Direct amperometric determination of lipase activity

Abstract

Lipases, triacylglycerol hydrolases (EC 3.1.1.3) that cleave triacylglycerols at the oil/water interface, have extensive applications in the food, paper, pharmaceutical, cosmetic, detergent, leather, and textile industries [1–5]. Widespread practical use of these enzymes requires fast and reliable analytical routines to assess their activity. Typical protocols for lipase assay include use of a pH-stat and back titrations, application of a Langmuir–Blodget film balance, spectrophotometric determination of para-nitrophenyl palmitate (pNPP) hydrolysis, etc. (for critical review, see [4]). Though commonly used, the above-mentioned techniques have several drawbacks: the methods of pH-stat and monolayer film balance are relatively cumbersome, expensive, and difficult to adapt for portable hand-held devices, while the relatively simple method of back titration does not provide continuous kinetic data. At the same time, the continuous spectrophotometric pNPP assay is limited to the transparent solutions, and it and the pH-stat titration are unusable under acidic pH conditions. The turbidity problem can be resolved by chemical or physical pretreatment of the solution. For example, with regard to the pNPP-based procedure, Gupta et al. [6] proposed to overcome this problem by using the nonionic surfactant Triton X-100. The electrochemical analysis techniques are practically insensitive to the presence of solid or/and liquid lightscattering microparticles in the solutions and could be