Engineering of a highly thermostable endoglucanase from the GH7 family of Bipolaris sorokiniana for higher catalytic efficiency

Applied Microbiology and Biotechnology
Shritama Aich, Supratim Datta

Abstract

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose.

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Methods Mentioned

BETA
protein folding

Software Mentioned

R
GraphPad
GraphPad Prism

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