林业工程学报2016，Vol.1Issue(5)：71-77,7.DOI:10.13360/j.issn.2096-1359.2016.05.013
南极嗜冷杆菌脂肪酶的原核可溶性表达优化及酶学性能表征
Optimized soluble expression and characterization of a cold-adapted lipase from psychrotrophic bacterium in Escherichia coli
摘要
Abstract
Lipase is any enzyme that catalyzes the hydrolysis of lipids, and is the subclass of the esterases. Given its ex⁃cellent functions, lipase plays an indispensable role in many industrial areas such as food processing and biodiesel prepa⁃ration. In this paper, in order to effectively improve the ulilization of lipase from microbes, a psychrophilic lipase gene from antarctic psychrotrophic bacterium, Psychrobacter sp. 7195 ( Lip7195) was cloned into the commercial plasmid pTrc99a by the methods of polymerase chain reaction ( PCR) and DNA restriction enzyme digestion as well as ligation. The recombinant lipase was chemically induced in the presence of isopropyl β⁃D⁃Thiogalactoside ( IPTG) and heterolo⁃gously expressed in Escherichia coli. The proportion of the soluble recombinant lipase was significantly enhanced by a se⁃ries of modifications at various levels including optimizing codon usage, incorporating polycationic amino acid tags. The engineered recombinant lipase showed the maximum activities at 40℃ and pH 9.0. The specific activity was determined as 10.9 U/mg. The thermal stability assay exhibited that Lip7195 was fairly thermally stable at the temperatures ranging from 30 to 40℃, and at the pH values from 8.0 to 10.0. The recombinant Lip7195 was chemically activated in the pres⁃ence of metal ion Co2+ under the assay conditions. The results indicate that these modifications can effectively decrease the formation of inclusion body in the protein biosynthesis process in E. coli. Apart from the decrease in the inclusion body of the desired protein, the biochemical property and catalytic ability of recombinant Lip7195 can be intactly main⁃tained after the relevant modifications.关键词
嗜冷脂肪酶/可溶性表达/多聚氨基酸标签/定点突变/酶学性质Key words
psychrophilic lipase/soluble expression/polycationic amino acid tags/site-directed mutagenesis/enzymatic property分类
生物科学引用本文复制引用
刘弘忍,王亮亮,何琦阳,王飞,李迅..南极嗜冷杆菌脂肪酶的原核可溶性表达优化及酶学性能表征[J].林业工程学报,2016,1(5):71-77,7.基金项目
国家林业局“948”项目（2014－4－37）；国家自然科学基金项目（31270612，31170537）；江苏高校品牌专业建设工程项目（ PPZY2015C22）；江苏高校优势学科建设工程资助项目（ PAPD）。 （）
