生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 281-291.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0307
收稿日期:
2023-04-06
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
郑菲,女,博士,讲师,研究方向:微生物代谢与酶工程;E-mail: zhengfei0718@bjfu.edu.cn作者简介:
杨俊钊,女,硕士研究生,研究方向:资源环境微生物学;E-mail: YJZbio@bjfu.edu.cn
基金资助:
YANG Jun-zhao(), ZHANG Xin-rui, SUN Qing-yang, ZHENG Fei()
Received:
2023-04-06
Published:
2023-10-26
Online:
2023-11-28
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B3cut突变体。结果显示,B3cut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B3cut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。
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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