Microbiome analysis of diverse natural niches by cultivation independent high throughput sequencing approach to identify potential carbohydrate active enzymes

Abstract

The microbes in extreme habitats are often rich in enzymes of high industrial importance which exhibiting wide range of thermal and/or pH stability, high catalytic efficiency etc. The carbohydrate active enzymes mined from these extreme niche could be employed for processing lignocellulosic biomass into value-added products. Cultivation-independent high throughput sequencing, i.e. metagenomics is a potential method for comprehensive survey of microbial diversity and their genomes. The present doctoral study was aimed at metagenomic investigation of the thermal aquatic habitat of extreme (98°C) to moderate (55°C) temperatures for gene mining and identification of novel carbohydrate related genes. The metagenomic investigation led to the identification of a variety of CAZymes. Further, the complete biochemical characterization of the metagenomically derived and#946;-glucosidase (BglM) was done. BglM also retained a significant catalytic potential at elevated levels of glucose and ethanol concentrations. In order to computationally design the thermostable sugar transferase, the whole genome of Leu. sp. LM10508 was analyzed and complete sequence of dextransucrase (sugar transferase) was mined. The industrial use of the dextransucrase enzyme from this strain is limited by weak thermal stability. Therefore, Molecular dynamics simulations of dextransucrase enzyme were performed at 30ºC and 40ºC. The calculated RMSF was analyzed to check the most fluctuating residues affected by increasing the temperature. Three mutants were generated- N335S, N335T, and Q345R. The bonding interaction between fluctuating sites and their neighboring residues provided an understanding of the mutants with improved thermostability. The improved thermal stability of the mutant was experimentally validated. newline