Strain improvement of Bacillus amyloliquefaciens SS35 for enhanced endoglucanase catalytic efficiency and identification of mutation causing the structure changes by cloning, expression and purification of glycoside hydrolase family 5 endoglucanase and its application in saccharification of Sorghum durra

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In bioethanol production, the enzymatic saccharification of lignocellulosic biomass for release of reducing sugars is the cost limiting step. Therefore, for reducing the production cost of bioethanol, strain improvement of cellulase producing microorganisms is important. Random mutagenesis by UV in microorganism resembles the natural evolution process. Therefore, it can be used for improvement of biochemical properties in industrial enzymes (cellulase). Wild-type strain of Bacillus amyloliquefaciens SS35 was exposed to UV irradiations to develop the UV2 mutant strain with improved endoglucanase catalytic efficiency and wide range pH stability. The gene encoding endoglucanase, BaGH5-WT and BaGH5-UV2 were amplified from wild-type and UV2 mutant of Bacillus amyloliquefaciens SS35, respectively, using degenerate primers for family 5 glycoside hydrolase (GH5) and were cloned in pET-28a(+) vector and expressed in E. coli BL21(DE3) pLysS cells. The recombinant mutant BaGH5-UV2 showed 22-fold higher catalytic efficiency and wider range pH stability than recombinant wild-type BaGH5-WT. The mutant enzyme, BaGH5-UV2 showed substitution mutation of residue, Asp256 to Gly256. This mutation was in loop connecting the β6 to α6 of (β/α)8 TIM-barrel fold. Molecular dynamics simulation studies showed more stable 3-D structure for BaGH5-UV2 than BaGH5-WT. Molecular docking results showed that BaGH5-UV2 gave maximum increase in Gibb’s free energy (ΔG°) against cellotetraose. Application of BaGH5-UV2 in saccharification of acid or base pretreated Sorghum durra stalk in cocktail with CtCBH5A and CtGH1 was explored for pretreatment specific customization of enzymes in mixture design. This report provides the information for protein engineering in GH5 endoglucanases for improving their biochemical properties and pretreatment specific optimization of enzymes in mixture for enzymatic saccharification.
Supervisor: Arun Goyal