Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

Abstract

Aims: The aims were to isolate a raw starch-degrading a-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 starchdegrading a-amylase gene baqA from B. aquimaris MKSC 6Á2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 a-amylase (BaqA) has no starchbinding domain, and together with a few putative a-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 a-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.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

“…: T1SIF2) as well as on previous bioinformatics analyses when the BLAST was performed with the Bacillus aquimaris α-amylase BaqA1935. The main criterion applied for the selection was the lack of at least one residue from the catalytic triad characteristic for the α-amylase family GH13.…”

Raw starch degrading α-amylases: an unsolved riddle

“…On the other hand, the role of domain C in raw starch adsorption has been proven by truncation and LangmuirHinshelwood adsorption experiments [61]. These findings lead recently to an establishment of a novel subfamily of GH family GH13 for α-amylases from Anoxybacillus species (ASKA and ADTA) [74], G. thermoleovorans (GTA [73], Pizzo [75], Gt-amyII [61]), and B. aquimaris (BaqA) [69]. Members of this new GH13 subfamily act on raw starch and are characterized by a C-terminus composed of five conserved aromatic residues, two tryptophan residues between CSR-V and CSR-II, and an LPDlx motif in CSR-V [76].…”

“…Raw starch digesting α-amylases described so far are predominantly originating from soil isolates, while significant number of reports also engage marine enzymes [27,[68][69][70] (Table 1). α-Amylase derived from unknown marine bacterium from marine metagenomic library (AmyP) displayed substrate specificity and high catalytic efficiency on the raw rice starch [27].…”

“…This has inspired other researchers to inspect the superimposed tertiary structures of barley AMY2 with the homology model of BaqA (RSDA from Bacillus aquimaris MKSC 6.2 without a separate SBD) to identify the two consecutive tryptophans in different parts of the structure [69]. This observation leads to the idea that Trp201 and Trp202 of BaqA might represent a fingerprint of the new GH13 α-amylase subfamily [69].…”

“…This has inspired other researchers to inspect the superimposed tertiary structures of barley AMY2 with the homology model of BaqA (RSDA from Bacillus aquimaris MKSC 6.2 without a separate SBD) to identify the two consecutive tryptophans in different parts of the structure [69]. This observation leads to the idea that Trp201 and Trp202 of BaqA might represent a fingerprint of the new GH13 α-amylase subfamily [69]. These two tryptophans are also conserved in Geobacillus thermoleovorans α-amylases GTA [73] and Gt-amyII [61] as W204 and W205 with an important difference that in these two cases W205 is not located on the surface of the protein but hidden within the structure.…”