Welcome to Chinese Journal of Biological Control,Today is

journal1 ›› 2015, Vol. 31 ›› Issue (6): 889-896.DOI: 10.16409/j.cnki.2095-039x.2015.06.011

Previous Articles     Next Articles

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

TONG Yuhang1,2, TAN Xiao1,2, DU Qian2, ZHANG Zhengkun2, XU Wenjing2, LU Yang2, LI Qiyun2   

  1. 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

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

CLC Number: