A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin.
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.
Biotecnología Industrial, CIATEJ A.C., 44270 Guadalajara, Jalisco, Mexico.
María de Los Angeles
Mde L
Camacho-Ruiz
Biotecnología Industrial, CIATEJ A.C., 44270 Guadalajara, Jalisco, Mexico.
Juan Carlos
JC
Mateos-Díaz
CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 13402 Marseille Cedex 20, France.
Frédéric
F
Carrière
Biotecnología Industrial, CIATEJ A.C., 44270 Guadalajara, Jalisco, Mexico.
Jorge A
JA
Rodriguez
D002210:Caprylates / Q000737:chemistry*; D004396:Coloring Agents / Q000737:chemistry; D057075:Enzyme Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D014280:Triglycerides / Q000737:chemistry*
D002210:Caprylates; D004396:Coloring Agents; D014280:Triglycerides; C003637:tricaprylin; D008049:Lipase; C518354:PNLIP protein, human; C005830:tributyrin
bile acids and salts; fatty acid; high-throughput screening; lipase inhibitors; lipids; microtiter plate assay; pH-dependent activity; triacylglycerol emulsion; triglycerides