Quantitative spectrophotometric assay for staphylococcal lipase.
We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.
Smeltzer
M S
MS
Department of Pathology and Microbiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506.
Hart
M E
ME
Iandolo
J J
JJ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008066:Lipolysis; D011549:Pseudomonas / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014280:Triglycerides
D001426:Bacterial Proteins; D003470:Culture Media; D014280:Triglycerides; D008049:Lipase; C005830:tributyrin