An active-site titration method for lipases

A method for active-site titration of lipases has been developed based on i rreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This met hod was applied to five lipases displaying from minor to pronounced interfa cial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of s ome of the immobilized preparations adsorbed part of the produced p-nitroph enolate, This problem could be solved by extracting the p-nitrophenolate af ter inhibition. The method was extended to apolar organic solvents in the c ase of immobilized lipase preparations. (C) 2000 Elsevier Science B.V. All rights reserved.