Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465

Abstract

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Exploration of computational approaches to predict the structural features and recent trends in α‐amylase production for industrial applications

“…The starch is preferably cheaper, easily available sizing, and strengthen agent applied on the threads. The starch paste is applied for the strengthening of threads during the weaving process in the textile industry (Al‐Amri et al, 2021). The starch‐coated clothes are treated with amylases to give smoothness before the dying process (Zafar et al, 2019).…”

“…The starch paste is applied for the strengthening of threads during the weaving process in the textile industry (Al-Amri et al, 2021).…”