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Raw starch–degrading α‐amylase from B acillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme
Author(s) -
Puspasari F.,
Radjasa O.K.,
Noer A.S.,
Nurachman Z.,
Syah Y.M.,
Maarel M.,
Dijkhuizen L.,
Janeček Š.,
Natalia D.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12025
Subject(s) - escherichia coli , biology , amylase , recombinant dna , subfamily , microbiology and biotechnology , starch , gene , open reading frame , enzyme , biochemistry , peptide sequence
Aims The aims were to isolate a raw starch–degrading α‐amylase gene baqA from B acillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in E scherichia 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 GH 13 subfamily most closely related to GH 13_1. Two consecutive tryptophans ( T rp201 and T rp202, B aqA numbering) were identified as a sequence fingerprint of this novel GH 13 subfamily. E scherichia coli cells produced the recombinant Baq A protein as inclusion bodies. The refolded recombinant B aq A 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 B aq A is proposed to be a member of new GH 13 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.