Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

作者：Kemin Lv, Xiaozhou Li, Kequan Chen, Bin Wu, Bingfang He, Gerhard Schenk · 发表于：ACS Catalysis · 年份：2024 · DOI：10.1021/acscatal.4c01083 · 被引用次数：31 · 研究领域：Biofuel production and bioconversion、Enzyme Catalysis and Immobilization、Carbohydrate Chemistry and Synthesis

Processive endoglucanase is a typical bifunctional biocatalyst for cellulose degradation. A GH5 processive endoglucanase from Bacillus subtilis BS-5 was previously identified and shown to exhibit highly efficient catalytic performance. To further augment its catalytic efficiency, both consensus mutagenesis and loop engineering were applied. Compared to the wild-type enzyme, a variant (M3-1) with the four point mutations, i.e., K91I, A198T, Q237D, and V240P, exhibits an 8.5- and 4.8-fold increase in catalytic efficiency toward the soluble substrate carboxymethyl cellulose-Na (CMC) and the insoluble phosphoric acid-swollen cellulose (PASC), respectively. Molecular dynamics simulations were employed to elucidate the conformational changes that led to the enhanced catalytic efficiency. Structural superpositions suggest that the mutations cause a swing in loop 230–241, which in turn affects enzyme–substrate affinity and recognition. Residues K91 and A198 are located distal from the active site. The mutations K91I and A198T influence key amino acids within the active pocket through residue interaction networks in the protein. Furthermore, dynamic cross-correlation matrices (DCCMs) indicate that variant M3-1 possesses a conformation that is more favorable than that of the wild-type enzyme, promoting an increased frequency of interactions between active site residues and substrate molecules and thereby enhancing catalytic efficiency. The combined results provide valuable insights int...