Abstract
An automated Cobas Fara method was developed determining the activity of recombinant M. thermophila laccase (rMtL). The chromogenic substrate used was syringaldazine. Under aerobic conditions, rMtL catalyses the oxidation of syringaldazine forming tetrametoxy-azo bis methylene quinone. The developed violet colour was measured kinetically at 530 nm as an expression of the enzyme activity, rMtL is a very sensitive oxidoreductase, therefore many factors had to be carefully controlled in order to get a robust analytical assay. In order to stabilize rMtL, PEG 6000 was added to the enzyme dilution medium. Furthermore, Triton X-I00 was included in the enzyme incubation solution. The analytical as well as technical conditions have been optimized, resulting in a method with good precision, sensitivity and speed of analysis. The Michaelis-Menten constant, Km, was determined to be 22μM syringaldazine. LOQ was determined to be 0.010 Uml-1, LOD to be 0.0002 Uml-1 The analytical range of the enzyme dilution curve was from 0.01 to 0.044 Uml-1 The repeatability was 1.9%, the reproducibility 3.1%. Testing the robustness of the method showed that the most sensible factors in the rMtL analysis in decreasing range were: incubation temperature, concentration of Triton X-I00, molarity and pH of the incubation buffer, and finally the concentration of syringaldazine.
Highlights
One LAMU unit is the amount of enzyme capable of converting lamol of syringaldazine per min under the given reaction conditions
A stock solution was composed of syringaldazine (0.56 mM dissolved in 96% ethanol)
As the sensitivity of the syringaldazine reaction was at a satisfying level, 30-C was preferred as the routine temperature during the reaction
Summary
Molecular oxygen is chemically inert, and a catalyst, e.g. rMtL, is needed in order to reduce dioxygen. Many compounds have been used as substrates in the activity determination of laccases, e.g. guajacol [13], 2,4 dimetoxyphenol [14], 2,2-azino-bis(3ethyl-benzothiazoline-6-sulphonate) (ABTS), ABTS combined with para-hydroxybenzoic acid/NaN3 [15, 16] and syringaldazine [17,18,19,20]. Most of these substrates are insoluble in water.
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