海洋微生物Cellulophaga algicola DSM 14237木聚糖酶的分子鉴定及酶学性质  

Molecular identification and enzymatic properties of marine microorganism Cellulophaga algicola DSM 14237 xylanase

作　　者：丁睿 谢会芳 韩正刚 杨江科[1] DING Rui;XIE Hui-fang;HAN Zheng-gang;YANG Jiang-ke(School of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生命科学与技术学院,武汉430023

出　　处：《武汉轻工大学学报》2022年第1期16-24,共9页Journal of Wuhan Polytechnic University

摘　　要：从海洋微生物中挖掘酶基因资源是获取新型工业酶的重要途径。从海洋微生物基因组资源库中发掘到一个来源于海洋微生物Cellulophaga algicola DSM 14237的木聚糖酶基因(Xyn14237)。通过氨基酸序列比对和三维结构模拟分析得知Xyn14237属于糖苷水解酶第10家族。将部分截短并密码子优化后的Xyn14237在大肠杆菌BL21内进行重组表达,通过镍柱亲和纯化获得重组Xyn14237,采用还原糖法测定其酶学性质。结果显示Xyn14237的最适催化温度和pH分别40℃和7.0。酶分子在pH 5~10之间稳定性较好,相对酶活均保持在90%以上。酶分子不耐热,当温度超过30℃时其活性急剧下降。以榉木木聚糖为底物,Xyn14237的最大反应速度为1627.31 U/mg,Km为3.15 mg/mL、kcat为975.98 s^(-1)。薄层层析分析显示Xyn14237水解木聚糖释放出的产物为木三糖、木二糖及木糖。从分子和酶学性质层面上分析了来自海洋微生物C.algicola的第一个木聚糖酶基因。Mining enzyme gene resources from marine microorganisms is an important way to obtain novel industrial enzymes.In this study,a xylanase gene(Xyn14237)derived from the marine microorganism Cellulophaga algicola DSM 14237 was discovered from the marine microbial genome resource bank.Through amino acid sequence alignment and three-dimensional structure simulation analysis,it was known that Xyn14237 belonged to the 10th family of glycoside hydrolases.The codon-optimized Xyn14237 was recombined and expressed in Escherichia coli BL21,and the pure recombinant Xyn14237 was obtained by affinity purification with a nickel column,and its enzymatic properties were determined by the reducing sugar method.The results showed that the optimal catalytic temperature and pH of Xyn14237 were 40°C and 7.0,respectively.Enzyme molecules had good stability between pH 5-10,and the retained relative enzyme activity was above 90%at this pH range.Xyn14237 was not heat resistant,and its activity dropped sharply after 1 h incubation at 30°C.Using beechwood xylan as the substrate,Xyn14237 showed maximum xylanase activity of 1627.31 U/mg,Km value of 3.15 mmol/L,and kcat value of 975.98 s^(-1).Thin-layer chromatography analysis showed that the products released by Xyn14237 hydrolyzing xylan were xylotriose,xylobiose,and xylose.This study analyzed the first xylanase gene from the marine microorganism C.algicola.

关 键 词：海洋微生物 木聚糖酶 10家族糖苷水解酶 酶学性质 原核表达 亲和纯化 蛋白质三维结构模拟 

分 类 号：Q814.9[生物学—生物工程]