Electrochemical method for the detection of lipase activity.
A novel electrochemical technique for the general assay of lipase activity is described. The method utilizes a solid-supported lipase substrate, which is formed by dripping and drying a small amount of an ethanol solution of 9-(5'-ferrocenylpentanoyloxy)nonyl disulfide (FPONDS) onto gold modified by a hexanethiol self-assembled monolayer. The redox ferrocene group of FPONDS generates the electrochemical signal, the intensity of which is proportional to the number of FPONDS molecules at the interface. Electrochemical and surface-enhanced infrared absorption spectroscopic data, as well as control experiments with an engineered, deactivated mutant enzyme, demonstrate that the wild-type lipase from Thermomyces lanuginosus is capable of cleaving the ester bonds of FPONDS molecules via an enzymatic hydrolysis mechanism, which includes the adsorption of the lipase onto the substrate surface. The hydrolysis liberates the ferrocene groups from the interface triggering a decay of the electrochemical redox signal. The rate of the electrochemical signal decrease is proportional to the lipase activity/concentration. These data suggest a general method for the direct measure of enzymatic activity of lipases.
Valincius
Gintaras
G
Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania. ginataras@bchi.lt
Ignatjev
Ilja
I
Niaura
Gediminas
G
Kazemekaite
Maryte
M
Talaikyte
Zita
Z
Razumas
Valdemaras
V
Svendsen
Allan
A
D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000075163:Metallocenes; D003904:Mitosporic Fungi / Q000201:enzymology; D009154:Mutation; D010084:Oxidation-Reduction
C518059:9-(5'-ferrocenylpentanoyloxy)nonyl disulfide; D004220:Disulfides; D005296:Ferrous Compounds; D000075163:Metallocenes; D008049:Lipase; C004998:ferrocene