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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.
Highlights
Alpha-amylase is a hydrolytic enzyme of primary metabolism that catalyses the breakdown of α-1,4glycosidic linkage of glucose in starch, liberating glucose and/or maltose
The recombinant alpha amylase was successfully produced in E. coli cells under T7 promoter system
Amylase are essential enzyme used in carbohydrate metabolism by the hydrolysis of glycosidic bonds
Summary
Alpha-amylase is a hydrolytic enzyme of primary metabolism that catalyses the breakdown of α-1,4glycosidic linkage of glucose in starch, liberating glucose and/or maltose. The α-amylases are important products obtained from the living organisms that have a wide range applications in clinical, pharmaceutical and other industries. These days, the chemical hydrolysis of starch on the industrial scale has been replaced by amylase catalyzed processing. Amylases have great value in the textile, fermentation, food, paper and other industries (Pandey et al, 2000) This is the most frequently used industrial enzyme which counts about 25% of total enzyme market worldwide
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Schedule a callMore From: Brazilian journal of biology = Revista brasleira de biologia
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