重组木聚糖酶XynH的C/N端及“Thumb”结构对其酶学性质的影响  

Effect of C/N terminus and“Thumb”structure of recombinant xylanase XynH on its catalytic properties

作　　者：朱澳迪 赵婷 徐锦 陈茂彬 方尚玲 李琴 ZHU Aodi;ZHAO Ting;XU Jin;CHEN Maobin;FANG Shangling;LI Qin(Cooperative Innovation Center of Industrial Fermentation(Ministry of Education&Hubei Province),Key Laboratory of Fermentation Engineering(Ministry of Education),Hubei Key Laboratory of Industrial Microbiology,School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068,China;College of Food Science,Sichuan Agricultural University,Ya′an 625014,China)

机构地区：[1]工业发酵省部共建协同创新中心,发酵工程教育部重点实验室,工业微生物湖北省重点实验室,湖北工业大学生物工程与食品学院,湖北武汉430068 [2]四川农业大学食品学院,四川雅安625014

出　　处：《食品与发酵工业》2024年第12期17-23,I0001-I0004,共11页Food and Fermentation Industries

基　　金：国家自然科学基金(31901634);四川省科技厅项目(2022NSFSC1739);湖北工业大学研究生教学改革研究项目资助(校2021057)。

摘　　要：木聚糖酶在造纸、饲料、能源、食品和医药等行业广泛应用,利用基因工程技术快速挖掘适用于工业生产需求的木聚糖酶资源具有重要意义。该文基于生物信息学分析,针对木聚糖酶XynH的C/N端及“Thumb”结构进行分子改造,成功构建突变体X-N、X-C、X-f。各突变体与原酶XynH相比,X-N的最适pH值为5.5,其他酶均是在pH 5.0时表现出最高酶活力;各突变体与XynH在pH 4.0~6.0的残余酶活力均在60%左右,具有良好的耐酸性。X-N的最适温度(60℃)与其他酶相比提高了5℃;在55℃下保温1 h后,X-N和X-f的残余酶活力高于XynH,X-N的耐热性表现更好。各突变体对榉木木聚糖的亲和力较弱,但表现出更高的转化率。该文利用基因工程手段对木聚糖酶进行分子改造,构建了3个突变体,均表现出较好的耐酸性,X-N的最适pH略高于原酶,而且在高温下稳定性更好。N端替换改善了木聚糖酶的热稳定性,C端替换可能涉及到催化结构域的一部分,从而导致酶活性的变化。而“Thumb”末端的改造可能使活性位点的裂缝变窄,阻止了底物的进入,导致酶的催化效率降低。突变体凸显出的与原酶酶学性质的差异,从中展现出的结构与功能的关系,为木聚糖酶的改良和应用提供了一定的思路和策略。Xylanase is widely used in papermaking,feed,energy,food,medicine,and other industries.It is of great significance to use genetic engineering technology to quickly tap xylanase resources suitable for industrial production.Based on bioinformatics analysis,we successfully constructed mutant X-N,X-C,and X-f based on molecular modification of the C/N terminal and“Thumb”structure of xylanase XynH.Compared with the proenzyme XynH,the optimal pH of X-N was 5.5,and the other enzymes showed the highest enzyme activity at pH 5.0.The residual enzyme activity of all mutants and XynH at pH 4.0-6.0 was about 60%,and they had good acid resistance.The optimum temperature of X-N(60℃)was increased by 5℃compared with other enzymes.After heat preservation at 55℃for 1 h,the residual enzyme activity of X-N and X-f was higher than that of XynH,and the heat resistance of X-N was better.All the mutants showed weak affinity to beech xylan,but showed higher conversion rate.In this paper,the molecular modification of xylanase by genetic engineering means was used to construct three mutants,all of which showed good acid resistance,the optimal pH of X-N was slightly higher than that of the original enzyme,and the stability was better at high temperature.N-terminal replacement improves the thermal stability of xylanase,and C-terminal replacement may involve a part of the catalytic domain,resulting in a change in enzyme activity.The modification of the end of"Thumb"may narrow the cracks at the active site,preventing the entry of the substrate,resulting in a reduction in the catalytic efficiency of the enzyme.The difference between the mutant and the original enzyme in enzymology and the relationship between structure and function revealed by the mutant provide some ideas and strategies for the improvement and application of xylanase.

关 键 词：木聚糖酶 生物信息学 分子改造 酶学性质 

分 类 号：Q814[生物学—生物工程] TQ426.97[化学工程]