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. Ltd.,
Instituto Tecnolgico del Altiplano de Tlaxcala, Km. 7.5 Carretera Federal San Martn-Tlaxcala, San Diego Xocoyucan, Tlaxcala, Mxico
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Centro de Investigacin en Biotecnologa Aplicada del Instituto Politcnico Nacional, Carretera Estatal Santa Ins Tecuexcomac-Tepetitla Km. 1.5, Tepetitla de Lardizbal, Tlaxcala, Mxico
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ABSTRACT
Oxidation of benzidine was studied in a concentration of 100 mg L-1, using a crude enzyme extract of Trametes versicolor. Oxidation reaction was achieved at 800 rpm, room temperature, and pH 5. Benzidine was oxided in 92.47% after 24 h without mediator and non-sterile condition. The kinetics oxidation was second order with a kinetics constant of 0.4606 L/mol s. Reaction product were analyzed by FT-IR, UV-Vis spectroscopy and HPLC, results show an oxide form of benzidine, which depend of enzymatic concentration. Crude enzyme extract from T.versicolor also was able to oxidize brown direct 2 (benzidine-based dyes) with 500 mgL-1 after 3 day. Results obtained suggest using crude enzyme extract in removal recalcitrant compound.
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Laccase Activity For these assays, samples were prepared as follow: total reaction volume was 1 mL, containing 100 uL of 1 M acetate buffer at pH 5, 700 uL distilled water, 100 uL crude enzyme extract and 100 uL 5 mM ABTS substrate. The samples were mixed and incubated at 25C. Laccase activity changes were measured spectrophotometrically with a Genesys 5 spectrophotometer, using 5 mM 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonate (ABTS) as substrate, and a molar extinction coefficient of 36 000/Mcm (Jung et al. 2002; Bourbonnais and Paice 1997). One activity unit (U) was defined as the amount of enzyme that oxidizes 1 mol of substrate per minute under system reacting conditions. Following this procedure, it was determined laccase activity of the crude enzyme extract. All tests were run by duplicate. UV-Vis Analyses Oxidation of benzidine and DB2 were followed by ultraviolet (UV)-Vis spectroscopy using Hewlett Packard HP8453 diode array UV/Vis spectrophotometer. Spectrums were recorded from 190 to 1100 nm. Cromatographic Analysis. Samples were filtered with 0.45 m membranes, and reaction products were analyzed in a high-performance liquid chromatography (HPLC) using Hewlett-Packard model 1100 series Agilent, coupled to diode array detection with C-18 column, at flow rate of 0.5 mL/min. The mobile phase was methanol/water (50/50 v/v), injection volume was 10 L, detection of 280 nm, column temperature was 26 C, and total run time was 10 minutes. FT- IR Analysis FT- IR spectra, using Attenuated Total Reflectance (ATR) technique, were recorded using Bruker Vertex 70 apparatus with a resolution of 4 cm1, in ranges of 600 4000 cm1, and room temperature. Samples of reaction product and standard of benzidine were solubilized in methanol, placed on a glass plate for solvent evaporation, and introduced as a thin film in the FT- IR.
RESULTS
Oxidation rate was measure by HPLC at 280 nm. The peak with retention time of about 7.8 min was identified as benzidine (Fig. 2). Peaks at 3.64 and 4.92 min can be attributed to crude enzyme extract, since in chromatogram of blank (5 mL of sterile water and 200 L of active CEE) were observed signals at 3.59 and 4.92 min (Fig. 3). In addition in standard benzidine chromatogram of (Fig. 2a) those peaks do not appear, that confirm which are produced by the EEC. After 24h (Fig. 2f), it also was observed new peak at 9.15 min, which could indicate the reaction product. The oxidation rate of benzidine was evaluated by calibration curve of peak area in different times. Kinetics oxidation corresponds to an irreversible reaction as a second-order kinetic reaction, estimated rate constant was 0.4606 L/mol s, with correlation coefficient 0.993. UV-Vis Analysis The UV-Vis spectrum of control had maximum absorbance at 316.9 nm while reaction product was 292.11 nm (Fig. 4) after 24 h; differences between absorbances it can be due to oxided form of benzidine. In case of BD2, it had decrease absorbance intensity to max absorbance (485 nm) from first day, which implies a discoloration by CEE. However, in Fig. 6, it can also be observed that after 3 day, UV-Vis profiles spectrum of reaction product changed respect to control, which could indicate a modification in chemical structure of DB2.
