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Fourier‐transform infrared assay of bile salt‐stimulated lipase activity in reversed micelles
Author(s) -
O'Connor Charmian J.,
Cleverly Douglas R.
Publication year - 1994
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.280610305
Subject(s) - lipase , salt (chemistry) , fourier transform infrared spectroscopy , micelle , chemistry , infrared , fourier transform , chromatography , biochemistry , enzyme , organic chemistry , physics , aqueous solution , optics , quantum mechanics
Abstract A Fourier‐transform infrared (FT‐IR) spectroscopic method has been developed for assaying the bile salt‐stimulated human milk lipase (BSSL, EC3.1) catalyzed hydrolysis of triolein in AOT reversed micelles in iso‐octane. At 37°C in 50 mmol dm −3 AOT the molar absorbtivities for the carbonyl stretching frequencies for triolein (at 1751 cm −1 ) and oleic acid (at 1714 cm −1 ) were 1646 dm 3 mol −1 cm −1 and 743 dm −3 mol −1 cm −1 , respectively. The rate was linearly dependent upon the concentration of enzyme in the water pool up to 10 mg cm −3 and maximum activity was observed at a ratio ( w 0 ) of [H 2 O]:[AOT] = 16·7. Using these conditions, and in the presence of 10 mmol dm −3 sodium taurocholate (TC), the derived Michaelis–Menten parameters V max and K m were 57·5 μmol min −1 mg −1 and 5·53 mmol dm −3 , respectively. These results are compared with those obtained in an oil‐in‐water microemulsion system and are discussed in terms of the relative partitioning of the enzyme and the substrate in the aqueous and oil phases and the interfacial concentration of the substrate in the two systems.