文章基本信息

标题：A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin
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作者：María de los Angeles Camacho-Ruiz ; Juan Carlos Mateos-Díaz ; Frédéric Carrière 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2015
卷号：56
期号：5
页码：1057-1067
DOI：10.1194/jlr.D052837
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.
关键词：bile acids and salts ; fatty acid ; lipids ; triglycerides ; high-throughput screening ; pH-dependent activity ; triacylglycerol emulsion ; microtiter plate assay ; lipase inhibitors