Feller G, Thiry M, Arpigny J L, Gerday C
University of Liege, Institute of Chemistry, Belgium.
Gene. 1991 Jun 15;102(1):111-5. doi: 10.1016/0378-1119(91)90548-p.
The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.
描述了嗜冷细菌基因在嗜温宿主中的克隆与表达。通过将Sau3AI酶切产生的DNA片段插入pSP73质粒载体的BamHI位点，克隆了来自南极嗜冷菌莫拉克斯氏菌TA144的三个脂肪酶（Lip）编码基因（lip）。为防止基因产物热变性，在含有乳化脂质的琼脂平板上进行筛选时，大肠杆菌重组菌落先在25℃培养，然后在0℃孵育。这三种重组脂肪酶（reLip）与细胞相关，对不同脂肪链长度的对硝基苯酯具有各自不同的特异性。这些克隆的reLip保留了野生型酶的主要特性，即活性最佳温度向低温显著偏移以及明显的热不稳定性。
