Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome.

Roma Garg,Girish Sahni,Subramanian Karthikeyan,Ritika Srivastava,Lata Verma,Vijaya Brahma

doi:10.1038/srep39634

Abstract

Cellulase catalyzes the hydrolysis of β-1,4-linkages of cellulose to produce industrially relevant monomeric subunits. Cellulases find their applications in pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. These industries always have great demand for cellulases that can work efficiently even in harsh conditions such as high salt, heat, and acidic environments. While, cellulases with high thermal and acidic stability are already in use, existence of a high halotolerant cellulase is still elusive. Here, we report a novel cellulase Cel5R, obtained from soil metagenome that shows high halotolerance and thermal stability. The biochemical and functional characterization of Cel5R revealed its endoglucanase activity and high halostability. In addition, the crystal structure of Cel5R determined at 2.2 Å resolution reveals a large number of acidic residues on the surface of the protein that contribute to the halophilic nature of this enzyme. Moreover, we demonstrate that the four free and non-conserved cysteine residues (C65, C90, C231 and C273) contributes to the thermal stability of Cel5R by alanine scanning experiments. Thus, the newly identified endoglucanase Cel5R is a promising candidate for various industrial applications.

Highlights

Endoglucanases are very diverse and are part of 14 glycosyl hydrolase (GH) families[12]
We report a novel endoglucanase, Cel5R, that belong to GH5 family, identified by soil metagenomic approach, which is tolerant to high salt conditions with moderate tolerance to temperature and pH
Functional screening of the library on LB plates containing 0.5% carboxymethyl cellulose (CMC) (Carboxymethyl cellulose) revealed a positive clone with an insert size of ~5 kb that showed a clear zone of hydrolysis on CMC plate

Summary

Introduction

Endoglucanases are very diverse and are part of 14 glycosyl hydrolase (GH) families[12]. Among the known strategies[13,14,15], metagenomics (culture independent approach) is a unique way to access the hidden information in unexplored microbial lineages and discover novel genes, metabolic pathways, and industrially important products[16,17] as only 0.1–1% of the microbes are culturable under laboratory conditions. We report a novel endoglucanase, Cel5R, that belong to GH5 family, identified by soil metagenomic approach, which is tolerant to high salt conditions with moderate tolerance to temperature and pH. The Cel5R shows thermostability up to 58 °C and pH stability from 5–9. The Cel5R shows halotolerance and extreme halostability in 4 M NaCl, 3 M LiCl and 2 M KCl which is higher than other known halostable cellulases[18,19]. The combination of extreme halostability with moderate thermal and pH stability makes Cel5R a potential candidate for industrial applications

Methods

Results

Conclusion