Identification And Characterization Of A Novel Thermo Stable And Oxidant Stable Protease From Chumathang Hot Spring Using Functional Metagenomics | 63569
Journal of Biotechnology & Biomaterials
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Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of
microorganism. The common strategies for metagenomics analysis include functional and sequence based approaches.
Functional metagenomics is a promising strategy for the exploration of the bio-catalytic potential of micro biomes in order to uncover
novel enzymes for industrial processes. However, such methods suffer from low hit rates of positive clones and hence, the discovery
of novel enzymatic activities from metagenomes is highly challenging. In the present study, functional metagenomics as a promising
approach was applied for exploring the potential of hot springs for various industrial enzymes. Hot spring metagenomics offers the
possibility of exploiting the potential of unique niches in order to unravel the functional aspects of the hidden micro biomes. Hot
spring metagenomic library of Chumathang-a hot spring of Ladakh region was constructed in E. coli using pUC 18 as cloning vector.
Functional screening of approximately 10,000 clones was done for protease activity on protease substrate plates. Screening of the
metagenomic library led to the identification of one clone with potent protease activity. The clone was designated as pCHpro1. The
protease positive clone (pCHpro1) derived from the Chumathang sediment metagenomic library showed 41% identity with subtilase
family (sediment metagenome) and 35% structural similarity with crystal structure of Pro-Tk SP from Thermococcus kodakaraensis.
MEROPS peptidase database analysis showed that it belonged to peptidase S8-S53 superfamily. The enzyme was purified to a final
specific activity of 84.51 IUmg-1 proteins with a yield of 15.4%. The purified enzyme had a molecular mass of about ~38 kDa as
revealed by SDS-PAGE. The present study indicates that metagenomics without doubt offers the possibility of exploring novel genes/
ORF’s which can be characterized and applied in various industrial processes.