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A SPECTROPHOTOMETRIC ASSAY FOR LACCASE USING o‐TOLIDINE
Author(s) -
MILLER R.,
KUGLIN J.,
GALLAGHER S.,
FLURKEY W.H.
Publication year - 1997
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1997.tb00199.x
Subject(s) - absorbance , chemistry , laccase , periodate , industrial and production engineering , hypochlorite , chromatography , biochemistry , organic chemistry , enzyme , electrical engineering , engineering
A spectrophotometric method for measuring laccase activity using o‐tolidine has been developed. Oxidation of o‐tolidine by laccase to a blue colored product corresponded with increases in absorbancies at 366 and 630 nm. This oxidation reaction and increases in absorbance at 366 and 630 nm could also be mimicked using hypochlorite, periodate and UV light in place of laccase. After a lag period, the assay was linear in absorbance with time, although the duration of linear region appeared to be affected by the pH. When assayed from 0.025 to 7 mM tolidine, maximum oxidation of substrate occurred using 3 mM o‐tolidine. Oxidation of o‐tolidine exhibited a pH dependency and showed an apparent pH optimum at approximately 5.0. The utility of this assay was shown by determining laccase activity in various fungal extracts.