摘要
目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌BL21(DE3)中实现高效表达,并对重组酶进行酶学性质研究。方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定。结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL。重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失。该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力。结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础。
Objective：A lipase gene lipA was cloned from Serratia marcescens and overexpressed in E.coli BL21（DE3）.Then the characterization of recombinant lipase was studied.Method：The gene encoded lipase from Serratia marcescens was amplified by PCR and the recombinant expression vector pET-lipA was constructed and transformed into E.coli strain,then was induced for expression.The expressed products of the recombinant strain was analyzed by SDS-PAGE and the characterization of the recombinant lipase was studied.Result：After optimization,the maximum lipase activities reached 104U/mL.The optimal temperature of the recombinant lipase was at 40-45℃,the optimal reaction pH value was at 7-7.5.The enzyme kept 80% of its activity when it was incubated for one hour at 50℃.The lipase activity was increased obviously by addition of Ca2＋ ,Sr2＋,Mn2＋and Mg2＋,especially the activity was enhanced more than double when Ca2＋ existed,while inhibited by Ni2＋,Fe2＋,Fe3＋ ,Cu2＋ ,Zn2＋ and Al3＋.There was no activity when Zn2＋ and Al3＋ existed.The enzyme was also tolerant to methanol.The lipase was incubated in 50% methanol solution and glycerol for 24h,there was still 84% activity remained.Conclusion：Because the recombinant lipase was good thermostability and methanol endurance,it was very useful in application as biocatalyst for biodiesel production.There will be the foundation of production for biodiesel by enzymatic methods of genetic engineering.
出处
《生物技术》
CAS
CSCD
北大核心
2010年第5期12-16,共5页
Biotechnology
基金
广西科技攻关项目("生产生物柴油共性键技术的研发-生物柴油生产用共性脂肪酶的技术和工艺研究"
桂科攻08950034-1)
广西科学院创新基金("基于广西优势资源甘蔗及木质纤维
木薯和油料作物等的生物质能源关键共性技术研究"
桂科院研0701)
广西科学院基金项目("粘质沙雷氏菌脂肪酶基因在大肠杆菌中的高效表达及分子改性"
09YJ17SW04)资助
