK3-4; ADTA, strain DT3-1; BLA, Bacillus licheniformis (P06278); BSTA, Bacillus stearothermophilus (P06279); TAKA, Aspergillus oryzae (P0C1B3); and AmyA, Anaerobranca gottschalkii (Q5I942). The seven highly conserved regions among amylases are boxed; the three active site residues are highlighted; and the different amino acids between ASKA and ADTA are shown in white text (PDF 74 kb)

Supplementary material 2 (PDF 22 kb)

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Chai, Y.Y., Rahman, R.N.Z.R.A., Illias, R.M. et al. 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 (2012). https://doi.org/10.1007/s10295-011-1074-9

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  • DOI: https://doi.org/10.1007/s10295-011-1074-9

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