Construction of metagenome libraries and characterization of a novel amylolytic enzyme

Abstract

The classical approach for isolating enzymes from environmental samples that includes enrichment of microorganisms in pure culture can analyze only subset of the total microbiota in nature since it has been estimated that less than 1% of microorganisms in nature can be cultivated by standard techniques. Metagenome was isolated directly from two different soil samples and metagenomic libraries were constructed using pUC19 vector to obtain useful microbial enzymes from uncultured soil microorganisms. The libraries were screened for amylase, cellulase, protease, DNase and lipase. Initial screening revealed that one clone from approximately 12,000 recombinant Escherichia coli strains had amylase activity. Sequencing of the clone revealed that the amylolytic enzyme was expressed from a novel gene. The putative amylase gene was overexpressed and purified for characterization. The amylase had 42˚C and pH 9.5 as the optimum temperature and pH conditions. Ca2+ was found to be the essential element for enzyme stability. The amylase hydrolyzed soluble starch, β-cyclodextrin to produce high level of maltose and pullulan was also hydrolyzed to produce panose. The enzyme showed high transglycosylation activity to make 1,4-linkages exclusively. These results suggest that the amylase is a novel enzyme conferring maltose producing α-amylase activity.