Isolation and Optimization of Laccase producing Marine Fungi Neurospora crassa AJAS1

Abstract

The study includes isolation and screening of laccase enzyme producing fungus from marine soil sample collected from Kovalam beach (8.4004˚N, 76.9787˚E), Kerala. In this study, 12 fungal strains were isolated by serial dilution technique on different complex media. For primary screening of laccase production, plate assay was performed where the fungal isolates were cultivated on potato dextrose agar supplemented with tannic acid, ABTS and guaiacol as indicator substrates respectively. Out of the 12 isolates, 6 showed positive results for laccase production and were designated as FL1-FL6. Further, laccase enzyme assay was performed. The potential strain with maximum enzyme production was found to be FL6. The strain was optimised in submerged fermentation under different sets of environmental parameters. The fungal growth was maximum at 37˚C and pH 5 with starch, yeast extract, glutamine, KH2PO4 and 3% NaCl concentration as the carbon, nitrogen, amino acid, and mineral salt source respectively. Furthermore, the enzyme stability was also checked for varying temperature and pH. Antimicrobial potential of the strain FL6 was assessed using the Kirby Bauer well diffusion method where it showed good results against Gram-negative bacteria than the Gram-positive organisms. FL6 was morphologically identified as belonging to genus Neurospora using the lactophenol cotton blue stain.28s rRNA sequencing of the potential strain was performed and identified as Neurospora crassa AJAS1.