Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity

Abstract

The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of - 64.964 KJ/mol for wild-type Cel5E and - 176.148, - 200.921, and - 120.038 KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity.

Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites

“…Rational engineering coupled with structural analysis and functional prediction is an efficient genetic approach to optimize enzyme properties [9,10]. To date, many thermostable endoglucanases from diverse origins and GH families have been engineered to improve their specific activity and thermal stability [11][12][13][14].…”

Engineering cellulases for conversion of lignocellulosic biomass

“…Two single mutations, Y30F and Y173F, increased the enzyme’s specific activity toward using carboxymethylcellulose sodium (CMC-Na) by 1.4- and 1.9-fold, respectively. Torktaz et al (2018) engineered Cel5E from Clostridium thermocellum by rational mutagenesis and found that individual mutations N94W, N94F, E133F and N94A improved activity on carboxymethyl cellulose (CMC) and barley β-glucan by 1.1- to 1.9-fold. As a final example, Aich and Datta (2020) reported a 2-fold increase in catalytic activity on CMC by engineering conserved residues in the substrate-binding tunnel and on the surface of a thermostable GH7 endoglucanase from Bipolaris sorokiniana .…”