Expression and Thermostability Modification of Alkaline Xylanase

doi:10.13304/j.nykjdb.2024.1030

Abstract

Abstract:

Xylanase （EC 3.2.1.8） is an important industrial enzyme， which is widely used in industries such as food， feed， papermaking， textile and biofuels. Pm10868 is derived from the genome of rumen ciliates， which could be expressed in yeast， but could not be expressed in Escherichia coli BL21. In order to increase the expression level of xylanase Pm10868 in Escherichia coli and further explore the relationship between soluble expression level and amino acid sequence in Escherichia coli， by utilizing the existing generation model MPB-MUT and expression level prediction model MPB-EXP in the laboratory， the top 3 xylanase Pm10868 mutants were screened out for the detection of enzymatic properties such as expression level， thermostability and catalytic efficiency. The results showed that the expression levels of 3 mutants in E. coli were significantly increased. Among them， the specific activity of Pm10868-117 （138.9 U·mg^-1） was approximately 2 folds that of the wild type expressed in yeast （62.5 U·mg^-1）. The k_cat/K_m of Pm10868-117 was 228.09 mL·min^-1·mg^-1， which was about 1.2 times that of wild type. When incubated at 45 ℃ for 1 h， the enzyme activity of wild-type Pm10868 was 27% of original enzyme activity， while the enzyme activity of Pm10868-117 was 81% of original enzyme activity， approximately 3 times that of the wild type. When incubated at 50 ℃ for 20 min， the enzyme activity of the wild-type Pm10868 was only 6% of original enzyme activity， while the enzyme activity of Pm10868-117 was 49% of original enzyme activity. In conclusion， the specific activity， catalytic efficiency and thermostability of Pm10868-117 were all greatly improved. Molecular dynamics simulation analysis of the wild type and mutant found that the CBM（cellulose-binding motif） fluctuation of Pm10868-117 was significantly reduced， which helped to improve the overall thermostability and binding ability with the substrate， thereby enhancing the catalytic efficiency. Above results had great significance for clarifying the structure-activity relationship of xylanase with different enzymatic properties， and layed a theoretical foundation for the molecular design of xylanase to meet the requirements of different application scenarios.

Key words: xylanase, expression level, thermostability, catalytic efficiency, mechanism analysis

摘要：

木聚糖酶（EC 3.2.1.8）是一种重要的工业酶，广泛应用于食品、饲料、造纸、纺织和生物燃料等行业。Pm10868是来源于瘤胃纤毛虫基因组的木聚糖酶，它能够在酵母中表达，但是在大肠杆菌BL21中无法表达。为了提高木聚糖酶 Pm10868 在大肠杆菌中的表达量，并深入探究在大肠杆菌中可溶性表达量与氨基酸序列之间的关系，利用实验室已有蛋白质表达量预测模型MPB-EXP和蛋白多点突变体生成模型MPB-MUT筛选到排名靠前的3条木聚糖酶，对其表达量、耐热性和催化效率等酶学性质进行检测。结果显示，3个木聚糖酶在大肠杆菌中的表达量均大幅提升。其中，木聚糖酶Pm10868-117（138.9 U·mg^-1）的酶比活约为酵母表达的野生型Pm10868（62.5 U·mg^-1）的2倍，其k_cat/K_m为228.09 mL·min^-1·mg^-1，是野生型Pm10868的1.2倍。在45 ℃条件下孵育1 h，野生型Pm10868的酶活为原始酶活的27%，而Pm10868-117的酶活为原始酶活的81%，约为野生型的3倍。在50 ℃下孵育20 min，野生型Pm10868的酶活仅为原始酶活的6%，而Pm10868-117的酶活为原始酶活的49%。综上，木聚糖酶Pm10868-117的酶比活、催化效率和耐热性方面都有较大提升。分子动力学模拟分析发现，Pm10868-117的CBM（cellulose-binding motif）结构域的波动明显减小，有助于增强突变体蛋白的整体热稳定性及其与底物的结合能力，从而提升催化效率。以上研究结果对阐明木聚糖酶具有不同酶学性质的构效关系具有重要意义，同时为木聚糖酶在不同应用场景需求的分子设计奠定理论基础。

关键词: 木聚糖酶, 表达量, 耐热性, 催化效率, 机制解析

CLC Number:

Yiyang ZHANG, Tuoyu LIU, Yuan WANG, Jian TIAN, Feifei GUAN. Expression and Thermostability Modification of Alkaline Xylanase[J]. Journal of Agricultural Science and Technology, 2025, 27(8): 73-79.

张铱洋, 刘拓宇, 王苑, 田健, 关菲菲. 碱性木聚糖酶表达及耐热性改造[J]. 中国农业科技导报, 2025, 27(8): 73-79.

Figures/Tables 7

Table 1 Screening criteria and mutation sites for wild-type and its mutant proteins

木聚糖酶Xylanase	突变位点Mutation site	模型评分Model scoring
Pm10868	—	0.26
Pm10868-117	T17V，T51E，V107Q，S124E，A300E，N437S，T493K	0.49
Pm10868-14	G15F，A214E，N246H，T324D，S464D	0.46
Pm10868-153	G24H，G41D，K43S，L45K，A218K，L419N，E445K	0.45

Fig. 1 Expression and crude enzyme activity of Pm10868 and its mutants in Escherichia coliA： SDS-PAGE analysis of soluble expression of Pm10868 and its mutants； B： Enzymatic activity of Pm10868 and its mutants

Fig. 2 Determination of expression of purification and relative activity of Pm10868-117 under different temperatures and pHA： Detection of Pm10868-117 enzyme solution； B： Relative activity of different temperatures； C： Relative activity of different pH

Fig. 3 Stability of Pm10868 and Pm10868-117 under different conditionsA： Temperature stability at 45 ℃； B： Temperature stability at 50 ℃； C： pH stability after incubation for 1 h

Fig. 4 Specific activity and kinetic parameter fitting curve of Pm10868 and Pm10868-117A： Enzyme specific activity under optimal condition； B： Kinetic parameter fitting curve of Pm10868； C： Kinetic parameter fitting curve of Pm10868-117

Table 2 Kinetic parameters of Pm10868 and Pm10868-117

参数Parameter	Pm10868	Pm10868-117
K_m/（mg·mL^-1）	3.48±0.50	3.63±0.18
k_cat/min^-1	663.87±22.25	828.33±6.75
k_cat/K_m/（mL·min^-1·mg^-1）	190.67±19.89	228.09±11.63

Fig. 5 Structural information and dynamic simulation analysisA： 3D structure of Pm10868-17 protein， purple is CBM structure， blue is binding pocket， black labels is fluctuation sites； B： RMSD analysis of Pm10868 and Pm10868-117； C： RMSF analysis of Pm10868 and Pm10868-117

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