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

Autor: Mujunqi Wu, Kemin Lv, Jiahuang Li, Bin Wu, Bingfang He
Rok vydání: 2022
Předmět:
Zdroj: Biotechnology and Bioengineering. 119:2105-2114
ISSN: 1097-0290
0006-3592
Popis: EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (K
Databáze: OpenAIRE
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