Yellow laccase produced by Trametes versicolor K1 on tomato waste: A comparative study with the blue one produced on semi-synthetic medium.

Laccase production by fungal growth on agrifood waste is still poorly studied. Trametes versicolor K1 isolated from palm bark produced a yellow non glycosylated laccase from tomato waste based medium (TMT) and a blue glycosylated laccase on glucose medium (GLU). Lignocellulosic biomass, such as pinecones (PIN), palm leaves (PLM), olive pomace (OLV), and alfa stems (ALF) have also been used as growth medium for T. versicolor K1. In these conditions, very low or no laccase production was observed. When peptone was supplied in TMT medium, the laccase activity increased from 4170 U/L to 8618 U/L. By increasing the culture volume up to 1 L, laccase production on TMT was 9929 U/L. The yellow laccase (TmtLac) was purified from the supernatant TMT medium and has shown similar characteristics with the blue laccase (GluLac) purified from the GLU medium. Their apparent protein size was 63 kDa. Catalytic activities of the yellow form were not very different from those of the blue form, but specific activity of the purified yellow laccase produced on tomato waste was much higher. The Km and Vm values for four substrates, ABTS, DMP, guaiacol, and pyrogallol were almost similar for both isoenzymes. The optimum pH and temperature were respectively 4.0 and 50 °C. Although the level of glycosylation is clearly different, the thermostability of TmtLac and GluLac are quite similar. TmtLac is even slightly more tolerant at 60 °C for 24 h than GluLac. Moreover TmtLac showed greater stability at alkaline pH after 24 h compared to that of GluLac.We demonstrate that activity of the yellow TmtLac is not significantly affected compared to the blue laccase and that tomato waste is a simple and interesting lignocellulosic substrate to the laccase producer Trametes sp.

High yield production in seven days of Coriolopsis gallica 1184 laccase at 50 L scale; enzyme purification and molecular characterization.

The optimization of culture conditions for high yield laccase production by white rot fungi has been extensively studied. However, to achieve short time laccase production remains a major challenge in several cases. The present study investigated an optimal process for production of Coriolopsis gallica 1184 laccase in a high yield of 200 900 Ul(-1) in 7 d by 50 L scale submerged fermentation. Coriolopsis gallica 1184 laccase appeared as a robust enzyme against downstream process; only 13.5 % of laccase activity was lost at the end of downstream procedure. The pure enzyme appeared as a one-species laccase, with a molecular mass of 66 kDa as determined by SDS-PAGE. The pH optimum for 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] oxidation ranged between 2.5 and 3.0 in 100 mM tartrate buffer. Optimum temperature for laccase activity was determined to be around 70 °C. The kinetic of laccase was investigated with four phenolic substrates. The lowest Km values (17 and 20 µM) were found for ABTS and guaiacol, respectively. Coriolopsis gallica 1184 laccase was characterized by mass spectrometry and shows that C. gallica 1184_LacI is very likely a new member of the AA1_1 subfamily. Our results clearly show high competitive potential of the robust extracellular C. gallica 1184 laccase to use it in different industrial processes.

Production of a high level of laccase by submerged fermentation at 120-L scale of Cerrena unicolor C-139 grown on wheat bran.

Submerged fermentation in a stirred bioreactor of the white rot fungus Cerrena unicolor C-139 was done at a 120-L scale in the presence of wheat bran as a cheap lignocellulosic substrate for fungus growth and laccase production. Enzyme monitoring showed that laccase production started after 2 days of cultivation, attaining a maximum activity of 416.4 U·mL(-1) at day 12 of fermentation. After treatment of culture liquid by successive micro- and ultrafiltration (5kDa), a liquid concentrate containing 22203176 units of laccase was obtained. Obtaining large amount of laccase is essential for various industrial applications, including detoxification of industrial effluents, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated area, stabilization of beverages, production of cosmetics, manufacture of anti-cancer drugs, and nanobiotechnology. The cultivation method and the fungal strain used here provided a substantial amount of enzyme produced at a price lower than 0.01 € cent/unit enzyme.

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants.

The white-rot fungus Cerrena unicolor C-139 produced 450 000 U l(-1) of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K(m) value (100 µmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 µmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 µmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.