土壤中脂肪酶产生菌的筛选、鉴定及脂肪酶基因的克隆  

The Screening,Identification and Lipase Gene Cloning of Soil Lipase Producing Strain

作　　者：玄国营[1] 陈芳[1] 李予霞[1] 高剑峰[1] 

机构地区：[1]石河子大学生命科学学院,石河子832003

出　　处：《生物技术通报》2009年第8期139-143,150,共6页Biotechnology Bulletin

基　　金：兵团博士资金项目

摘　　要：以橄榄油为惟一碳源进行富集培养、以罗丹明B为指示剂的平板进行初筛,摇瓶复筛得到产脂肪酶菌株;利用滴定法、平板扩散法以及转酯反应试验,最终筛选出具有较高转酯活性脂肪酶的菌株B2。通过对B2进行形态学观察,生理生化测定以及16S rDNA特征片段比较分析,初步确定B2为克雷伯氏菌属(Klebsiella sp.)。通过特定引物PCR扩增得到B2菌株两个脂肪酶基因K1和K2。利用丙酮法和(NH)2SO4沉降法得到B2菌株体内的胞内酶,以叔丁醇为溶剂,催化棉籽油与甲醇反应,经过薄层层析和高效液相检测产物为生物柴油。Using olive oil as sole carbon source in enrichment culture, and Rhodamine B as indicator to screening, one lipaseproducing strain B2 with higher lipase activity were screened. B2 strain was identified as Klebsiella sp. according to morphological ob- servation, determination of physiological and biochemical. The part 16S rDNA was amplified by PCR and sequenced. By designing specific primers, two lipase genes K1 and K2 in B2 strain was amplified by PCR and sequenced. The lipase can catalyze cotton oil and methanol into biodiesel, when tert-butyl alcohol as solvent and acetone and （NH）2SO4 sedimentation method involved in.

关 键 词：生物柴油 克雷伯氏菌 脂肪酶 脂肪酶基因 

分 类 号：S154.3[农业科学—土壤学]