Partial purification and characterization of ligninolytic enzymes produced by Pleurotus ostreatus during solid state fermentation

Abstract

The production of ligninolytic enzymes including laccase, manganese peroxidase and lignin peroxidase from Pleurotus ostreatus was studied under different parameters using solid state fermentation. Maximum production of enzymes was observed after 7 days in solid state fermentation (SSF) medium containing 5 g wheat straw (66% w/w moisture) in a still culture SSF. Different parameters had a significant effect on enzyme production. Maximum laccase (455.11 U/mL), manganese peroxidase (210.77 U/mL) and lignin peroxidase (54.50 U/mL) were produced when wheat straw (5 g) at 66% moisture (w/w) was used with 4 mL inoculum at pH 4.5 and 30°C in the presence of 1% (v/v) glycerol as carbon source, 0.2% w/w urea as nitrogen source, 1% (w/v) 2, 2 azinobis 3-ethylbenzthiazoline 6 sulphonate (ABTS) as an inducer for laccase and 1% (w/v) MnSO4 for manganese peroxidase,1% (w/v) CuSO₄ as metal ion for laccase and Mn⁺ for manganese peroxidase. Both enzymes laccase and manganese peroxidase produced by Pleurotus ostreatus were partially purified by ammonium sulphate precipitation followed by gel filtration chromatography. Additionally, the protein content of the recovered supernatants was also noted. Purification results showed an increase in purity up to 3.37 and 3.07 fold for laccase and manganese peroxidase, respectively. The Michaelis constant, K_M was 62 and 33 μM for laccase and manganese peroxidase, respectively. Lignin peroxidase was not produced during solid state fermentation of ligniocellulosic material by P. ostreatus because this fungus is a laccase producer. The activators ABTS and MnSO₄ proved good for laccase and MnP production. This shows that SSF parameters had a significant influence on catalytic activity of ligninolytic enzymes produced by P. ostreatus under still conditions.

Key words: Ligninolytic enzymes, Pleurotus ostreatus, 2,2 azinobis 3-ethylbenzthiazoline 6 sulphonate (ABTS), solid state fermentation.