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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1273 -1281 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1275 H 2 O 2 for varying times at 37°C. The reaction was terminated by boiling at 100°C for 10 min. Dye decolorization was monitored by measuring the difference at the maximum absorbance for each dye (λ max for RB 15 and RR 4 are 675nm & 517nm respectively) as compared with control experiments without enzyme on UV-visible spectrophotometer (JASCO V-550, Japan). Untreated dye solution (excluding the enzyme) was used as control (100%) for the calculation of percent decolorization. The dye decolorization was calculated as the ratio of the difference of absorbance of treated and untreated dye to that of treated dye and converted in terms of percentage (14). % decolorization = Initial absorbance - Observed absorbance Initial absorbance x 100 Five independent experiments were carried out in duplicate and the mean was calculated. Effect of redox mediator on T. dioica peroxidase mediated reactive dye decolorization : Each of the two reactive dyes (5.0 mL) were incubated with (PGP) (0.45 EUmL -1 ) in the presence of each redox mediators viz., syringaldehyde, guaiacol, 1-hydroxybenzo-triazole (HOBT), vanillin, bromophenol and quinol (0.5 mM) and 0.75 mM H 2 O 2 in 0.1 M glycine HCl buffer, pH 4.0 for 1 h at 37°C. The reaction was terminated by boiling the sample at 100°C for 10 min. The absorbance of the dye solutions at the respective λ max for each dye was recorded against untreated dye as control (100%). To find out the optimum concentration of HOBT a similar set of experiment as mentioned above was performed in the presence of varying concentrations of HOBT (0.05 to 2.0 mM). The reaction was terminated by boiling the sample at 100°C for 10 min. The absorbance of the dye solutions at the respective λ max for each dye was recorded against untreated dye as control (100%) in all the subsequent sets of experiments. Reactive dye decolorization with varying concentration of Enzyme (PGP) and H 2 O 2 : Each of the two reactive dyes were incubated with increasing concentrations of PGP (0.065 to 0.50 EUmL -1 ) and H 2 O 2 (0.2 to 1.8 mM) in 0.1 M glycine HCl buffer, pH 4.0 in the presence of 0.75 mM H 2 O 2 for 1 h at 37°C. HOBT used as a redox mediator at concentrations of 1.0 mM for both the dyes. The reaction was stopped by boiling the sample at 100°C for 10 min. Reactive dye decolorization as a function of temperature and pH : Each of the two reactive dyes was incubated with PGP (0.45 EUmL -1 ) at different temperatures (20°C to 90°C). Other reaction conditions were common. The reaction was stopped by boiling the sample at 100°C for 10 min. The reactive dye solutions were made in different buffers each of 100mM and in the range of pH 2.0 to pH 10.0. The buffers were glycine- HCl (pH 2.0, 3.0 and 4.0), sodium acetate (pH 5.0), sodium phosphate (pH 6.0, 7.0 and 8.0), and Tris-HCl (pH 9.0 and 10.0). Each of the two dye was treated with PGP (0.45 EUmL -1 ) in buffers of varying pH and in the presence of 1.0 mM H 2 O 2 for 1 h at 37°C. The decolorization of each dye RB15 and RR4 was also performed in the presence of 1.0 mM HOBT. The reaction was stopped by boiling the sample at 100°C for 10 min. T. dioica mediated reactive dye decolorization as a function of time : The individual reactive dye was treated with PGP (0.45 EUmL -1 ) in the presence of 1.0 mM H 2 O 2 in 0.1 M glycine HCl buffer, pH 4.0 at 37°C for varying time intervals. HOBT was used as a redox mediator at concentrations of 1.0 mM. The reaction was terminated by boiling the sample at 100°C for 10 min. Simple approach to reactive dye decolorization
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1273 -1281 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1276 Results Effect of different redox mediators on the extent of reactive dye decolorization : The effect of different redox mediators on dye decolorization by PGP is shown in Fig. Ia. Among the six different redox mediators studied for dye decolorization, HOBT was most effective in decolorizing the dyes. The extent of decolorization was 98.6% and 68.2% for RB15 and RR4 respectively in the presence of HOBT. The other redox mediators were relatively less effective. Fig.1b shows the effect of increasing concentrations of HOBT (0.05 to 2.0 mM) on RB15 and RR4. With increasing concentration of HOBT there was an increase in the extent of decolorization of both dyes. Maximum decolorization was achieved in the presence of 1.0 mM HOBT. At concentration above 1.0mM there was insignificant change in percent decolorization of the dyes. Effect of enzyme concentration and pH on the extent of reactive dye decolorization : Fig.2 shows the extent of decolorization of RB15 and RR4 with varying concentrations of PGP. The maximal decolorization for these two dyes was observed at PGP concentration of 0.45EUmL -1 . At concentrations above this, there was no remarkable change in dye decolorization. Fig.3 shows the percent decolorization improved with Reactive Blue 15 Reactive Red 4 HOBT Conc. (mM) Fig.1a. Reactive dye decolorization as a function of different redox mediators. The concentration of each redox mediator viz., syringaldehyde, guaiacol, 1- hydroxybenzotriazole (HOBT), vanillin, bromophenol, and quinol was 0.5mM; other conditions were 0.8mM H 2 O 2 , 100mM glycine HCl buffer, pH 4.0 for 60 min at 37°C. (λ max for RB 15 and RR 4 are 675nm & 517nm respectively). Fig.1b. Reactive dye decolorization as a function of different concentrations of HOBT (0.05 to 2.0 mM). % Dye decolorization % Dye decolorization Enzyme in Eu/ml Reactive Blue 15 Reactive Red 4 Fig. 2. Enzyme activity plot. Reactive dye decolorization as a function of different enzyme (PGP) concentrations (0.065 to 0.50 EUmL -1 ). H 2 O 2 Conc. in mM Reactive Blue 15 Reactive Red 4 Fig. 3. Hydrogen peroxide activity plot. Reactive dye decolorization at different concentrations of H 2 O 2 (0.2 to 1.8 mM). Farrukh Jamal
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(December 7-9, 2011) Venue: Karunya
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