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

• Autor(a) principal: Al-Amri,A.
• Data de Publicação: 2022
• Outros Autores: Al-Ghamdi,M. A., Khan,J. A., Altayeb,H. N., Alsulami,H., Sajjad,M., Baothman,O. A., Nadeem,M. S.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Brazilian Journal of Biology
• Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842022000100184
• topic: amylase; Geobacillus; cloning; properties; in silico

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.