Enhancement of the catalytic activity of thermostable Endo-1,4-β-glucanase B (TnCelB) from Thermotoga neapolitana by error-prone PCR

Endo-1,4-β-glucanase plays a crucial role in converting cellulose from lignocellulosic biomass into fermentable sugars for biofuel production. However, its commercial utility is hindered by poor catalytic performance under extreme conditions. This study enhanced the catalytic activity of the endo-1,4-β-glucanase ( Tn CelB) from Thermotoga neapolitana through error-prone PCR directed evolution. After screening >4000 colonies, three mutants with enhanced activity were obtained. Mutants Tn CelB Y88F , Tn CelB A233T , and Tn CelB W219R displayed 52.14 U/mg, 44.90 U/mg, and 34.70 U/mg of specific activity on CMC, respectively, which is 1.9, 1.7, and 1.3 times higher than that of the wild-type (26.74 U/mg), correspondingly. Likewise, their enzyme activity on barley β-D-glucan increased by 3.5, 2.2, and 1.8 times, respectively. Notably, Tn CelB Y88F maintained over 90 % activity after 60 mins at high temperatures (80–100 °C), indicating an exceptional thermostability. Protein docking revealed that Tn CelB Y88F had higher binding affinity, aligned with kinetic studies. Tn CelB was capble of released more non-oxidized sugars from the hydrolysis of regenerated amorphous cellulose (RAC) by synergy with auxiliary action family 10 ( AA 10), which is potential in development of efficient lignocellulosic saccharification. This study can provide useful insights for the future engineering of other endoglucanases in the glycoside hydrolases family 12.