Identification of Periplasmic α-Amlyase from Cow Dung Metagenome by Product Induced Gene Expression Profiling (Pigex)

A metagenomic library was constructed by cloning cow dung metagenomic DNA fragments into pGX-1 vector containing green fluorescent protein. The library was screened for the clones expressing GFP on maltose induction using fluorescence activated cell sorter. One positive clone was isolated from the library that fluoresced in response to maltose. Sequence analysis of the positive clone revealed the presence of 2031 bp ORF designated as amy1, which encodes for periplasmic alpha-amylase. The ORF was found to be poorly expressed under its native malt promoter and hence the ORF was cloned and over expressed in Escherichia coli Rosetta using T7 expression system. The over expressed recombinant amylase AMY1 was purified to homogeneity by Ni-NTA chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular mass of 76.5 kDa. The purified enzyme also showed a clear band of starch hydrolysis on zymogram analysis.