Vandana et al.

Laccase assay

Laccase activity was determined by measuring the oxidation of guaiacol at 530

nm. The reaction mixture was containing 10 mM guaiacol and 100 mM acetate

buffer (pH 5). Absorbance for blank was measured at 470 nm while that of test

samples was measured at 530 nm. The change in the absorbance of the reaction

mixture with guaiacol was monitored for 10 min of incubation. Enzyme activity

was measured in U/ml which is defined as the amount of enzyme catalyzing the

production of one micromole of coloured product for min per ml (Jhadav et al.,

2009).

Calculation

∆ A 530 nm/min x V t x dilution factor

Enzyme activity (u/ml)

€ x V s

Where,

Vt = final volume of reaction mixture (ml)

Vs = sample volume (ml)

€ = extintion co-efficient of guaiacol = 6740/M/cm.

Partial purification of Laccases

Partial purification of laccase enzyme was done through Ammonium sulfate

precipitationandDialysis.Proteincontentwasestimatedateachstepofpurification.

Ammonium sulfate precipitation

The crude laccases was filtered through Whatman No.1 filter paper and the

resulting filtrate was concentrated by freeze-drying and kept at 4ºC. Protein was

precipitated by ammonium sulfate from the filtrate obtained. Solid ammonium

sulfate was added slowly to the crude extract isolate to give 70% saturation and the

solution was stirred gently for at least 1 h at 4ºC and was left to stand overnight.

The precipitate was collected by centrifugation at 8000 rpm for 1 h at 4ºC. The

supernatant was discarded and the pellet was dissolved in 0.01 M phosphate buffer

(pH 7) (Barda and David, 1949).

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International Journal of Bioinformatics and Biological Sciences : v.2 n.3&4. Dec. 2014