ISOLATION CHARACTERIZATION AND GENETIC IMPROVEMENT OF XYLANASE PRODUCING THERMOPHILIC MICROORGANISMS FROM HOT SPRINGS OF HIMACHAL PRADESH

Abstract

ABSTRACT newlineThermophilic bacteria are a unique source of highly active xylanase enzyme with significant activity at high temperature. In this study, thermophilic xylanase (EC 3.2.1.8) producing microbes were screened from the soil of diverse thermal sites such as Manikaran and Tattapani of Himachal Pradesh. Among 200 bacterial isolates, 13 were selected for enzyme activity (xylanase). The single isolate which showed maximum xylanase activity was named as TP28. It was isolated from Tattapani hot water springs located in Mandi district of Himachal Pradesh. It showed maximum xylanase activity at 60°C. Isolate TP28 was confirmed as Bacillus sp. on the basis of colony morphology, stained preparation, biochemical tests as well as 16S rRNA sequencing and showed 99% similarity to Bacillus aestuarii. Response Surface Methodology was used to generate a process model for obtaining optimal conditions for selected nitrogen sources, carbon sources and metal ions for maximum xylanase activity. newlineThis study also incorporates mutational studies. Strain improvement was performed using ethyl methane sulfonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). High level of xylanase activity was obtained by mutant strain Bacillus sp. SC-2014 EMS200 after chemical treatment followed by EMS, which was significantly higher than wild strain Bacillus sp. SC-2014. Mutant strain was further optimized to enhance its enzyme activity. As compared to wild strain, Bacillus sp. SC-2014 EMS200 showed 3.06 folds increase in its enzyme activity. Both the wild and mutant strains show appreciable xylanase activity over a wide range from neutral to alkaline pH thereby making them amenable for industrial applications. The xylanase gene from wild and mutated strain was cloned by using Invitrogen Zero Blunt® TOPO® PCR Cloning Kit. The individual changes at the gene level were also recorded and a correlation was established between corresponding genotypic and phenotypic changes by employing various bioinformatics tools. Thermophilic and alkaline nature of mutant strain Bacillus sp. SC-2014 EMS200 are of great significance in biofuel and pulp paper industries. Keywords: Xylanase, Bacillus, 16S rRNA, Biofuel, Response Surface Methodology, Mutation, EMS, MNNG, Bacillus sp. SC-2014, Bacillus sp. SC-2014 EMS200, Bioinformatics. newline