粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

被引:11
作者
朱绮霞 [1 ]
陈英 [2 ]
张搏 [1 ]
黄日波 [1 ,2 ]
机构
[1] 广西科学院国家非粮生物质能源工程技术研究中心
[2] 广西大学生命科学与技术学院
关键词
粘质沙雷氏菌; 脂肪酶; 克隆; 表达;
D O I
暂无
中图分类号
Q785 [转化及克隆];
学科分类号
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。
引用
收藏
页码:12 / 16
页数:5
相关论文
共 22 条
[1]  
Molecular cloning: a laboratory manual. Sambrook,J.,Fritsch,E. F.,Maniatis,T. Cold Spring Harbor Laboratory Press . 2001
[2]   Enhancing functional expression of heterologous lipase in the periplasm of Escherichia coli [J].
Xu, Yali ;
Yasin, Amrita ;
Wucherpfennig, Thomas ;
Chou, C. Perry .
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, 2008, 24 (12) :2827-2835
[3]  
Enzymatic conversion of waste cooking oils into alternative fuel—Biodiesel[J] . Guanyi Chen,Ming Ying,Weizhung Li. &nbspApplied Biochemistry and Biotechnology . 2006 (1)
[4]  
Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures[J] . Oriol Cos,David Resina,Pau Ferrer,José Luis Montesinos,Francisco Valero. &nbspBiochemical Engineering Journal . 2005 (2)
[5]  
Purification and partial characterization of a lipase from Bacillus coagulans ZJU318[J] . Tang Lianghua,Xia Liming. &nbspApplied Biochemistry and Biotechnology . 2005 (2)
[6]  
Cloning, characterization and expression of the extracellular lipase gene from Aureobasidium pullulans HN2-3 isolated from sea saltern[J] . Zhiqiang Liu,Xiaoyu Li,Zhenming Chi,Lin Wang,Jing Li,Xianghong Wang. &nbspAntonie van Leeuwenhoek . 2008 (2)
[7]   Biodiesel fuel production by transesterification of oils [J].
Fukuda, H ;
Kondo, A ;
Noda, H .
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2001, 92 (05) :405-416
[8]  
Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63[J] . Jiangke Yang,Daoyi Guo,Yunjun Yan. &nbspJournal of Molecular Catalysis. B, Enzymatic . 2007 (3)
[9]  
Enzymatic approach to biodiesel production. Akoh CC,Chang SW,Lee GC,Shaw JF. Journal of Agricultural and Food Chemistry . 2007
[10]   产脂肪酶粘质沙雷氏菌发酵产酶条件的优化 [J].
朱绮霞 ;
张搏 ;
陆雁 ;
王青艳 ;
黄日波 .
生物技术, 2009, 19 (05) :61-65