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

• Published in: Journal of applied microbiology 2013-01, Vol.114 (1), p.108-120
• Main Authors: Puspasari, F., Radjasa, O.K., Noer, A.S., Nurachman, Z., Syah, Y.M., Maarel, M., Dijkhuizen, L., Janeček, Š., Natalia, D.
• Format: Article
• Language: English
• Subjects: alpha-Amylases - genetics; alpha-Amylases - metabolism; Amino Acid Sequence; B. aquimaris MKSC 6.2; Bacillus; Bacillus - enzymology; Bacillus - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Computational Biology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; GH13 subfamily; Manihot esculenta; Microbiology; Molecular Sequence Data; Open Reading Frames; Protein Folding; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; raw starch degrading; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Starch - metabolism; α‐amylase
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.12025

Aims The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. Methods and Results A 1539 complete open reading frame of a starch–degrading α‐amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α‐amylase (BaqA) has no starch‐binding domain, and together with a few putative α‐amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. Conclusions A novel raw starch–degrading B. aquimaris MKSC 6.2 α‐amylase BaqA is proposed to be a member of new GH13 subfamily. Significance and Impact of the Study This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.