New method for amylase production using metagenomics applications

Abstract

The traditional method of isolating enzymes from pure cultures only allows for the studying of a subset of the total naturally occurring microbiota in microorganism-rich environmental samples. Conventional methods are known to be able to cultivate less than 1% of all microorganisms found in nature. With the aim of isolating new enzymes and creating a special library for the amylase enzyme that has special capabilities from previously transplanted organisms, the metagenome technology has been directed. Soil DNA was isolated and the pUC19 vector was used for a metagenomic library creation. The resulted libraries were tested for enzymatic hydrolysis, and 8 clones out of 177 recombinant Escherichia coli clones found to have amylase activity by the fast traditional method for amylase screening. The libraries were examined for enzymes capable of breaking down starch. The best clones were selected to produce amylase. Two clones gave the highest amylase activity using quantitative methods. The inserted gene into clones were sequenced, and turned out to be an amylolytic enzyme that is new generated by a novel gene. For characterization, the amylase gene (SNSY) was over expressed and purified. The best Amyl SNSY enzyme activity was found at pH (8.0), temperature (55°C) and resistance to NaCl up to 3 M. This research provided a simple and reliable method for cloning recent amylase genes from ecological metagenomes. In the future, the Metagenomics guide method may be useful in increasing the collection of novel amylase from the environment.