海洋中产胆固醇氧化酶细菌的筛选及其发酵条件优化  

Screening of cholesterol oxidase-producing bacteria in marine and its fermentation conditions optimization

作　　者：迟乃玉[1,2] 希伦 刘洋 于爽 李美玉[1,2] 张庆芳 CHI Naiyu;XI Lun;LIU Yang;YU Shuang;LI Meiyu;ZHANG Qingfang(College of Life Science and Technology,Dalian University,Dalian 116622,China;Liaoning Marine Microbial Engineering Technology Research Center,Dalian 116622,China)

机构地区：[1]大连大学生命科学与技术学院,辽宁大连116622 [2]辽宁省海洋微生物工程技术研究中心,辽宁大连116622

出　　处：《中国酿造》2019年第4期36-41,共6页China Brewing

基　　金：国家高技术研究发展计划"863计划"(No.2018YFC0311100);国家高技术研究发展计划"863计划"项目(No.2014AA093512)

摘　　要：利用菌落染色法从海洋中分离、筛选高产胆固醇氧化酶的菌株,所得菌株经分子生物学技术进行鉴定,采用单因素试验对其产酶条件进行优化,并利用硫酸铵沉淀、凝胶过滤层析法对酶进行分离纯化。结果表明,以胆固醇为唯一碳源,筛选出具有高胆固醇氧化酶活性的菌株XLH059,酶活力为0.407 U/m L;菌株XLH059被鉴定为蜡样芽孢杆菌(Bacillus cereus);菌株XLH059的最佳产酶条件为发酵周期24 h,发酵温度26℃,接种量10%,初始pH值7.0,装液量100 m L/250 m L,转速200 r/min,最佳碳源和氮源分别为葡萄糖和蛋白胨,胆固醇含量0.5%。在此优化条件下,胆固醇氧化酶活力为0.72 U/m L,是优化前的1.76倍;经分离纯化获得分子质量约为59 kDa的胆固醇氧化酶。The high-yield cholesterol oxidase strain was isolated and screened from marine by colony staining method,and identified by molecular biological technique.The enzyme production conditions were optimized by single factor tests,and the cholesterol oxidase was separated and purified by ammonium sulfate precipitation and gel filtration chromatography.The results showed that the strain XLH059 with high cholesterol oxidase activity was screened with cholesterol as sole carbon source,and the cholesterol oxidase activity was 0.407 U/ml.The strain XLH059 was identified as Bacillus cereus.The optimal enzyme production conditions for strain XLH059 were fermentation cycle 24 h,temperature 26℃,inoculum 10%,initial pH 7.0,liquid loading volume 100 ml/250 ml,and rotation speed 200 r/min.The optimal carbon source and nitrogen source were glucose and peptone,respectively,and the cholesterol content was 0.5%.Under the optimized condition,the cholesterol oxidase activity was 0.72 U/ml,which was 1.76 times as much as before optimization.The cholesterol oxidase with molecular weight of about 59 kDa was obtained by separation and purification.

关 键 词：胆固醇氧化酶 细菌 筛选 鉴定 发酵条件优化 分离纯化 

分 类 号：TQ925.2[轻工技术与工程—发酵工程]