2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and 2-4-6-trichlorophenol

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V (max) values are 243.9 and 10.55 Um min(-1) mg(-1), respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.

Topics: Ascomycota; Benzothiazoles; Biodegradation, Environmental; Chlorophenols; Chromatography, High Pressure Liquid; Copper; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Genetic Vectors; Guaiacol; Indicators and Reagents; Ions; Isoelectric Point; Kinetics; Laccase; Molecular Weight; Recombinant Proteins; Substrate Specificity; Sulfonic Acids