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类产碱假单胞菌耐热碱性脂肪酶基因的克隆
引用本文:翁丽星,胡志浩,邓子新,李阜棣,施巧琴,吴松刚.类产碱假单胞菌耐热碱性脂肪酶基因的克隆[J].微生物学报,1997,37(6):434-437.
作者姓名:翁丽星  胡志浩  邓子新  李阜棣  施巧琴  吴松刚
作者单位:华中农业大学生命科技学院;福建师范大学生物工程学院;福建师范大学生物工程学院 武汉 430070 福建师范大学生物工程学院 福州
基金项目:国家自然科学基金委员会杰出青年科学基金,国家教委跨世圮人才基金,福建师大资助课题
摘    要:将类产碱假单胞菌(Pseudomonas pseudoalcaligene)总DNA经Sau3AI部分酶解后的35~50kbDNA片段与经BamHI线性化及CIAP处理过的粘粒pIJ285连接,以大肠杆菌LE392为受体,构建类产碱假单胞菌的基因文库。通过三丁酸甘油酯平板和橄榄油平板法检测克隆子,获得一株具有耐热碱性脂肪酶活性的菌株LE392(pHZ1401)。随后将pHZ1401上的外源DNA片段进行亚克隆,从而获得了具有脂肪酶活性的菌株HB101(pHZ1402)和HB101(pHZ1403),它们分别携带有2.9kb和3.0kb的外源片段。两外源片段约有2kb的重叠区。HB101(pHZ1403)所分泌的脂肪酶活性比HB101(pHZ1402)高4倍,是出发菌的5倍。

关 键 词:类产碱假单胞菌    耐热碱性脂肪酶    基因克隆

CLONING AND EXPRESSION IN ESCHERICH1A COLI OF AN ALKALINE AND THERMOSTABLE EXOLIPASE FROM PSEVDOMONAS PSEUDOALCALIGENES
Weng Lixing Hu Zhihao Deng Zixin Shi Qiaoqin Wu Songgang Li Fudi.CLONING AND EXPRESSION IN ESCHERICH1A COLI OF AN ALKALINE AND THERMOSTABLE EXOLIPASE FROM PSEVDOMONAS PSEUDOALCALIGENES[J].Acta Microbiologica Sinica,1997,37(6):434-437.
Authors:Weng Lixing Hu Zhihao Deng Zixin Shi Qiaoqin Wu Songgang Li Fudi
Institution:Huazhong Agriculture University, Wuhan 430070.
Abstract:A gene coding for an alkaline and thermostable exolipase of Pseudomonas pseudoalcaligenes was cloned into Escherichia coli LE392 by inserting Sau3 AI-generated DNA fragments into the BamHI site of pIJ285. Four colonies with esterase and lipase activities on the tributyrin agar plate were isolated by screening the constructed pseudomonas pseudoalcaligenes genomic library. Only one out of the four positive colonies showed lipase activity on the agar plate contatining olive oil and Rhodamine-B. Subclones of the 45 kb insert carrying lipase gene was obtained in E. coli HB101 using pUC118 as a vector, two of which (HB101 (pHZ1402)and HB101 (pHZ1403)), retained lipase activity, but the level seems to be different. They carried 3.0 kb and 2.9 kb inserts with a 2 kb overlapping sequence. The lipase activity of HB101 (pHZ1403) is about 4 times higher than that of HB101 (pHZ1402),as 5 times as that of original strain P. pseudoalcaligenes.
Keywords:Pseudomonas pseudoalcaligenes  Alkaline thermostable exolipase  Cloning
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