链霉菌769胆固醇氧化酶基因克隆、表达及生物学活性分析

doi:10.16409/j.cnki.2095-039x.2015.06.011

中国生物防治学报 ›› 2015, Vol. 31 ›› Issue (6): 889-896.DOI: 10.16409/j.cnki.2095-039x.2015.06.011

链霉菌769胆固醇氧化酶基因克隆、表达及生物学活性分析

佟雨航^1,2, 谭笑^1,2, 杜茜², 张正坤², 徐文静², 路杨², 李启云²

1. 吉林农业大学农学院, 长春 130118;
2. 吉林省农业科学院植物保护研究所/农业部东北作物有害生物综合治理重点实验室/吉林省农业微生物重点实验室, 公主岭 136100

收稿日期:2015-03-04 出版日期:2015-12-08 发布日期:2015-12-17
通讯作者: 路杨, 李启云
作者简介:佟雨航(1988-),女,硕士研究生,E-mail：tongyuhang@163.com
基金资助:
吉林省科技厅青年基金(20130522064JH);吉林省农业微生物研究与利用研究创新团队(20121812);吉林省人力资源与社会保障厅博士后基金(0010406);吉林省农业科学院博士启动基金(c4207010305)

Cloning, Expression and Activity Analysis of a Cholesterol Oxidase Gene from Streptomyces ahygroscopicus 769

TONG Yuhang^1,2, TAN Xiao^1,2, DU Qian², ZHANG Zhengkun², XU Wenjing², LU Yang², LI Qiyun²

1. College of Agriculture, Jilin Agricultural University, Changchun 130118, China;
2. Key Laboratory of Integrated Pest Management on Crops in Northeastern China, Ministry of Agriculture/Jilin Key Laboratory of Agricultural Microbiology/ Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China

Received:2015-03-04 Online:2015-12-08 Published:2015-12-17

摘要/Abstract

摘要： 胆固醇氧化酶(cholesterol oxidase,EC 1.1.3.6)是胆固醇降解代谢过程中的关键酶,在食品开发、医疗保健、临床检测、生物农药等方面具有广泛的应用价值。通过PCR方法从链霉菌Streptomyces ahygroscopicus 769的基因组中扩增得到769_ChOA基因(GenBank accession No. KF290994)。该基因全长为1578 bp,编码一个由525 aa组成的蛋白质,蛋白理论分子量为57 kDa,等电点为6.29。769_ChOA与来源于S. natalensis的胆固醇氧化酶基因的同源性为91%。构建了硫氧还蛋白－769_ChOA融合的表达载体pET32a::769_ChOA,以Escherichia coli Origami B(DE3)为表达宿主,获得了能可溶性表达的胆固醇氧化酶重组菌。经Ni亲和层析纯化得到的酶蛋白比活力为5.90 U/mg。酶学性质分析表明,该酶对胆固醇的催化活性最高,对测定的7种胆固醇类似物也都具有一定的催化能力。酶的最适温度和pH范围为20～40 ℃和pH 6.0～8.5,其活性均高于80%,且在30 ℃和pH 7.0时表现出最大的活性,酶活分别达到6.83和6.44 U/mg。酶在低于40 ℃和弱碱性条件下具有很好的稳定性。该酶对鳞翅目害虫亚洲玉米螟Ostrinia furnacalis(Guenée)和水稻二化螟Chilo suppressalis(Walker)幼虫具有很强的杀虫活性,半致死浓度分别为27.4和17.1 mg/mL。

关键词: 胆固醇氧化酶, 克隆, 表达, 活性分析

Abstract: Cholesterol oxidase is a key enzyme involved in the degradation of cholesterol, with wide applications in food processing, health care, clinical diagnosis, and biopesticides. A novel cholesterol oxidase gene, designated as 769_ChOA (GenBank accession No. KF290994) was cloned from Streptomyces ahygroscopicus 769. 769_ChOA had a nucleotide sequence of 1578 bp, encoding a 525 amino acids protein with a predicted molecular weight of 57 KDa and an isoelectric point of 6.29. The deduced amino acid sequence of 769_ChOA had 91% similarity with cholesterol oxidase from S. natalensis. The thioredoxin gene fusion expression vector pET32a(+) and Escherichia coli Origami B (DE3) were used to express 769_ChOA. The recombinant 769_ChOA mainly existed in soluble form and reached a specific activity of 5.90 U/mg after purification. The purified 769_ChOA exhibited oxidase activity against various homologous cholesterols. The optimal temperature and pH of the enzyme were 20―40 ℃ and 6.0—8.5, respectively, and the relative activity were higher than 80%. At 30 ℃ and pH 7.0, the specific activity were 6.83 U/mg and 6.44 U/mg, respectively. The enzyme was fairly stable at temperatures below 40 ℃ and in weak alkaline conditions. The purified enzyme exhibited highly efficient insecticidal activity against lepidopteran pests, including Asian corn borer Ostrinia furnacalis (Guenée) and rice-stem borer Chilo suppressalis (Walker), with 50% lethal concentration (LC₅₀) of 27.4 mg/mL and 17.1 mg/mL for the two insect pests, respectively.

Key words: cholesterol oxidase, cloning, heterologous expression, activity analysis

中图分类号:

S476.12

佟雨航, 谭笑, 杜茜, 张正坤, 徐文静, 路杨, 李启云. 链霉菌769胆固醇氧化酶基因克隆、表达及生物学活性分析[J]. 中国生物防治学报, 2015, 31(6): 889-896.

TONG Yuhang, TAN Xiao, DU Qian, ZHANG Zhengkun, XU Wenjing, LU Yang, LI Qiyun. Cloning, Expression and Activity Analysis of a Cholesterol Oxidase Gene from Streptomyces ahygroscopicus 769[J]. journal1, 2015, 31(6): 889-896.

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链霉菌769胆固醇氧化酶基因克隆、表达及生物学活性分析

Cloning, Expression and Activity Analysis of a Cholesterol Oxidase Gene from Streptomyces ahygroscopicus 769

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