CLONING AND EXPRESSION OF GENES BELONGING TO GH FAMILY OF ENZYMES IN BACILLUS SUBTILIS AND ASPERGILLUS ORYZAE

Abstract

GH enzymes are one of the most important and oldest industrial enzymes. These enzymes are of great significance in present day biotechnology with applications ranging from food, fermentation, pharmaceutical, and detergent, textile and paper industries. GH enzymes are used in detergents to facilitate the removal of starch containing stains. This experimental study aimed at generating recombinant Bacillus subtilis variants expressing significant amount of amylase enzyme. Site directed mutagenesis and site saturation libraries were used to impart mutations in various backbones (wild type strains). This method consists of C-fragment generation and giga PCR (mega primer based PCR). Mutated amylase genes were cloned in Bacillus subtilis and E.coli and transformed colonies were selected based on the presence of zone of clearance. The amylase gene of these clones was amplified by colony PCR and sent for DNA sequencing. The sequence confirmed variants were then fermented in different media for optimization. Expression study of GH enzymes was done using Sodium Dodecyl PolyAcrylamide Gel Electrophoresis (SDS PAGE). Change in the level of expression in different amylase backbones with respect to change in temperature and media volume was also studied. We have used a method for the fast and efficient cloning of GH enzyme genes from various sources by combining the ability of Bacillus subtilis to clone amplify and express the respective genes and of Aspergillus oryzae to efficiently express the heterologous genes. Recombinant protein production was screened in Aspergillus oryzae. Based on the characterization of the enzymes by expression analysis, purification and assay studies, large scale production may be undertaken.