生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (5): 26-.doi: 10.3969/j.issn.2095-1736.2024.05.026

• 研究报告 • 上一篇    下一篇

牛瘤胃微生物β-葡萄糖苷酶Bgls3的基因克隆、酶学性质及活性机理

夏　婷1， 张　萌1,3， 卢向阳1， 田　云1,3， 杨　辉1,2   

  1. 1. 湖南农业大学 生物科学技术学院， 长沙 410128； 2. 湖南省农业科学院茶叶研究所， 长沙 410125；
    3. 湖南甜蔓生物科技有限公司， 永州 425900
  • 出版日期:2024-10-18 发布日期:2024-10-14
  • 通讯作者: 田云，博士，教授，研究方向为生物资源挖掘与开发利用，E-mail:tianyun@hunau.edu.cn；杨辉，博士，研究方向为茶树植保，E-mail:yanghui2048@aliyun.com;田云和杨辉为共同通信作者
  • 作者简介:夏婷，硕士，研究方向为微生物学，E-mail:xiating@stu.hunau.edu.cn
  • 基金资助:
    湖南省科技人才托举工程-中青年优秀科技人才培养计划（2023TJ-Z04）

Gene cloning, enzymatic properties and activity mechanism of β-glucosidase Bgls3 from rumen microbiota of cattle

XIA Ting1, ZHANG Meng1,3, LU Xiangyang1, TIAN Yun1,3, YANG Hui1,2   

  1. 1. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China;
    2. Tea Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China;
    3. Hunan Sweetmax Inc., Yongzhou 425900, China
  • Online:2024-10-18 Published:2024-10-14

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摘要： β-葡萄糖苷酶因分离提取困难、酶活性低等因素限制其工业应用。从芒草驯养的牛瘤胃微生物宏基因组中克隆得到一个β-葡萄糖苷酶编码基因Bgls3并进行生物信息学分析，构建pET30a(+)-Bgls3于大肠杆菌BL21(DE3)中成功异源表达，并对Bgls3酶学性质进行研究，进一步通过同源建模、分子对接以及分子动力学分析Bgls3的活性机理。结果表明：β-葡萄糖苷酶编码基因Bgls3由2340个碱基构成，编码779个氨基酸，Bgls3蛋白具有β-葡萄糖苷酶的结构域特征，经SDS-PAGE电泳测定Bgls3蛋白分子质量为85.27 ku；Bgls3酶适应温度和pH范围宽泛，酶活力最高达到306.2 U/mg；同源建模获得Bgls3的结构模型，其符合糖苷水解酶家族3的结构特征；分子对接结果显示底物pNPG结合在Bgls3蛋白区域Ⅰ和蛋白区域Ⅱ连接的口袋位置，Bgls3中参与水解pNPG的关键氨基酸有D88、K193、E294、Y240、R160、W273、S395、E486和H194；分子动力学分析结果显示，氢键、疏水作用、电荷之间作用力以及水桥分子作用是β-葡萄糖苷酶Bgls3与底物pNPG结合的基本作用力，并确定与Bgls3活性相关的氨基酸位点。研究结果为β-葡萄糖苷酶的研究与应用增加酶源储备，也为进一步理性改造Bgls3提供理论依据。

关键词: β-葡萄糖苷酶, 异源表达, 酶学性质, 生物信息学

Abstract: Due to difficulties in the separation and extraction of β-glucosidase, as well as its low enzymatic activity, the industrial applications of β-glucosidase have been restricted. β-glucosidase geneBgls3was cloned from the metagenome of rumen microorganisms domesticated fromMiscanthus sinensisand subjected to bioinformatics analysis. The recombinant plasmid pET30a(+)-Bgls3 was constructed and successfully expressed inEscherichia coliBL21(DE3). Subsequently, the enzymatic properties of Bgls3 were investigated, and the catalytic mechanism of Bgls3 was analyzed by homology modeling, molecular docking, and molecular dynamics. The results showed that the gene encoding β-glucosidase,Bgls3, consists of 2340 base pairs, which encodes 779 amino acids. Bgls3 possesses structural domain characteristics of β-glucosidase. Through SDS-PAGE electrophoresis, the molecular weight of Bgls3 was determined to be 85.27 ku. Bgls3 enzyme showed good activity in a wide temperature and pH range, and the maximum enzyme activity can reach 306.2 U/mg. Homology modeling was used to obtain the structural model of Bgls3, which exhibited structural features consistent with those of glycoside hydrolase family 3. The molecular docking results revealed that the substrate pNPG binds to the pocket region between protein domains Ⅰ and Ⅱ of Bgls3. Key amino acids involved in the hydrolysis of pNPG by Bgls3 were identified as D88, K193, E294, Y240, R160, W273, S395, E486 and H194. Molecular dynamics analysis showed that hydrogen bonding, hydrophobic interactions, electrostatic interactions, and water bridge interactions were the fundamental forces involved in the binding of Bgls3 with pNPG, and amino acid residues relevant to the activity of Bgls3 were determined. The results of this study expanded the enzyme resources for the study and application of β-glucosidase and provided a theoretical basis for rational modification of Bgls3.

Key words: β-glucosidase, heterologous expression, enzymatic properties, bioinformatics

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