脂肪酶基因在枯草芽孢杆菌中的表达及表达产物性质的研究  

Expression of Lipase Genes in Bacillus subtilis and Characterization of the Recombinant Lipase

作　　者：贾建波[1] 李相前[1] 胡敏[1] 

机构地区：[1]淮阴工学院生物工程系,淮安223003

出　　处：《中国生物工程杂志》2008年第1期25-29,共5页China Biotechnology

基　　金：江苏省教育厅自然科学基金资助项目(06KJB180008)

摘　　要：从环境中筛选到了脂肪酶高产菌株金黄色葡萄球菌JH,依据NCBI上发表的原核微生物脂肪酶基因序列的多序列比对,发现它具有很强的序列保守性。利用PCR从金黄色葡萄球菌JH基因组中扩增得到了脂肪酶基因,利用基因重组技术将其整合到质粒pC194中,并导入到枯草芽孢杆菌中进行表达。应用选择抗药性筛选重组子,利用硫酸铵沉淀法和离子交换层析分离纯化重组脂肪酶,并用SDS-PAGE进行纯度鉴定,确定其相对分子量约为32kDa。通过对其酶学特性的研究发现,重组脂肪酶在反应温度为41℃,pH为8.0时具有最大活性,其Km和Vm各自为0.34mmol/L和308μmol/mg·min,Ca2+、K+、Mg2+能激活这种酶的活性,而Fe2+、Cu2+、Co2+则抑制它的活性。A Staphylococcus aureus JH strain which can produce lipase was obtained from environment. According to polysequence comparision of prokaryote＇ s lipase gene published on NCBI, its sequence is very conservative. Lipase gene was obtained by PCR from genome DNA of Staphylococcus aureus JH, and then it was incorporated into plasmid pC194 and transformed into B. subtilisH11 . The recombinant lipase precipitated by （ NH4 ） 2SO4, purified by ion exchange chromatography and was identified by SDS-PAGE. It was revealed that the molecular mass of the recombinant lipase is 32kDa; the recombinant lipase show maximum activity at 41℃, pH8.0 ;the values of Km and Vm were found to be 0.34mmol/L and 3081μmol/mg. rain;The lipase can be activated by the metal ions Ca^2＋ ,K ^＋ and Mg^2＋and be inhibited by Fe^2＋ ,Cu^2＋ ,and Co^2＋.

关 键 词：金黄色葡萄球菌JH 脂肪酶基因 枯草芽孢杆菌H11 表达 酶学特性 

分 类 号：Q78[生物学—分子生物学]