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FT-IR Spectrum FT-IR spectrum of benzidine (Fig. 5) shows strong absorption bands at 821, 1265, 1505, 1607, 3034, 3182, 3281, and 3400 cm-1. Set of the bands centered in 1607 and 1505 cm-1 correspond to aromatic ring vibrations (Coates, 2000). The band at 1264 cm-1 is assigned to stretching vibration C-N. The peak at 3030 cm-1 could be related to aromatic vibration CH stretch. The peaks at 3281 and 3400 cm-1are assigned to symmetric associated aromatic primary amine N-H stretch; whereas in spectrum of reaction product, signal at 3281 cm-1 disappeared and peak at 3400 cm-1 decrease; for other hand, new band at 3328 cm-1 was observed, which suggest formation secondary amine in benzidine structure instead primary amine, that is possible since benzidine possesses two readily oxidized amino. Therefore benzidine could be oxided to 1,1bi(cyclohexa-2,5-diene)-4,4-diimine. Concentration of Lacasse Activity Oxidation benzidine is affected by concentration of laccase activity; since for 0.02, 0.04 and 1.04 laccase activity unite for mg benzidine, it was obtained 66.77, 71.33 and 92.35 % oxidation after 30 min.
DISCUSSIONS
Enzymatic oxidation of benzidine can be explained by presence of peroxidases and oxidases enzymes from CEE, such as laccases and Manganese Peroxidase (MnP) contained in T. versicolor (Martnez et al. 2005); these enzymes have oxidation potential about 0.5 to 0.8 V for laccases (Sedighi et al. 2009) and 1.5 V in the case MnP (Sedighi et al. 2009; Dvila and Vzquez-Duhalt 2006) their values are slightly higher than those of benzidine (reported as 0.72-0.92V) (Hung et al. 2005). Therefore benzidine can be oxided by laccase and MnP, exhibited by intracellular and associated enzymes to mycelia from CEE. Laccase activity value of CEE were 0.1046 U/mL and oxidation was affected by concentration of laccase activity; since for 0.02, 0.04 and 1.04 laccase activity unite for mg benzidine, it was obtained 66.77, 71.33 and 92.35 % oxidation after 30 min. Which confirm laccase enzyme oxide to benzidine, moreover in FT-IR spectrum it was observed new band at 3328 cm-1, which correspond to secondary amine. For other hand, UV-Vis spectrum of control had maximum absorbance at 316.9 nm while reaction product was 292.11 nm (Fig. 4) after 24 h; differences between absorbances suggest that every each spectrum correspond to benzidine molecule and their oxidaded form. Oxidation benzidine could form intermediate species such as Mn-peroxo complexes, since this intermediate oxide is dark blue at first, and turns out black later on (Principle of Washburn Method) similar to product obtained in this work. In order to observe how stirring affects the reaction rate, other series of experiments were performed, keeping the same conditions except the stirring; the value obtained from oxidation after 24 hours without agitation, was 92.1 % while with stir was 92.4 %. The closeness between values means that the oxidation mechanism could be controlled by a homogeneous catalysis rather than diffusive processes. For other hand, kinetic oxidation corresponds to an irreversible reaction which produced insoluble aggregates that can be removed by decantation or filtration. Is important to mention that some dyes are metabolized to benzidine during discoloration proccess (Dong et al., 2007; Kalme et al., 2007; Bafana et al., 2007). Therefore, CEE could be used to removal benzidine in their oxide form, without presence of mediator versus to reported by Karim, (2012). In the case of DB2 oxidation, absorption UV-Vis spectra were completely different to the DB2 solution (control) after 3 day in contact with CEE; it is attributed to degradation of BD2 by enzymatic activity. Discoloration of DB2 by CEE provide faster discoloration than anaerobic process; for example, activated sludge that needed 90 days to discolor a solution of 1800 mg/L DB2 (Isik and Sponza 2007); with a less concentration (120 mg/L DB2) Pazarlioglu et al. (2005) required 6 days to discolor a solution using Phanerochaete Chrysosporium. Therefore, crude enzyme extract from T. versicolor have ability to efficiently oxidize benzidine molecules and bezidine based-dye, such as DB2.
