Chuang Yin Ching, Chiou Shu Fen, Su Jer Horng, Wu Mei Li, Chang Ming Chung
Division of Infectious Diseases, Department of Internal Medicine, National Cheng Kung University Medical College and University Hospital, Tainan, Taiwan, Republic of China.
Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Microbiology (Reading). 1997 Mar;143 ( Pt 3):803-812. doi: 10.1099/00221287-143-3-803.
The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79-9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites: one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli. The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C10 to C12 for ester hydrolysis and C8 to C10 for triacylglycerol hydrolysis.
编码嗜水气单胞菌MCC-2胞外脂肪酶的结构基因被克隆，并发现其利用自身启动子在大肠杆菌中表达。当克隆基因（lip）在大肠杆菌微小细胞中表达时，鉴定出一种80 kDa的蛋白质。对携带lip基因的大肠杆菌进行亚细胞分级分离表明，Lip蛋白主要与膜部分相关。核苷酸序列分析显示该基因长2253 bp，编码一个79-9 kDa的蛋白质，估计等电点为10.36。推导的蛋白质含有两个假定的信号肽切割位点：一个是典型的信号肽酶切割位点，另一个与已知的脂蛋白前导序列非常相似。当在大肠杆菌中表达时，[3H]棕榈酸的放射性被掺入Lip蛋白中。推导的蛋白质含有一段VHFLGHSLGA序列，在脂肪酶中非常保守。它与嗜水气单胞菌菌株H3和JMP636的脂肪酶氨基酸序列的总体同一性分别为67%和65%，但与其他脂肪酶的同源性很低。Lip蛋白从嗜水气单胞菌和重组大肠杆菌中均被纯化至同质。在使用纯化酶水解对硝基苯酯和三酰甘油时，底物上酰基部分的最佳链长对于酯水解为C10至C12，对于三酰甘油水解为C8至C10。
