蜡样芽孢杆菌W-3木聚糖酶xynA基因原核表达及其酶学性质研究  

Study on Prokaryotic Expression and Enzymatic Properties of Xylanase xynA Gene in Bacillus cereus W-3

作　　者：闫丽欢 肖遥 赵健 赵锦鹏 薛智权[1] 荆小院[1] 冯焱[1] YAN Lihuan;XIAO Yao;ZHAO Jian;ZHAO Jinpeng;XUE Zhiquan;JING Xiaoyuan;FENG Yan(College of Life Science,Shanxi Agricultural University,Taigu 030801,China;Beijing Viewsolid Biotechnology Company,Beijing 102200,China)

机构地区：[1]山西农业大学生命科学学院,太谷030801 [2]北京唯尚立德生物科技有限公司,北京102200

出　　处：《中国畜牧兽医》2023年第10期4114-4124,共11页China Animal Husbandry & Veterinary Medicine

基　　金：山西省自然科学基金(20210302124018);国家自然科学基金(32272829);山西省科技厅转化项目(J241942022)。

摘　　要：【目的】克隆蜡样芽孢杆菌(Bacillus cereus)W-3菌株的木聚糖酶基因xynA并进行生物信息学分析,探究其异源表达及酶学特性。【方法】采用同源扩增法克隆xynA基因,构建原核表达载体pCola-xynA,转化大肠杆菌BL21(DE3)感受态细胞进行异源表达,通过镍柱亲和层析分离纯化并通过SDS-PAGE鉴定重组蛋白,利用在线软件对xynA蛋白进行生物信息学分析。以榉木木聚糖为底物探究xynA在不同温度、不同pH条件下的酶学性质。【结果】xynA基因全长642 bp,含1个完整的开放阅读框,编码213个氨基酸。序列比对结果显示,xynA和解淀粉芽孢杆菌(Bacillus methylotrophicus)亚种FZB42木聚糖酶(AJD80562.1)相似性为96%,和类芽孢杆菌(Paenibacillus macerans)NBRC15307木聚糖酶(AAZ17386.1)及高地芽孢杆菌(Bacillus altitudinis)同家族木聚糖酶(WP-007407578.1)相似性为94%。xynA蛋白N-端含1个信号肽,理论等电点为9.42,分子质量为23.3 ku,蛋白结构稳定,为亲水性蛋白;存在5个潜在的糖基化位点、38个磷酸化位点,属于糖苷水解酶11家族。xynA酶活性为733.826 U/mg,其最适温度为70℃,最适pH为9.0。在60~80℃、pH 8.0~11.0时均具有较好的稳定性,80℃保温1 h可保留50.3%酶活,70℃和pH 9.0条件下保温1 h仍保留94.63%酶活。【结论】本研究成功从天然微生物蜡样芽孢杆菌W-3菌株中克隆木聚糖酶xynA基因,其编码蛋白为稳定的亲水蛋白,属于糖苷水解酶11家族。xynA表现出耐热、耐碱、高酶活特性,对温度、pH适应范围较广。研究结果为xynA开发利用奠定理论基础。【Objective】The objective of this study was to clone the xynA gene of Bacillus cereus W-3 and analyze its bioinformatically.The allogeneic expression and enzymatic characteristics were studied.【Method】xynA gene was cloned by homologous amplification,prokaryotic expression vector pCola-xynA was constructed,and heterologous expression was performed using Escherichia coli BL21(DE3).The recombinant protein was isolated and purified by nickel column affinity chromatography and identified by SDS-PAGE.The bioinformatic analysis of xynA protein was carried out by online software.The enzymatic properties of xynA at different temperature and pH were studied using beech xylanan as substrate.【Result】xynA gene was 642 bp in length and contained a complete open reading frame,encoding 213 amino acids.Amino acid sequence comparison showed that the similarity between xynA and Bacillus methylotrophicus subspecies FZB42 xylanase(AJD80562.1)was 96%,the similarity of NBRC15307 xylanase with Paenibacillus macerans(AAZ17386.1)and Bacillus altitudinis with the family xylanase(WP-007407578.1)was 94%.The N-terminal of xynA protein contained a signal peptide with a theoretical isoelectric point of 9.42 and a molecular weight of 23.3 ku.The protein was a hydrophilic protein with stable structure,5 potential glycosylation sites and 38 phosphorylation sites,and belongs to the glycoside hydrolase 11 family.The enzyme activity of xynA was 733.826 U/mg,the optimum temperature was 70℃,and the optimum pH was 9.0.It had good stability at 60-80℃and pH 8.0-11.0,50.3%enzyme activity could be retained at 80℃for 1 h,and 94.63%enzyme activity could be retained at 70℃and pH 9.0 for 1 h.【Conclusion】Xylanase xynA gene was successfully cloned from the natural microorganism Bacillus cereus W-3 strain.xynA protein was a stable hydrophilic protein,belonging to the glycoside hydrolase 11 family.xynA showed the characteristics of heat resistance,alkali resistance,high enzyme activity,and wide adaptability to temperature and pH.The results l

关 键 词：蜡样芽孢杆菌 木聚糖酶 原核表达 生物信息学 酶学性质 

分 类 号：Q814.9[生物学—生物工程]