Automated colorimetric determination of recombinant fungal laccase activity in fermentation samples using syringaldazine as chromogenic substrate

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 stabilise rMtL, PEG 6000 was added to the enzyme dilution medium. Furthermore, Triton X-100 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 Michealis-Menten constant Km, was determined to be 22 Um, 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 reproducability 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-100, molarity and pH of the incubation buffer, and finally the concentration of syringaldazine.