Optimization, purification and characterization of laccase from lignocellulolytic fungi Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535

Abstract

In the present study, lignocellulolytic fungi were isolated by lignocellulolytic waste (Banana waste) soil from the Bhilai-Durg region of Chhattisgarh, India, which was capable to produce laccase enzyme. The laccase producing potent organisms was identified as Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 using molecular characterization by Internal Transcribed Spacer (ITS). For the better production of laccase enzyme physical (incubation period, temperature and pH) and nutritional (carbon, nitrogen and vitamin source) parameters optimized by one factor-at-a time and response surface methodology approach were used. Laccase enzyme was partially purified by ammonium sulphate precipitation and dialysis. The partially purified laccase was characterized by the effect of substrate concentration, temperature, pH, metal ions and inhibitors. Results showed that out of 5 positive cultures only 2 cultures showed the best laccase production after the quantitative screening. ITS-rDNA gene sequencing and BLAST analysis were used to characterize the isolated fungal species and they were found to be D. funicola NFCCI 4534 and A. padwickii NFCCI 4535. The temperature 24 °C and 32 °C and pH 6.0 and 6.5 were suitable for production of D. funicola and A. padwickii, respectively. It was found that Ferric chloride and ferrous sulphate exhibited maximum laccase activity as compared to other metal ions. However, EDTA and l-arginine inhibited D. funicola and A. padwickii, respectively.