Cloning and extracellular expression of a raw starch digesting α-amylase (Blamy-I) and its application in bioethanol production from a non-conventional source of starch

The aim of this study was to clone and efficiently express a raw starch‐digesting α‐amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non‐conventional raw starch for bioethanol production. A raw starch digesting α‐amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α‐amylase (Blamy‐I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS‐PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy‐I revealed the presence of an extra Ca2+‐binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy‐I as compared to its production under non‐optimized conditions. Blamy‐I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non‐conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy‐I and Saccharomyces cerevisiae also proved to be highly promising.