Mining of Thermostable Alpha-amylase Gene from Geothermal Springs using a Metagenomics Approach

Abstract

The geothermal springs are said to contain the greatest diversity of undiscovered microorganisms, making them the best source for enzymes with economic significance. The untapped microbial diversity living in the geothermal springs can be mined for novel genes, bioactive substances, and industrially significant biocatalysts using the metagenomics technique. Metagenome was extracted from soil samples of various geothermal springs of India. Metagenome was screened for various carbohydrate degrading enzymes (amylase, cellulase, xylanase, amylopullulanase) using degenerate primers-based Polymerase chain reaction amplifications. Further amplicons were cloned, sequenced and analysis of data was done using various bioinformatics tools, e.g., Blast analysis, Protparam and phre2 program. We have isolated numerous enzymes, including cellulase, amylase, amylopullulanase, and xylanase, from diverse geothermal spring in different parts of India using sequence and function-based metagenomics. In this study, we describe the metagenomics-based isolation of a thermostable amylase from the geothermal spring of Odisha. The amylase gene (1503 bp) was amplified using the metagenome as a template using degenerate primers and cloned into the linearized T vector. The putative gene was likely to encode a protein of 469 amino acids with a molecular weight of 53895.05 Da with pI-7.78. Sequence analysis showed its maximum identity of 98.95% with Bacillus licheniformis alpha-amylase gene. Homology modeling of the amylase protein was done using the phyre2 program, which shows it belongs to the (trans) glycosidase superfamily and contains the catalytic TIM alpha/beta-barrel fold. Hence, we can conclude that geothermal springs are hotspots for the mining of industrially robust biocatalysts.