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Title: Methods for inhibition of residual lipase activity in colorimetric assay: A comparative study
Authors: Kanwar, Shamsher Singh
Kaushal, Rajeev Kumar
Jawed, Arshad
Gupta, Reena
Chimni, Swapandeep Singh
Keywords: Bacillus coagulans MTCC-6375
Microbial and commercial lipases
Chilling
Ethanol:acetone mixture
Denaturing/reducing/chelating agents
PMSF
Issue Date: Aug-2005
Publisher: CSIR
IPC CodeC12N1/00; C12N9/00; G01N33/52
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.
Page(s): 233-237
ISSN: 0301-1208
Source:IJBB Vol.42(4) [August 2005]

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