Methods for inhibition of residual lipase activity in colorimetric assay: a comparative study.
A comparative study of various treatments for inhibition of the residual activity of a lipase (obtained from Bacillus coagulans MTCC-6375) in a colorimetric assay using p-nitrophenyl palmitate (pNPP) was made. Direct chilling of contents of reaction mixture or addition of chilled mixture of ethanol : acetone (1:1) decreased the residual lipase activity by 94.0 and 95.0% respectively, as compared to lipase incubated at 45 degrees C for 20 min (control). Amongst various ionic and non-ionic detergents, Triton X-100 (0.07%, v/v) and sodium lauryl sarcosine or SLS (0.25%, w/v) partially, and SDS (0.05%, w/v) completely blocked the residual lipase activity of B. coagulans lipase in colorimetric assay. Addition of a serine protease inhibitor, PMSF (15 mM) or EDTA (200 mM) inhibited residual lipase activity by 99.5 and 100%, respectively. However, addition of reducing agents viz., 2-mercaptoethanol and dithiothreitol caused decomposition of chromogenic substrate (pNPP) thus rendering the colorimetric method unfit for lipase assay. EDTA (200 mM) and SDS (0.05%, w/v) were also highly effective in inhibiting the residual activities of lipases of Pseudomonas aeruginosa MTCC-4713, P. cepacia and commercial grade lipolytic preparations such as lipozyme, lipolase and porcine pancreatic lipase. However, PMSF (15 mM) completely inhibited the residual activity of lipase of P. aeruginosa.
Department of Biotechnology, Himachal Pradesh University, Summer-Hill, Shimla 171 005, India. kanwarss2000@yahoo.com
Shamsher Singh
SS
Kanwar
Rajeev Kumar
RK
Kaushal
Arshad
A
Jawed
Reena
R
Gupta
Swapandeep Singh
SS
Chimni
D001407:Bacillus / Q000187:drug effects / Q000201:enzymology*; D001681:Biological Assay; D002614:Chelating Agents / Q000494:pharmacology; D003124:Colorimetry; D003902:Detergents / Q000494:pharmacology*; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology*; D019163:Reducing Agents / Q000494:pharmacology
D002614:Chelating Agents; D003902:Detergents; D019163:Reducing Agents; D008049:Lipase