Prim N, Bofill C, Pastor F I J, Diaz P
Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain.
Biochimie. 2006 Jul;88(7):859-67. doi: 10.1016/j.biochi.2006.02.011. Epub 2006 Mar 29.
Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.
菌株假单胞菌属CR-611，先前从添加三丁酸甘油酯的平板上的亚热带森林土壤中分离得到，其表型和生理特性与荧光假单胞菌所描述的一致。然而，经过16S rDNA比较后，未发现与该物种完全匹配的结果。对该菌株的酶谱分析揭示了一个复杂的脂肪分解系统，显示存在至少两种对MUF-丁酸有活性的酶。荧光假单胞菌脂肪酶/酯酶编码序列的比对使得能够设计出针对VI型脂肪酶的特异性引物，并分离和克隆了由此产生的基因estA6。获得的重组克隆在脂肪酸衍生物底物上显示出高活性，表明该菌株的一种脂肪分解酶已被克隆。然后对该酶（命名为EstA6）进行纯化和表征，结果表明在高温和中性pH条件下，它对短链长度底物具有最大活性。EstA6与其他VI型酯酶的氨基酸序列比对使得能够鉴定出VI型脂肪酶中一个高度保守的β-/γ-原核生物簇，EstA6属于该簇。
