Stuer W, Jaeger K E, Winkler U K
J Bacteriol. 1986 Dec;168(3):1070-4. doi: 10.1128/jb.168.3.1070-1074.1986.
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.
脂肪酶（三酰基甘油酰基水解酶，EC 3.1.1.3）由铜绿假单胞菌PAC1R在对数生长后期分泌。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对无细胞培养上清液进行表征，结果显示存在大量脂多糖，其中一部分似乎与脂肪酶紧密结合。通过超滤浓缩培养上清液后，用EDTA- Tris缓冲液处理并随后在两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐存在下进行超声处理，使脂肪酶-脂多糖复合物解离。通过在含有相同去污剂的琼脂糖凝胶中进行等电聚焦来纯化溶解的脂肪酶；脂肪酶活性出现在一个单一峰中对应于银染凝胶中的一条明显条带。等电点为5.8。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和扫描对纯化的脂肪酶进行分析，结果显示其表观分子量为29,000，比活性为760微卡特/毫克蛋白质。基于这些数据的估计表明，单个铜绿假单胞菌细胞分泌约200个脂肪酶分子，每个分子的分子活性为每秒2.2×10⁴。
