Biodegradation of Dyes by Laccase from Isolated …
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2.2 Laccase Enzyme Production by Solid-State Fermentation
Agricultural waste substrate ribbed gourd peel was moistened with water and autoclaved in 250 ml Erlenmeyer flasks up to 7 days with 7-day-old culture at 30 °C.
After fermentation, the supernatant was collected, stored and used as enzyme source
of laccase.
2.3 Enzyme Assay
Free laccase activity was determined using the process as described by Desai et al.
(2011) where guaiacol was used as substrate. Laccase activity was expressed in U/ml.
2.4 Effect of Laccase and Dye Concentrations
on Decolorization Efficiency
The effect of laccase concentration on decolorization efficiency was determined by
incubating the synthetic dyes with varying concentrations of laccase (20–100 U/ml)
for 24 h. To study the effect of dye concentrations with varying concentrations (5, 10,
15, 20, 25 µM) were used for 24 h. Immediate after incubation the dye degradation
efficiency was expressed in percentage (%).
2.5 Effect of Temperature, PH, Incubation Time and Laccase
Mediator on Dye Decolorization Efficiency
During optimization of dye decolorization efficiency by laccase, the enzyme and dyes
were incubated at varying temperatures (25–45 °C), pH (4.0–8.0) and incubation time
(0–24 h). 1-hydroxybenzotriazole (HBT) (1 mM) was added in the reaction mixture
as redox mediator and observed up to 24 h. Dye degradation efficiency was expressed
in percentage (%).
2.6 Box–Behnken Response Surface Methodology
The effects of dye concentration (mM), enzyme concentration (U/ml), pH, HBT (%)
on dye decolorization efficiency were evaluated. These factors were chosen as they
showed influencing effects in one variable at a time (OVAT) optimization. Levels
111
2.2 Laccase Enzyme Production by Solid-State Fermentation
Agricultural waste substrate ribbed gourd peel was moistened with water and autoclaved in 250 ml Erlenmeyer flasks up to 7 days with 7-day-old culture at 30 °C.
After fermentation, the supernatant was collected, stored and used as enzyme source
of laccase.
2.3 Enzyme Assay
Free laccase activity was determined using the process as described by Desai et al.
(2011) where guaiacol was used as substrate. Laccase activity was expressed in U/ml.
2.4 Effect of Laccase and Dye Concentrations
on Decolorization Efficiency
The effect of laccase concentration on decolorization efficiency was determined by
incubating the synthetic dyes with varying concentrations of laccase (20–100 U/ml)
for 24 h. To study the effect of dye concentrations with varying concentrations (5, 10,
15, 20, 25 µM) were used for 24 h. Immediate after incubation the dye degradation
efficiency was expressed in percentage (%).
2.5 Effect of Temperature, PH, Incubation Time and Laccase
Mediator on Dye Decolorization Efficiency
During optimization of dye decolorization efficiency by laccase, the enzyme and dyes
were incubated at varying temperatures (25–45 °C), pH (4.0–8.0) and incubation time
(0–24 h). 1-hydroxybenzotriazole (HBT) (1 mM) was added in the reaction mixture
as redox mediator and observed up to 24 h. Dye degradation efficiency was expressed
in percentage (%).
2.6 Box–Behnken Response Surface Methodology
The effects of dye concentration (mM), enzyme concentration (U/ml), pH, HBT (%)
on dye decolorization efficiency were evaluated. These factors were chosen as they
showed influencing effects in one variable at a time (OVAT) optimization. Levels