Raw starch-degrading alpha-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme.

摘要

Aims. The aims were to isolate a raw starch-degrading alpha-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 alpha-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 alpha-amylase (BaqA) has no starch-binding domain, and together with a few putative alpha-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 alpha-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. copyright 2012 The Society for Applied Microbiology.