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CONCLUSIONS
Intracellular and associate enzymes to mycelia of crude enzyme extract from Trametes versicolor, have ability to efficiently oxidation benzidine and direct brown 2. The oxided form of benzidine can be 1,1-bi(cyclohexa-2,5-diene)-4,4diimine; direct brown 2, was discolored after 1 day. Results obtained suggest using crude enzyme extract in removal recalcitrant compound.
ACKNOWLEDGEMENTS
The authors are thankful to CIBA-IPN and M. Solis for their technical support.
REFERENCES
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14. Husain Q and Kulshrestha Y (2007). Decolorization and degradation of acid dyes mediated by partially purified turnip (Brassica rapa) peroxidase. Toxicol Environ Chem 89: 255-267 15. Lynn R K, Donielson A M, Ilias J M, Kennish K, Wong K, and Matthews H B (1980). Metabolism of bisazobiphenyl dyes derived from benzidine, 3,3-dimethylbenzidine or 3,3-dimethoxybenzidine to carcinogenic aromatic amines in the dog and rat. Toxicol Appl Pharmacol 56(2): 248-58. 16. Martnez-Salgado M, Pedrosa-Rodrguez A, Rodrguez-Vzquez R, and Rosas-Acosta J (2005). Efecto de la glucosa y nitrato de amonio sobre las enzimas ligninolticas producidas por Trametes versicolor inmovilizado en espuma y la decoloracin de un efluente papelero en un biorreactor de lecho fluidizado. Rev Fac Ciencias, 10, 27-36 17. Pazarlioglu N K, Urek R O and Ergun F (2005). Biodecolourization of Direct Blue 15 by immobilized Phanerochaete chrysosporium. Process Biochem 40:19231929 18. Sainos E, Daz G. Montiel-Gonzlez A M and Loera O (2006). Growth of Pleurotus ostreatus on wheat straw and wheat grain-based media: biochemical aspects and preparation of mushroom inoculum. Appl Microbiol Biotechnol 72: 812-815 19. Sedighi M, Karimib A, and Vahabzadeh F (2009). Involvement of ligninolytic enzymes of Phanerochaete chrysosporium in treating the textile effluent containing Astrazon Red FBL in a packed-bed bioreactor. Jal of Hazard Mat 169:88-93 20. Skadauskiene, O. P., & Skadauskas, J. S. (2001). Kinetic Determination of Iodide by the Oxidation Reaction of Benzidine with Chloramine B. J Anal Chem 56: 170-172 21. Tannenbaum S R (2000). Bladder cancer in workers exposed in aniline. J Natl Cancer Inst Bd 83: 1507- 1508 22. Weber E J, Colon D and Baughman G L (2001). Sediment-associated reactions of aromatic amines. 1. Elucidation of sorption mechanisms. Environ. Sci. Technol 35: 24702475 23. Won K, Kim Y H, An E S, Lee Y S, and Song B K (2004). Horseradish peroxidase catalyzed polymerization of cardanol in the presence of redox mediators. Biomacromolecules 5:1-4 24. Zoheb K, Qayyum H, and Rohana A (2012). Redox-mediated polymerization and removal of benzidine from model wastewater catalyzed by immobilized peroxidase. Afr J Biotechnol 11: 12053-12062 Figure Caption Fig. 1 Chemical structure; (a) Benzidine; (b) Direct Brown 2 Fig. 2 Chromatograms of benzidiene reacted with CEE in different times; (a) control (benzidine), (b) 30 min; (c) 1 h; (d) 3 h; (e) 9 h;
(f) 24 h Fig. 3 Chromatograms blank (5 mL of sterile water and 200 L of active CEE). Fig. 4 UV-Vis spectral of control and sample of reaction after 24 h; condition reaction: 5 mL of benzidine solution and 200 L of CEE. Reactions were carried out in duplicate at 800 rpm pH 5 and room temperature Fig. 5 FT-IR spectra of benzidine, and reaction product
Fig. 6 UV-Vis spectrum of the DB2 with 500 mg L-1, with CEE at different times at 800 rpm, pH 5, at room temperature
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Figure 1
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