The Role of Trp201 in Bacillus aquimaris MKSC 6.2 \(\alpha\)-Amylase for Starch Binding
Ayra Ulpiyana, Diandra Sekar Annisa, Josephine Claudia Tan, Fernita Puspasari, Reza Aditama, Ihsanawati & Dessy Natalia

Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung

Abstract

\(\alpha\)-Amylases (\(\alpha\)-1,4-glucan-4-glucanohydrolase- EC 3.2.1.1) catalyze the hydrolysis of \(\alpha\)-1,4 glycosidic bonds in starch and glycogen to produce linear and branch oligosaccharides. \(\alpha\)-Amylase has a wide spectrum of industrial applications such as in the food, pharmaceutical, textile, paper, and bioethanol industries. A marine bacteria Bacillus aquimaris MKSC 6.2 produces \alpha-amylase BaqA which is a member of a new glycosyl hydrolase 13 subfamily characterized by a sequence fingerprint of two consecutive tryptophan residues (Trp201 and Trp202, BaqA numbering). These residues are located in the helix \(\alpha\)3 of the catalytic (\(\beta\)/\(\alpha\))\(_{8}\)-barrel and are predicted to have a function in the starch binding site based on in silico analysis. The aim of this study was to investigate the role of Trp201 in starch hydrolysis by employing molecular biology, molecular docking, and molecular dynamics approach. BaqA\(\Delta\)C which is a truncated form of BaqA has been previously generated and was designated as ^wildtype^. In this study, the BaqA\(\Delta\)C Trp201Ala variant has been constructed by the site-directed mutagenesis method. Both BaqA\(\Delta\)C and BaqA\(\Delta\)C W201A were expressed in Escherichia coli ArcticExpress (DE3) and purified with Ni-NTA affinity column chromatography. The BaqA\(\Delta\)C and BaqA\(\Delta\)C Trp201Ala were produced as soluble proteins with a molecular weight of ~58 kDa based on SDS-PAGE analysis. The ability of BaqA\DeltaC Trp201Ala in soluble starch hydrolysis was found to be only 2.5% of BaqA\(\Delta\)C. Further investigation through molecular docking and molecular dynamics studies showed that BaqA\(\Delta\)C W201A has lower stability and shorter ligand interaction time in the substrate binding site compared to that of BaqA\(\Delta\)C. Taken together, these results indicate that Trp201 has important in starch binding.

Keywords: \(\alpha\)-amylase, BaqA\(\Delta\)C, binding, starch, tryptophan

Topic: Biochemistry