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

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DS-PAGE进行纯度鉴定，确定其相对分子量约为32000Da。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0.34mM和308μmol•mg-1min-1，Ca2+、K+、Mg2+能激活这种酶的活性鳩e2+、Cu2+、Co2+则抑制它的活性。

Overexpression of lipase genes in Bacillus subtilis and characterization of the recombinant lipase

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 ，we found that 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.subtilis ．We selected the recombinant strain which is antipenicillin．The recombinant lipase was purified by ammonium sulfate fractionation and ion exchange ．SDS-PAGE was used to identify the recombinant lipase ．It was revealed that the molecular mass of the recombinant lipase is 24kDa；the recombinant lipase show maximum activity at 41℃ and pH8.0；the values of Km and Vm were found to be 0.34mM and 308 μmol•mg-1•min-1；and the metal ions Ca2+,K+and Mg2+ can activate the lipase, wheras Fe2+,Cu2+,and Co2+inhibit it.