Affecting Mechanism of Loop B3 on the Function of GH7 Endoglucanase

doi:10.13560/j.cnki.biotech.bull.1985.2023-0307

Abstract

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

YANG Jun-zhao, ZHANG Xin-rui, SUN Qing-yang, ZHENG Fei. Affecting Mechanism of Loop B3 on the Function of GH7 Endoglucanase[J]. Biotechnology Bulletin, 2023, 39(10): 281-291.

Figures/Tables 7

Fig. 1 Structural simulation（A）and amino acid sequence alignment（B）of MtCel7b A: Modeled structure of MtCel7b, loop structure in red, E194, D196 and E199 are catalytic triplets. B: The Loop region are marked by a yellow box. The strain source and PDB ID of the alignment sequence are HiCel7B（H. insolens, 6YOZ）; ReCel7B（R. emersonii CBS 394.64, 6SU8）; FoCel7B（F. oxysporum, 1OVW）; TrCel7B（T. reesei, 1EG1）; ThCel7B（T. harzianum CBS 226.95, 5W0A）

Fig. 2 SDS-PAGE analysis of wild-type MtCel7b and mutant B3cut M: Protein molecular weight standard; 1: purified protein of wild-type MtCel7b; 2: purified protein of mutant B3cut

Fig. 3 Enzymatic properties of wild-type MtCel7b and mutant B3cut A: Optimal temperature-activity profile. B: Optimal pH. C: pH stability. D: Thermostability at 70℃. E: Thermostability at 80℃. F: Thermostability at 90℃

Fig. 4 Halo tolerant（A）and halo stability（B）of wild-type MtCel7b and mutant B3cut

Fig. 5 Specific viabilities of wild-type MtCel7b and mutant B3cut

Fig. 6 RMSD and RMSF of wild-type MtCel7b and mutant B3cut A: RMSD of wild-type MtCel7b; B: RMSD of mutant B3cut; C: RMSF of wild-type MtCel7b; D: RMSF of mutant B3cut

Fig. 7 Trajectory principal component analysis plots and average structure of molecular dynamics simulations of wild-type MtCel7b and mutant B3cut A and B: Principal component analysis of the molecular dynamic simulation trajectory of wild-type MtCel7b and mutant B3cut. C and D: Protein surface of wild-type MtCel7b and mutant B3cut. E and F: Amino acid residues around the catalytic cleft of wild-type MtCel7b and mutant B3cut

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