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

Abstract

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.

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS‐5

“…MD simulations have been widely used to study the conformational changes of the protein (Liu et al, 2019), and a 50 ns MD simulation was conducted on WT EG5C‐1 and variant K51T/L93T. The root‐mean‐square deviation (RMSD) is an essential parameter to assess the quality of MD simulation and was also used for analyzing the different conformations from its initial structure to its final position (Singh et al, 2020). As shown in Figure 4a, the RMSD values of the WT and variants stabilized after 35 ns, suggesting that an equilibrium state was reached, with average RMSD values of 0.16 and 0.17 nm for WT EG5C‐1 and K51T/L93T, respectively.…”

“…Although efficient hydrolysis of cellulose requires the synergistic action of multiple enzymes, processive cellulases form the core component of cellulolytic systems (Zheng & Ding, 2013). Hitherto, the high cost and low efficiency of cellulases are the major obstacles that hinder the biochemical conversion of cellulose in industrial applications (Singh et al, 2020). Therefore, it is essential to reduce the high dosages of cellulases and improve the efficiency of cellulolytic enzymes, especially the activity of processive cellulases.…”

Heterologous Expression and Biochemical Characterization of a Thermostable Endoglucanase (MtEG5-1) from Myceliophthora thermophila

“…The two catalytic residues in GH5 enzymes are both glutamic acids: one is a nucleophile and the other is a catalytic proton donor [34]. The carbohydrate-active enzyme database (http://cazy.org (accessed on 8 May 2023)) lists 53 sub-families, with approximately 80% of all known GH5 sequences falling into one of these subfamilies [35]. MtEG5-1 belongs to the GH5-1 subfamily.…”