Isolation and characterization of a putative amylase from a soil metagenomic library

Abstract

Most microorganisms in the environment cannot be cultured by standard methods. It has been estimated that less than 1% of microorganism in nature can be cultured by standard technique. To crack down the unculturability, metagenome, the collective genomes of environmental microorganisms, was studied as the target of environmental microbiology research. DNA was isolated from ranch manure samples directly and metagenomic library was constructed in E.coli using BAC vectors. To detect clones having activities of amylase, cellulase, protease, lipase, phytase, screening was done with the constructed libraries. One putative amylase gene was obtained from 10,000 recombinant E.coli clones. The clone contained novel amylase gene that showed no homology to the genes deposited in the GenBank. The putative amylase gene was expressed using pET28 and purified for characterization. Since the expressed protein produced inclusion body, the putative amylase was purified under denaturing conditions. After refolding, the protein showed amylase activity in DNS assay. The optimal temperature and pH for the enzyme was 37¡É, pH 7.0 respectively.