Cloning and expression of α-Amylase from novel specie, Enterobacter xiangfangensis; locally isolated from Pakistani flora

Living in an era of industrialization, there is an utmost need to replace chemical catalysts with biocatalysts owing their cost effectiveness, milder reaction conditions, eco-friendly nature and specificity. α-amylase is one of the  top commercial enzymes of industrial importance. It hydrolyzes internal α (1, 4) glycosidic linkages in polysaccharides and yields oligosaccharides of varying length. The aim of present study was to clone and express α-amylase from a novel specie Enterobacter xiangfangensis. Amplified product was cloned by A and T overhangs and transformed into cloning host; E. coli DH5α cells. Gene encoding α-amylase (1488bp) was sequenced to confirm the adequate amplification, digested with BamH1 and Xho1, cloned into expression vector; pET28b (+) and transformed into expression host; E. coli strain BL21 CodonPlus (DE3) cells.             The expression of α-amylase was induced by IPTG and optimized with varying IPTG concentrations and its induction intervals. The maximum expression was observed after 8 hours of 1mM IPTG induction. The results showed that α-amylase from novel specie, Enterobacter xiangfangensis has a potential to expresses itself in heterologous expression system so this recombinant α-amylase is ray of hope for enormous downstream applications.