Decolorization of synthetic dyes by Citrus peroxidase

Abstract

The objective of the current project was to establish an economical method utilizing peels of kinnow (Citrus reticulata) and lemon (Citrus limon) for decolorization of synthetic dyes and industrial effluents. Firstly, both peroxidases extracted from Citrus reticulata (CRP) and Citrus limon (CLP) were characterized in terms of kinetic and thermodynamic parameters. The optimum pH for both the enzymes was determined to be 6.0 whereas the temperature optima were 55 and 45 0 C, respectively for CRP and CLP. The values of K m and V max for guaiacol oxidation for CRP were recorded to be 0.66 and 380 μmol/mL/min, whereas for CLP they were recorded as 2.70 mM and 2222 μmol/mL/min, respectively. Results regarding thermostability showed that both enzymes were fairly stable at 60 0 C but at 80 0 C, CRP retained 58 % while CLP retained 37 % of its activity after 60 min. The energy of activation for thermal denaturation was found to be 95.85 and 77.27 kJ/mol for CRP and CLP, respectively. Metal ions like, Mg 2+ , Cd 2+ , Cu 2+ , Al 3+ and one of the surfactants, Lemon max behaved as activator whereas Co 2+ , Ni 2+ , Hg 2+ , Triton X-100, Tween-80, SDS, Brite total, Surf excel, Rin behaved as an inhibitor for both CRP and CLP. Besides this, inhibitory effect of urea, sodium azide and EDTA was also reported for both CRP and CLP. Secondly, both the enzymes were utilized for degradation of synthetic dyes. Using CRP, 88.92 % decolorization was achieved for Remazol brilliant blue R (RBBR) at pH, temperature, enzyme dose, H 2 O 2 , dye and p-coumaric acid concentarion of 4.0, 35 0 C and 18 U/mL, 0.125 mM, 50 mg/L, 0.1 mM, respectively within 5 min. Whereas for Golden yellow PRA (GYPRA) pH of 2.0, temperature of 40 0 C, enzyme does of 24 U/mL, H 2 O 2 concentration of 0.375 mM dye concentration of 100 mg/L and vanillin concentration of 0.05 mM were determined as optimum to attain 95.98 % decolorization within 5 min. On the other hand, CLP decolorized 85.97 % of Brilliant yellow (BY) and 96.34 % of Crystal violet (CV) respectively at pH 5.0 and 4.5, temperature 50 and 45 0 C, enzyme dose 24 and 42 U/mL, dye concentration 18.75 and 7.5 mg/L, syringaldehyde 0.025 mM and p-coumaric acid 0.5 mM, at 0.25 mM H 2 O 2 with in 10 and 5 min. The degradation products of BY and CV were also identified by LC/MS analysis. The optimizations and interactive effects of pH, enzyme dose and dye concentration for % decolorization of all four dyes were also investigated by RSM through CCD. The CLP was also employed for complete degradation of two effluents under the optimized conditions of pH 2.0 and 3.0, temperature 45 and 40 0 C, enzyme dose 18 and 24 U/mL and time of incubation of vi20 and 60 min, respectively for Effluent 1 and 2. Finally, phytotoxicity of the effluents and their degraded samples was also evaluated for Zea mays. It was observed that the degraded effluent samples were less toxic than the original ones.