Studies on production, purification and partial characteristics of the extracellular laccase from Armilliria mellea / 生物工程学报

ABSTRACT

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).