PURIFICATION, GENE CLONING, AMINO-ACID-SEQUENCE ANALYSIS, AND EXPRESSION OF AN EXTRACELLULAR LIPASE FROM AN AEROMONAS-HYDROPHILA HUMAN ISOLATE
PURIFICATION, GENE CLONING, AMINO-ACID-SEQUENCE ANALYSIS, AND EXPRESSION OF AN EXTRACELLULAR LIPASE FROM AN AEROMONAS-HYDROPHILA HUMAN ISOLATE
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DOI：
10.1128/aem.59.8.2411-2417.1993
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发表时间：
1993-08-01
影响因子：
4.4
通讯作者：
NAHARRO, G
中科院分区：
文献类型：
--
作者：
ANGUITA, J;APARICIO, LBR;NAHARRO, G
利用pBR 322作为载体，在大肠杆菌中克隆了编码从女性住院患者分离的嗜水气单胞菌H3中的胞外脂肪酶（EC3.1.1.3）的结构基因。从A. Escherichia培养上清和E.在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，含有初始克隆（质粒pLA 2）中的lip决定簇的大肠杆菌显示M（r）为67，000，这与Sephacryl S-200色谱法测定的M（r）一致。关于底物特异性，对于酯水解，酰基部分的最佳链长为C6，对于三酰甘油水解，为C6和C-8。序列分析显示主要开放阅读框为2，052 bp，预测多肽的M（r）为71，804。发现该多肽含有在脂肪酶中高度保守的氨基酸序列（V-H-F-L-G-H-S-L-G-A）。
A structural gene which codes for an extracellular lipase (EC 3.1.1.3) in Aeromonas hydrophila H3, which was isolated from a female hospitalized patient, was cloned in Escherichia coli by using pBR322 as a vector. Lipase purified from both A. hydrophila culture supernatant and the periplasmic fluids of E. coli containing the lip determinant in the original clone (plasmid pLA2) showed an M(r) of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the M(r) determined by Sephacryl S-200 chromatography. Regarding substrate specificity, the optimum chain lengths for the acyl moiety were C6 for ester hydrolysis and C6 and C-8 for triacylglycerol hydrolysis. Sequence analysis showed a major open reading frame of 2,052 bp, which predicts a polypeptide with an M(r) of 71,804. The polypeptide was found to contain an amino acid sequence (V-H-F-L-G-H-S-L-G-A) which is highly preserved among lipases.