北华大学学报（自然科学版）2016，Vol.17Issue(2)：186-190,5.DOI:10.11713/j.issn.1009-4822.2016.02.009
脂肪酶产生菌的筛选及基因的克隆表达
Isolation of Lipase-producing Strain and the Cloning Expression of Pseudomonas Peli
摘要
Abstract
Objective To isolate lipase-producing strains from mud,to clone and characterize the lipase gene, and to determine the optimum temperature and pH.Method High activity lipase-producing strains were screened using tributyrin as substrate by transparent ring method.Lipase gene sequence of Pseudomonas peli (PP)was obtained by PCR amplification using the genomic DNA as template.The expression plasmid pET-28a-PP(rePP)was constructed and expressed in Escherichia coli BL21(DE3).Lipase enzyme was purified by Ni column,and the optimum temperature and pH were determined according to the activity curve.Results A high lipase activity strain was isolated, and was preliminarily identified as Pseudomonas peli.The fragment obtained from PCR amplification was 801 bp,which shared 100% sequence identity with that from PP.The protein of PP was expressed in E.coli BL21(DE3)with the molecular of 29 KD.The purified recombinant enzyme showed high lipolytic activity at temperature 55 ℃ and pH 7.0.Conclusion The PP gene was obtained, and the over expression protein with high lipolytic activity was obtained in this study,which maybe widely used in industry.关键词
脂肪酶/筛选/基因克隆/纯化Key words
lipase/isolation/gene cloning/purification分类
生物科学引用本文复制引用
赵雪,宋文刚,兰英..脂肪酶产生菌的筛选及基因的克隆表达[J].北华大学学报（自然科学版）,2016,17(2):186-190,5.基金项目
中国科学院院地合作项目 （）
国家高技术研究发展计划(863计划)项目(2012AA02A702). （863计划）
