Modulation of the catalytic activity and thermostability of a thermostable GH7 endoglucanase by engineering the key loop B3.

The loop B3 of glycoside hydrolase family 7 (GH7) endoglucanases is confined into long and short types. Tt Cel7 is a thermophilic GH7 endoglucanase from Thermothelomyces thermophilus ATCC 42464 with a long loop B3. Tt Cel7 was distinct for the excellent thermostability (>30 % residual activity after 1-h incubation at 90 °C). The catalytic efficiency was reduced by removing the disulfide bond in loop B3 (C220A) and truncated the loop B3 (B3cut). However, B3cut exhibited improved thermostability, the remaining enzyme activity increased by 39 %–171 % compared to Tt Cel7 when treated at 70–90 °C for 1-h. Based on the analysis of molecular dynamics simulation, both loops B1 and A3 of B3cut swing toward the catalytic center, which contributed to the reduced cleft-space and increased structure-rigidity. Conversely, the deletion of disulfide bond resulted in a reduction of structural rigidity in C220A. Through structure-directed enzyme modulation, this study has identified two structural elements that are related to the catalysis and thermostability of Tt Cel7. The loop B3 of Tt Cel7 possibly stretches the catalytic pocket, thereby increases the openness of the catalytic tunnel and enhancing flexibility for efficient catalysis. Additionally, the disulfide bond within loop B3 serves to enhance structural stability and maintain a heightened level of activity. • A novel thermophilic GH7 endoglucanase (Tt Cel7) was identified and characterized. • Variants were designed to study the role of loop B3 in catalytic efficiency and thermostability. • Loop B3 in Tt Cel7 stretches the catalytic pocket more openly, enhancing protein flexibility for efficient catalysis. • Disulfide bond in loop B3 serves to enhance structural stability and maintain a heightened level of activity. [ABSTRACT FROM AUTHOR]