Loop B3对GH7内切纤维素酶功能的影响机制

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

摘要/Abstract

摘要：

糖苷水解酶第七家族（GH7）包含内切和外切两类纤维素酶，其中对外切纤维素酶的研究较为成熟，但对内切纤维素酶的研究相对较少。本研究从嗜热真菌Myceliophthora thermophila的基因组中鉴定出一个新型GH7内切纤维素酶MtCel7b，其在60℃，pH 5.0时表现出最佳酶活力。在90℃孵育1 h后，MtCel7b仍能保留40%以上的活性。经过序列统计分析发现，MtCel7b loop B3存在长链型和短链型的进化差异，为了探究loop B3对内切纤维素酶结构和功能的影响，将MtCel7b的长链型loop B3进行截短，构建了B3^cut突变体。结果显示，B3^cut突变体在高温下的稳定性较野生型提高了约9%-44%，而其对3种纤维素底物的比活性降低了34%-74%。借助分子动力学模拟进一步分析显示，在突变体B3^cut中，其loop B3的截短导致催化裂隙两端的loop A3和loop B1发生了显著位移，缩小了催化口袋的空间结构，加强了催化位点周围的氢键作用网络，从而导致酶在高温下更加稳定。本研究阐明了loop B3在GH7内切纤维素酶中的重要作用，为酶分子的改良工作提供了新的参考。

关键词: GH7, 内切纤维素酶, loop B3, 嗜热

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

杨俊钊, 张新蕊, 孙清扬, 郑菲. Loop B3对GH7内切纤维素酶功能的影响机制[J]. 生物技术通报, 2023, 39(10): 281-291.

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.

图/表 7

图1 MtCel7b结构模拟（A）及氨基酸序列对比（B） A：MtCel7b结构模拟图（红色区域为loop结构，E194、D196和E199为催化三联体）；B：Loop B3区由黄色方框标记

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）

图2 野生型MtCel7b和突变体B3cut的SDS-PAGE分析 M：蛋白分子质量标准；1：野生型MtCel7b纯化蛋白；2：突变体B3cut纯化蛋白

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

图3 野生型MtCel7b及突变体B3cut的酶学性质A：最适温度；B：最适pH；C：pH稳定性；D：70℃下温度稳定性；E：80℃下温度稳定性；F：90℃下温度稳定性

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℃

图4 野生型MtCel7b及突变体B3cut的耐盐性（A）与盐稳定性（B）

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

图5 野生型MtCel7b及突变体B3cut的比活值

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

图6 野生型MtCel7b及突变体B3cut的均方根偏差和均方根波动图 A：野生型MtCel7b的均方根偏差图；B：突变体B3cut的均方根偏差图；C：野生型MtCel7b的均方根波动图；D：突变体B3cut的均方根波动图

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

图7 野生型MtCel7b及突变体B3cut分子动力学模拟的轨迹主成分分析及平均结构图 A、B：野生型MtCel7b及突变体B3cut分子动力学模拟轨迹的主成分分析；C、D：野生型MtCel7b及突变体B3cut的蛋白质分子表面图；E、F：野生型MtCel7b及突变体B3cut催化裂隙周围氨基酸残基

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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