Abstract:

Glycoside hydrolase family 7（GH7）contains two kinds of cellulases, cellobiohydrolase and endoglucanase, of which the study of cellobiohydrolase is more mature, however few studies on endoglucanase. A thermophilic endoglucanase of GH7 from the genome of the M. thermophila, MtCel7b, was identified and characterized with the maximum activity at 60℃, pH 5.0. After 1 h incubation at 90℃, MtCel7b retained over 40% activity. The loop B3 of MtCel7b, can be divided into short- and long-chains based on amino acid sequence comparison. In order to explore the effect of loop B3 on the structure and function of, endoglucanase, the mutant B3^cut was formed by truncating 14 amino acids on long-chain B3 loop of MtCel7b. At high temperatures, the activity of B3^cut was 9%-44% higher than that of MtCel7b; however, the hydrolysis ability of B3^cut to different substrates was reduced by 34%-74%. Additional analysis with the assistance of molecular dynamics simulations demonstrated that in the mutant B3^cut, the truncation of its B3 loop led to a significant displacement of loop A3 and loop B1 at both ends of the catalytic cleft, narrowing the spatial structure of the catalytic pocket and strengthening the network of hydrogen bonding interactions around the catalytic site, resulting in a more stable enzyme at high temperatures. This study sheds light on the role of loop B3 in GH7 endoglucanase and provides insight into the improvement of the enzyme molecule.

Key words: GH7, endoglucanase, loop B3, thermostability