Role of glycine 256 residue in improving the catalytic efficiency of mutant endoglucanase of family 5 glycoside hydrolase from Bacillus amyloliquefaciens SS35.

Wild-type, BaGH5-WT and mutant, BaGH5-UV2 (aspartate residue mutated to glycine), endoglucanases belonging to glycoside hydrolase family 5 (GH5), from wild-type, and UV2 mutant strain of Bacillus amyloliquefaciens SS35, respectively, were earlier cloned in pHTP0 cloning vector. In this study, genes encoding BaGH5-WT or BaGH5-UV2 were cloned into pET28a(+) expression-vector and expressed in Escherichia coli BL-21(DE3)pLysS cells. BaGH5-UV2 showed 10-fold (43.6 U/mg) higher specific activity against carboxymethylcellulose sodium salt (CMC-Na), higher optimal temperature by 10°C at 65°C, and 22-fold higher catalytic efficiency against CMC-Na, than BaGH5-WT. BaGH5-UV2 showed stability in wider acidic pH range (5.0-7.0) unlike BaGH5-WT in narrow basic pH range (7.0-7.5). BaGH5-UV2 displayed a mutation, Asp256Gly in L11 loop, connecting β6 -sheet with α6 -helix, near active site toward the domain surface of (α/β)8 -TIM barrel fold. Molecular dynamics simulation studies showed more stable structure, accessibility of substrate for a catalytic site, and increased flexibility of loop L11 of BaGH5-UV2 than the wild type, suggesting enhanced catalysis by BaGH5-UV2. Molecular docking analysis displayed enhanced hydrogen bond interactions of cello-oligosaccharides with BaGH5-UV2, unlike BaGH5-WT. Thus, Gly256 residue of loop L11 plays an important role in enhancing catalytic efficiency, and pH stability of GH5 endoglucanase. Therefore, these results help in protein engineering of GH5 endoglucanase for improved biochemical properties.