Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal 741246, India.
School of Energy Science and Engineering, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
J Phys Chem B. 2024 Sep 12;128(36):8628-8640. doi: 10.1021/acs.jpcb.4c03464. Epub 2024 Sep 2.
The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate -nitrophenyl β-d-glucopyranoside (NPGlc) than on the natural substrate, cellobiose. We report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg), a 1.5-fold increase in (340.8 ± 27 s), and a 3-fold increase in catalytic efficiency (236.65 mM s) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase β-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.
木质纤维素原料经纤维素酶转化为葡萄糖是生物燃料生产的关键步骤。β-葡萄糖苷酶催化纤维素分解的最后一步，生成葡萄糖，通常是生物质水解的限速步骤。大多数天然和工程化的β-葡萄糖苷酶在人工底物-硝基苯-β-D-葡萄糖苷（NPGlc）上的比活性高于天然底物纤维二糖。我们报道了一种经过工程改造的β-葡萄糖苷酶（Q319A H0HC94），其比活性（366.3±36μmol/min/mg）提高了 1.8 倍，（340.8±27s）提高了 1.5 倍，催化效率（236.65mM s）提高了 3 倍，而 H0HC94（WT）对纤维二糖的催化效率则提高了 3 倍。分子动力学模拟和蛋白质结构网络分析表明，与 WT 相比，Q319A H0HC94 的活性位点口袋发生了显著重塑，导致酶构象发生变化，纤维二糖在活性口袋内的可及性更好，酶活性更高。本研究表明，通过减少位于门控区附近的活性口袋残基与纤维二糖之间的拥挤相互作用，以及增加口袋体积和表面积，合理工程化非保守残基可显著提高β-葡萄糖苷酶的底物可及性和催化效率。因此，对以前表征的酶进行合理工程化可能是提高纤维素水解的一种极好策略。
