Production of efficient lignin degrading enzymes by newly isolated Cerrena species

Abstract

White rot fungi have attracted global attention because of their potential application in biotechnology industry. Of all ligninolytic exoenzymes produced by white rot fungi, laccases are one of the most investigated enzymes related to a variety of green oxidation processes. The study of ligninolytic enzymes has been hampered by the difficulty in producing these enzymes in sufficient quantities due to a large number of factors contribute to the development of morphological forms that will influence the enzymes production optimization. In this study, the identification of white rot fungi producing ligninolytic enzymes from local soils, optimization study, purification and characterization work aiming to laccase were carried out. Eight isolates white rot fungi were identified as potentially useful over 119 isolates screened. Cerrena sp. WICC F39 was chosen based on the high laccase activity of enzyme production. Optimization was carried out using one-factor-at-time (OFAT) and statistical approach using response surface methodology (RSM). Laccase enzymes produced in selected unoptimized screened medium composed of (g L-1): mannitol, 10.0; ammonium tartrate, 2.0; potassium dihydrogen phosphate, KH2PO4, 0.8; dipotassium hydrogen phosphate, K2HPO4, 0.6; magnesium sulphate heptahydrate, MgSO4·7H2O, 0.5; copper sulphate pentahydrate, CuSO4·5H2O, 0.1 mM; Manganese sulphate pentahydrate, MnSO4·5H2O, 0.1 mM and yeast extract, 3.0 (pH 6.0), which produced highest laccase at 199.67 U ml-1. The medium optimized using OFAT composed of (g L-1): rice straw, 200.0; starch, 5.0; peptone, 1.5; ammonium tartrate, 2.0; potassium dihydrogen phosphate, KH2PO4, 0.8; K2HPO4, 0.6; MgSO4, 0.5; CuSO4 (1 mM); MnSO4 (1 mM) produced laccase of 552.31 U ml-1. Next, optimization using RSM contributed to maximum laccase production of 496.89 U ml-1 with optimum medium concentration (in g L-1): rice straw, 179.3; starch, 11.8; peptone meat, 3.5; ammonium tartrate, 0.1 and KH2PO4, 0.2. Further cultivation of Cerrena sp. WICC F39 was carried out using batch mode in a 5-L bioreactor. Using OFAT optimized medium, copper added resulted maximum laccase production 478 U ml-1 after 72 hours cultivation while non-copper added medium produced 189 U ml-1 after 48 hours cultivation. Laccase from Cerrena sp. WICC F39 was purified by anion-exchange chromatography and gel filtration chromatography resulted with the fold of purification about 5834.68 times and 158.6% recovery and molecular weight of 62 kDa. The calculated Km and Vmax value of the enzyme using ABTS as substrate were 0.107 mM and 77101.00 S-1 mM-1, respectively. The optimum pH, optimum temperature, pH stability and thermal stability of laccases were 2.5, 60°C, 4–6, 20-80°C, respectively. Sodium azide was an inhibitor for laccases from Cerrena sp. WICC F39. In accordance to the results showed in this study, such high level secretion of laccase and other ligninolytic enzymes make Cerrena sp. WICC F39 as a potential candidate for enhanced bioremediation.