Expression of thermophilic two-domain laccase from Catenuloplanes japonicus in Escherichia coli and its activity against triarylmethane and azo dyes

Background Two-domain laccases are copper-containing oxidases found in bacteria in the beginning of 2000ths. Two-domain laccases are known for their thermal stability, wide substrate specificity and, the most important of all, their resistance to so-called «strong inhibitors» of classical fungal laccases (azides, fluorides). Low redox potential was found to be specific for all the two-domain laccases, due to which these enzymes lost the researchers’ interest as potentially applicable for various biotechnological purposes, such as bioremediation. Searching, obtaining and studying the properties of novel two-domain laccases will help to obtain an enzyme with high redox-potential allowing its practical application. Methods A gene encoding two-domain laccase was identified in Catenuloplanes japonicus genome, cloned and expressed in an Echerichia coli strain. The protein was purified to homogeneity by immobilized metal ion affinity chromatography. Its molecular properties were studied using electrophoresis in native and denaturing conditions. Physico-chemical properties, kinetic characteristics, substrate specificity and decolorization ability of laccase towards triphenylmethane dyes were measured spectrophotometrically. Results A novel two-domain recombinant laccase CjSL appeared to be a multimer with a subunit molecular mass of 37 kDa. It oxidized a wide range of phenolic substrates (ferulic acid, caffeic acid, hydroquinone, catechol, etc.) at alkaline pH, while oxidizing of non phenolic substrates (K4[Fe(CN)6], ABTS) was optimal at acidic pH. The UV-visible absorption spectrum of the purified enzyme was specific for all two-domain laccases with peak of absorption at 600 nm and shoulder at 340 nm. The pH optima of CjSL for oxidation of ABTS and 2, 6-DMP substrates were 3.6 and 9.2 respectively. The temperature optimum was 70 °C. The enzyme was most stable in neutral-alkaline conditions. CjSL retained 53% activity after pre-incubation at 90 °C for 60 min. The enzyme retained 26% activity even after 60 min of boiling. The effects of NaF, NaN3, NaCl, EDTA and 1,10-phenanthroline on enzymatic activity were investigated. Only 1,10-phenanthroline reduced laccase activity under both acidic and alkaline conditions. Laccase was able to decolorize triphenylmethane dyes and azo-dyes. ABTS and syringaldehyde were effective mediators for decolorization. The efficacy of dye decolorization depended on pH of the reaction medium.

Preparation of Menthol-Based Hydrophobic Deep Eutectic Solvents for Extraction of Triphenylmethane Dyes: Quantitative Properties and Extraction Mechanism

A series of natural, environmentally friendly and low-cost menthol-based hydrophobic deep eutectic solvents (DES) were synthesized to extract and concentrate solutes from dilute aqueous solution, especially triphenylmethane (TPM) dye micropollutants....

Validation of an UHPLC-MS/MS Method for the Determination of Malachite Green, Leucomalachite Green, Crystal Violet, and Leucocrystal Violet in Shrimp, Fish, and Salmon Muscle Using a Modified QuEChERS Approach

A quantitative and confirmatory method for detecting the presence of triphenylmethane dyes in shrimp muscle using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction approach was validated. The method exhibited linearity and selectivity and the coefficient of determination (R2) was higher than 0.95 for all studied analytes. Limits of detection (LODs) varied from 0.32 to 0.44 µg kg-1 and the limit of quantification (LOQ) was determined to be 0.5 µg kg-1 for all studied analytes. The trueness, precision, decision limits (CCα), detection capability (CCβ) and uncertainty presented adequate performance. In addition to the validation in shrimp muscle, fish and salmon muscle were also satisfactory validated as an extension of scope. The suitability of the proposed method was also evaluated through an interlaboratory proficiency test, in which satisfactory results were obtained. The fully validated method is thus suitable for the analysis of triphenylmethane dyes in shrimp, fish, and salmon muscle.