6.Benzidine

International Journal of Bio-Technology and Research (IJBTR) ISSN 2249-6858 Vol.2, Issue 3 Dec 2012 43-50 © TJPRC Pvt. Ltd.,

BENZIDINE OXIDATION BY ENZYMES FROM FUNGI MARIBEL CANO-HERNÁNDEZ1, JOEL DÍAZ-REYES2 & JOSÉ ALBERTO ARIZA-ORTEGA3 1

Instituto Tecnológico del Altiplano de Tlaxcala, Km. 7.5 Carretera Federal San Martín-Tlaxcala, San Diego Xocoyucan, Tlaxcala, México 2

Centro de Investigación en Biotecnología Aplicada del Instituto Politécnico Nacional, Carretera Estatal Santa Inés Tecuexcomac-Tepetitla Km. 1.5, Tepetitla de Lardizábal, Tlaxcala, México 3

Universidad CUP Xolotzingo No.1.Col. San José Xolotzingo. Puebla, México

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.

KEYWORDS: Benzidine, Oxidation, Azo Dye, Trametes Versicolor INTRODUCTION Benzidine is a moderately persistent pollutant in the environment (Bi et al., 2003; Zoheb, 2012). Release of these compounds into the environment is important because of their carcinogenic and toxic nature (Harden et al., 2006).

Benzidine-based dyes have the characteristic diazotized benzidine nucleus (4,4 diaminofenil) (Fig. 1), which is used as intermediate in the production of azo dyes, salts, color, naphthols and other compounds (Weber et al. 2001). Also it found in wasterwater from tannery dyeing (Cao et al. 2007). In the environment, dyes can be metabolized releasing benzidine which forms adducts with hemoglobin (Choudhary 1996), and carcinogenic products (Tannenbaum, 2000; Harden et al.

2006). Exposure to benzidine-based dyes is equivalent to receive an equimolar dose of benzidine (Lynn et al. 1980).

Studies of benzidine removal from contaminated sites, are based on adsorption processes (Chen and Nyman 2007; Weber et al. 2001; Colon et al. 2002). Other studies are focused on benzidine oxidation with Chloramine B, catalyzed by iodide (Skadauskiene and Skadauskas, 2001). Chemical oxidation also it was studied using chlorine dioxide (Cao et al.

2007). Other works using oxidative degradation in the presence of some redox mediators (Won et al. 2004; Kulshrestha and Husain, 2007; Zoheb, 2012). However, these processes have some disadvantages such as toxicity of chemicals or their high costs. On the other hand, direct brown 2 contain benzidine nucleus and two azo groups which widely used in textile industry and it is not permitted for commercial use in the USA. However, in many other countries these dyes are produced and commercialized without any restriction. Therefore, it is important to develop efficient methods for the discoloration and degradation of dyes in industrial effluents. Oxidation of recalcitrant organic as benzidine or direct brown 2 compounds by crude enzyme extract of Trametes versicolor can be a viable technique for the wastewater decontamination.

The aim of this study is an approach to benzidine oxidation catalyzed by a crude enzyme extract (CEE) from Trametes versicolor and application in brown direct 2 with benzidine nucleus.

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Maribel Cano-Hernández, Joel Díaz-Reyes, José Alberto Ariza-Ortega

MATERIALS AND METHODS Chemicals Malt extract, dextrose and potato were purchased from Bioxon (México). Analytical grade reagent was 2,2Azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS), benzidine was purchased from Fluka with 98 % purity percentage; while acetone, methanol and water were HPLC grade. Direct brown 2 (CAS number 2429825) was purchased from Dalaquimia, México.

Microorganism and Growth Medium Trametes versicolor (Tv) ATCC 8273 was a gift from Dr. Rafael Vázquez Duhalt, from Instituto de Biotecnología (UNAM, México). Fungi were grown first in Petri dishes at 30 °C using a media containing: 10 g glucose/L, 3 g malt extract/L, 5 g soybean peptone/L, 3 g yeast extract/L, and 40 g dextrose and potato agar/L. T. versicolor were inoculated in an Erlenmeyer flask with 150 mL of a previously sterilized liquid medium prepared with extract obtained from wheat straw chopped (0.5 cm) (100 g L-1) and malt extract (20 g L-1) boiled at 90 °C for 1h, as reported by Sainos et al. (2006). After inoculation flasks were incubated at 30 °C for 10 days in stationary conditions.

Crude Enzymes Extract (CEE) Liquid medium cultured fungi produced 127.28 +- 0.007 g biomass/L, from this a 39.5 g weight biofilms were separated from liquid medium, and milled with 90 mL of sterile water in a sterile blender. The biomass was centrifuged at 10 000 rpm for 20 minutes, at 25 °C. Supernatant was separated from biomass and used in bioassays.

Benzidine Reference Solution In order to obtain complete benzidine solubilization, it was mixed with 15 % v/v acetonitrile for 100 mg L-1 of benzidine.

Oxidation Benzidine Bioassays were performed in beakers of 10 mL, placing 5 mL of benzidine solution with 100 mg L-1 and 200 µL of CEE. Oxidation reactions were carried out in duplicate at 800 rpm, pH 5 and room temperature. Reaction evolution was monitored at 0, 0.5, 3, 9 and 24 hours by HPLC. The volume used in each analysis was returned to the reaction vessel to maintain concentration. For these set of reacting systems, it was considered to include a control (5 mL of benzidine with 200 µL of inactive CEE, sterilized at 15 psi and 121 °C for 15 min), and a blank (5 mL of sterile water over 200 µL of active CEE).

The quantification of the oxidation was determined using a calibration curve, with concentrations of 10, 25, 50, 75 and 100 mg L-1 of benzidine with 15 % acetonitrile in water, and 200 µL of CEE sterilized for 20 minutes 121 °C and 15 psi. All samples were covered with plastic lids to prevent water loss through evaporation. Reaction product was centrifuged (10000 rpm for 15 min and at 4 °C); the supernatant was filtrated and analyzed by UV-VIS, HPLC, FT- IR, techniques.

Discoloration of Direct Brown 2 Dye Direct brown 2 (DB2) is a molecule based benzidine, CEE was essays with DB2 with 500 mgL-1 under same experimental condition that benzidine bioessays. Discoloration was measured at the initial dye absorbance maximum (485 nm) by UV-Vis spectrophotometric analysis.

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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 25°C. Laccase activity changes were measured spectrophotometrically with a Genesys 5 spectrophotometer, using 5 mM 2,2-azino–bis (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 µmol 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 µm 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 µL, detection λ 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 cm−1, in ranges of 600– 4000 cm−1, 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 µL 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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Maribel Cano-Hernández, Joel Díaz-Reyes, José Alberto Ariza-Ortega

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 1,1’bi(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 (Martínez 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; Dávila and Vázquez-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)-4,4´diimine; 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.

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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. Martínez-Salgado M, Pedrosa-Rodríguez A, Rodríguez-Vázquez R, and Rosas-Acosta J (2005). Efecto de la glucosa y nitrato de amonio sobre las enzimas ligninolíticas producidas por Trametes versicolor inmovilizado en espuma y la decoloración 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 40:1923–1929 18. Sainos E, Díaz G. Montiel-González 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: 2470–2475 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 µL 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 µL 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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Benzidine Oxidation by Enzymes from Fungi

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Maribel Cano-Hernández, Joel Díaz-Reyes, José Alberto Ariza-Ortega

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