Automated colorimetric determination of recombinant fungal laccase activity in fermentation sarples 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 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.

Purification and Partial Characterization of a Thermostable Laccase from Pycnoporus sanguineus CS-2 with Ability to Oxidize High Redox Potential Substrates and Recalcitrant Dyes

“…This parameter has been relatively little studied in thermotolerant laccases. The calculated E a value (16.2 kJ/mol) for P. sanguineus CS 2 laccase is similar to the values reported for other thermotolerant laccases, as that for Myceliophora thermophila (19 kJ/mol) [36] and for the recombinant laccase from Coprinus cinereus (14 kJ/mol) [37], but smaller than those calculated in this report for other laccases, which apparently did not show a direct relationship between thermotolerance and the magnitude of E a . On the other hand, the function showed a change in slope in the high temperature zone (50-70 °C) before the enzyme denaturing breaking zone.…”

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

“…These experiments indicate that free copper ions are a factor in the reconstitution process. The observation that PEG6000 acts as a stabilizing agent for the laccase activity assay [31,46,47] was the motivation for testing this as an additive in the experiment. As seen when comparing solution B and E (Fig.…”

“…The elution was performed by a stepwise gradient of Buffer A to Buffer B (Buffer A without ammonium sulfate). Fractions of 10 mL were collected and evaluated for laccase activity using syringaldazine as reducing substrate [30,31]. Fractions showing laccase activity were concentrated by ultrafiltration using a 30 kDa cut-off filter on an Amicon 8400-system (both from Millipore Corporation).…”

“…The laccase activity was assayed using syringaldazine as reducing substrate according to Bauer and Ruper and to Holm et al [30,31] using ε 530 = 65 mM −1 cm −1 [30]. One laccase unit was defined as the amount of laccase catalyzing the turn-over of 1 μmol of syringaldazine per min.…”

“…One laccase unit was defined as the amount of laccase catalyzing the turn-over of 1 μmol of syringaldazine per min. Syringaldazine was dissolved in 96% ethanol to a concentration of 0.56 mM and was diluted 1:1 in dH 2 O prior to use [31]. The assay was composed of 100 μL diluted enzyme solution, 4000 μL assay buffer and 300 μL syringaldazine.…”