PURIFICATION AND CHARACTERIZATION OF A RECOMBINANT LACCASE FROM Kluyveromyces lactis GG799

MOLINA A; SGROPPO S; MILDE LB; ZAPATA,P.D.; FONSECA, MI.

Congreso; Red Argentina de Tecnologia Enzimática; 2021

Laccases (EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of many aromatic substrates. Fungal laccases have many advantages, such as directly oxidizing many phenolic compounds, using molecular oxygen as the final electron acceptor and showing a considerable level of stability. The properties of laccases allow their application in food, delignification, degradation of recalcitrant compounds, bioremediation, dye discoloration, biosensors and other. These enzymes have been found in fungi isolated in the province of Misiones, one of them is Phlebia brevispora BAFC633. Our group has successfully expressed the laccase enzyme from the white rot fungus Phlebia brevispora BAFC633 in the yeast Kluyveromyces lactis. The aim of this work was to purify and characterize a novel laccase enzyme expressed in K. lactis. For this, the previously standardized culture conditions were used (YPGal: 20% yeast extract, 10% peptone, 20% galactose in a final volume of 20 ml) incubated for four days at 28ºC at 100 rpm supplemented with 0.5 mmol l-1 of iron sulfate. For purification, culture broth was centrifuged at 6000 g for 10 min. The supernatant was filtered by 0.22 µm polyvinylidene fluoride membrane, obtaining a yeast-free supernatant. Then, an ultrafiltration with 100 kDa membranes was performed using centrifuge devices. Laccase activity was measured at 30°C using 5 mmol l-1 of 2,6-dimethoxyphenol (DMP) as substrate in 0,1 mol l-1 of sodium acetate buffer (pH 3,6). The absorbance increase in the reaction mixture was monitored at 469 nm. Enzyme activity was measured in U ml-1 which is defined as the amount of enzyme catalyzing the production of one micromole of colored product per min per ml.The purity of the enzyme was analyzed by silver stained SDS-PAGE. The apparent molecular mass of the purified laccase was determined by molecular marker comparison. Laccase activity was confirmed by zymogram analysis on native PAGE. Staining was carried out after native PAGE by incubating the gel for 5 min in 0.1 mol l-1 of sodium acetate buffer containing 5 mmol l-1 of DMP. To determine optimum pH the purified laccase extract was assayed at 30°C in different pH buffers in a range from 3.6 to 6. For pH stability, the extract was incubated in various pH buffers from 3.6 to 6 for 6 h at 30°C, and the residual enzyme activity was then analyzed. To determine optimal temperature, the enzyme activity was measured at different temperatures in a range from 30 to 60°C. Thermal stability was investigated by measuring the residual activity after incubating the purified enzyme at different temperatures in a range from 20 to 60°C for 6 h. Km and Vmax values of purified laccase were determined by measuring enzyme activity at various concentrations (0.01 to 5 mmol l-1) of 2,6-DMP as substrate, at optimal pH. Kinetic constants were calculated by Michaelis?Menten method using nonlinear regression.SDS-PAGE analysis silver staining showed an single band at 180 kDa. The zymogram revealed laccase with DMP. The optimum pH for this purified laccase was 4. Laccase enzyme showed high pH stability, maintaining a constant activity after 30 min of incubation at pH 3,6 maintaining 90% of activity after an hour of incubation and 85% of activity after 2 h of incubation. The optimum temperature was 40ºC. As to the thermal stability, the enzyme had maximum activity at pH 4 at 40ºC maintaining 100% of activity after half an hour of incubation. At pH 3.6 the laccase kept 89% of activity after an hour of incubation and 75% after 2 h of incubation. The kinetic parameters Km and Vmax of purified laccase were found to be 435 µmol l-1 and 39.46 µmol l-1 min-1 respectively.The analysis of the biochemical characteristics of the laccase enzyme will allow a biotechnological application and future investigations of the laccase enzyme for its application in different processes.