Lonon M K, Woods D E, Straus D C
Department of Microbiology, Texas Tech University Health Sciences Center, Lubbock 79430.
J Clin Microbiol. 1988 May;26(5):979-84. doi: 10.1128/jcm.26.5.979-984.1988.
Ten clinical isolates of Pseudomonas cepacia from the sputum of cystic fibrosis patients were examined for the ability to produce lipase. Lipase substrates used included egg yolk agar, four different polyoxyethylene sorbitans (Tweens), and p-nitrophenylphosphorylcholine, a chromogenic substrate used to assay for phospholipase C. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in either tryptose minimal medium or chemically defined medium. Lipase activity increased in the filtrates if the cultures were allowed to proceed into the stationary phase. None of the isolates produced phospholipase C. Lipase activity on Tween 20 ranged from 41.6 X 10(-3) to 640.0 X 10(-3) U/micrograms of protein. The activity was similar or slightly lower when Tween 40, 60, or 80 was used as the substrate. There was no correlation between lipase activity on Tween and that demonstrated on egg yolk agar. Lipase activity increased as pH increased from 7.0 to 9.0. Boiling for 5 min resulted in 66% loss of enzyme activity. The remaining activity continued to decrease with increasing boiling time. The enzyme was purified by gel filtration on Sephadex G-200, and the resultant preparation, when subjected to polyacrylamide gel electrophoresis, resulted in a single protein band (molecular weight, approximately 25,000) from which lipase activity could be eluted. The purified lipase was not cytotoxic to HeLa cells, nor was it toxic when injected intravenously into mice.
对从囊性纤维化患者痰液中分离出的10株洋葱伯克霍尔德菌临床菌株进行了脂肪酶产生能力检测。所用的脂肪酶底物包括蛋黄琼脂、四种不同的聚氧乙烯山梨醇酐（吐温）以及对硝基苯磷酰胆碱（一种用于检测磷脂酶C的显色底物）。在胰蛋白胨基本培养基或化学限定培养基中生长至指数期的菌株滤液中检测到了脂肪酶活性。如果培养物进入稳定期，滤液中的脂肪酶活性会增加。所有分离株均未产生磷脂酶C。在吐温20上的脂肪酶活性范围为41.6×10⁻³至640.0×10⁻³U/μg蛋白质。以吐温40、60或80为底物时，活性相似或略低。在吐温上的脂肪酶活性与在蛋黄琼脂上所显示的活性之间没有相关性。随着pH从7.0增加到9.0，脂肪酶活性增加。煮沸5分钟导致酶活性丧失66%。随着煮沸时间的增加，剩余活性继续下降。通过在Sephadex G - 200上进行凝胶过滤对该酶进行了纯化，所得制剂在进行聚丙烯酰胺凝胶电泳时，产生了一条单一的蛋白带（分子量约为25,000），可从中洗脱脂肪酶活性。纯化的脂肪酶对HeLa细胞无细胞毒性，静脉注射到小鼠体内时也无毒。
