Process optimization, purification and characterization of alkaline stable white laccase from Myrothecium verrucaria ITCC-8447 and its application in delignification of agroresidues

Abstract

The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation. The media components were optimized by response surface methodology (CCD-RSM). The nutritional components (glucose and peptone) and physical parameters (pH and temperature) were optimized by response surface methodology for enhanced laccase production by Myrothecium verrucaria ITCC-8447. The enzyme activity under optimum condition exhibited 1.45 fold increases in laccase activity. The white laccase was subjected to ion exchange chromatography with 6 fold purification. The molecular weight of white laccase was ~63-75kDa as estimated by SDS-PAGE followed by the activity staining with ABTS where green bands confirmed the presence of laccase. The enzyme was stable over an alkaline pH range of 7-9 and the temperature range of 30-40°C. The characterization of white laccase was done by CD spectra, UV-visible absorption, FTIR and XRD. The K_m and V_max values of the purified laccase were 2.5mM and1818.2μmol/min/L. The delignification capability of the white laccase was determined by reduction in Kappa number (58.8%) and Klason lignin (64.7%) of wheat straw after 12h of incubation. Further the delignification was confirmed FTIR and XRD.

Keywords: CCD-RSM; Delignification; Myrothecium verrucaria ITCC-8447; White laccase.