Methods for inhibition of residual lipase activity in colorimetric assay: A comparative study

Abstract

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 45oC 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.