来源于红球菌胆固醇氧化酶ChOG的异源表达、纯化及催化反应结构分析

doi:10.13523/j.cb.20170607

中国生物工程杂志

2017, Vol. 37

Issue (6): 43-49 DOI: 10.13523/j.cb.20170607

研究报告

来源于红球菌胆固醇氧化酶ChOG的异源表达、纯化及催化反应结构分析

张玉富¹, 王建文¹, 李松涛¹, 朱张亮¹, 路福平^1,2,3, 毛淑红^1,2,3, 秦慧民^1,2,3

1. 天津科技大学生物工程学院天津 300457;
2. 天津科技大学工业发酵微生物教育部重点实验室天津 300457;
3. 工业酶国家工程实验室天津 300457

The Expression, Purification and Structural Analysis of Cholesterol Oxidase ChOG

ZHANG Yu-fu¹, WANG Jian-wen¹, LI Song-tao¹, ZHU Zhang-liang¹, LU Fu-ping^1,2,3, MAO Shu-hong^1,2,3, QIN Hui-min^1,2,3

1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;
2. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China;
3. National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, China

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摘要： 胆固醇氧化酶是胆固醇代谢过程中的关键酶，临床上用胆固醇氧化酶作为检测血清胆固醇含量的应用潜力巨大。将来源于红球菌Rhodococcus ruber的胆固醇氧化酶ChOG，分别转化到大肠杆菌宿主BL21（DE3）和Rosetta（DE3）中，在不同条件下进行诱导表达，结果表明：BL21（DE3）菌株在诱导温度为16℃、IPTG浓度为0.1 mmol/L时，ChOG可溶性表达量最高（0.49 mg/ml）。ChOG的最适反应温度为30℃，最适反应pH为7.5。最适反应条件下，酶活性达到8.0 U/mg。利用TLC、HPLC对ChOG催化产物胆甾-4-烯-3-酮进行了鉴定分析。三维结构及定点突变分析表明Glu406及Arg408、Glu261在进行胆固醇C3羟基的脱氢、质子传递，以及底物异构化方面发挥着重要作用。

关键词： 分离纯化; 结构分析; 酶活力测定; ChOG; 胆固醇氧化酶

Abstract: Cholesterol oxidase is a key enzyme during cholesterol metabolism. It could be potential used to detect the cholesterol level in serum in clinical trials. The cholesterol oxidase from Rhodococcus ruber (ChOG) was transformed into BL21(DE3) and Rosetta(DE3) for protein expression, and was induced at different conditions. The results showed that ChOG was overexpressed at 16℃ and 0.1 mmol/L IPTG. The activity reached 8.033 U/mg after purification with Ni-NTA superflow. The product cholest-5-en-3-one was characterized by TLC and HPLC, respectively. The structural analysis showed that Glu406, together with Arg408 and Glu261 played an important role during the dehydrogenation of cholesterol C3-OH, proton transfer, and isomerization.

Key words: ChOG Structural analysis Activity assay Cholesterol oxidase

收稿日期: 2016-12-16 出版日期: 2017-06-25

ZTFLH:

Q819

基金资助: 天津市科技支撑计划（14ZCZDSY00012）、国家高技术研究发展计划项目（2011AA02A211-06）、国家自然科学基金（21206127）资助项目

通讯作者: 毛淑红, 秦慧民 E-mail: shuhongmao@tust.edu.cn;huiminqin@tust.edu.cn

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引用本文:

张玉富, 王建文, 李松涛, 朱张亮, 路福平, 毛淑红, 秦慧民. 来源于红球菌胆固醇氧化酶ChOG的异源表达、纯化及催化反应结构分析[J]. 中国生物工程杂志, 2017, 37(6): 43-49.

ZHANG Yu-fu, WANG Jian-wen, LI Song-tao, ZHU Zhang-liang, LU Fu-ping, MAO Shu-hong, QIN Hui-min. The Expression, Purification and Structural Analysis of Cholesterol Oxidase ChOG. China Biotechnology, 2017, 37(6): 43-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170607 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I6/43

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