Laccase production by Galerina sp. and its application in dye decolorization

The laccase producing fungus HC1 was isolated from a sample collected in the Bolivian Amazon region. Analysis of 5.8S and 28S rDNA, and internal transcribed spacer 1 and 2 sequences revealed that isolate HC1 belongs to genus Galerina. High production of laccase was achieved in basal salt medium supplemented with 30 g L-1 glucose, 10 g L-1 yeast extract and 0.01 g L-1 copper sulphate. The enzyme production was further improved by addition of 2,5-xylidine, α-benzoin oxime and p-coumaric acid, but strongly repressed by sinapyl alcohol, 3-(dimethylamino)benzoic acid, hydroquinone, 1-naphthale-neacetic acid, 2,6-dichlorophenol and 3-methyl-2-benzothiazolinone hydrazone. Cultivation under optimum conditions in the presence of 2 mmol L-1 2,5-xylidine and 0.01 g L-1 copper sulphate, resulted in the enzyme yield of over 26 000 U L-1. Galerina sp. HC1 could also produce laccase in media composed of orange peels both in submerged- and solid-state fermentations. The laccase (0.5 U ml-1) from Galerina sp. was able to decolorize over 60% of 35 μmol L-1 Congo red in 2 h in the presence of 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as mediator, and 65% of 30 µmol L-1 Coomassie Brilliant Blue G-250 in 5 min using syringaldehyde as mediator. The laccase was immobilized on cross-linked chitosan and used efficiently to decolorize the dyes.   Key words: Oxidase, inducer, immobilized enzyme, enzyme production, solid-state cultivation.