An active-site titration method for lipases.
A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial 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 some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.
Rotticci
D
D
Department of Chemistry, Organic Chemistry, Royal Institute of Technology, SE-100 44, Stockholm, Sweden.
Norin
T
T
Hult
K
K
Martinelle
M
M
D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry; D004800:Enzymes, Immobilized; D006536:Heptanes; D008049:Lipase / Q000032:analysis* / Q000037:antagonists & inhibitors; D012694:Serine / Q000737:chemistry; D012996:Solutions; D017186:Titrimetry / Q000379:methods*; D014867:Water
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D006536:Heptanes; D012996:Solutions; D014867:Water; D012694:Serine; D008049:Lipase