A novel strategy to evaluate biodegradation products by capillary zone electrophoresis and MALDI-TOF

ANA RINALDI; EMANUEL GRASSI; LAURA LEVIN; ROMINA CARBALLO

Ciudad Autonoma de Buenos Aires

Simposio; The 18th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2012; 2012

LACE

Azo dyes (-N=N-) are the largest group of colorants used in industry for color printing, textile dyeing, etc 1. However, the production and use of azo dyes result in environmental pollution due to the color visibility and the toxicity of certain dyes. The main disadvantages of the chemical degradation methods include high cost, secondary pollution or low efficiency. By far the single class of microorganism most efficient in breaking down synthetic dyes is white rot fungi 2. The nonspecific nature of the lignin-degrading systems of white rot fungi is an advantage for degradation of azo dyes by oxidation, in contrast to the reduction pathway in bacterial decolorization that generates amines, many of which are mutagenic and/or carcinogenic. Furthermore, knowledge of mechanisms and metabolites of fungal degradation of dyes is lacking 3,4. Thus, effective analytical techniques are needed to identify the metabolites of azo dyes and understand their biodegradation mechanism. In particular, the combination of CE and MALDI-TOF mass spectra, which also employs small sample quantities, is a good alternative for identification of metabolic degradation pathway of these azoic dyes. In the present study the ability to decolourise Xylidine and Methyl Orange by crude culture filtrates from Trametes trogii with outstanding laccase (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) activity, was assessed. T. trogii is a worldwide distributed white-rot basidiomycete. It demonstrated to be a good producer of laccases and other ligninolytic enzymes including LiP and MnP. Our main objective was to evaluate degradation products of two azo dyes by ecofriendly techniques. This analysis was performed in a P/ACE MDQ (Beckman Coulter), equipped with a UV-vis photodiode array detector. For all experiments, the CE system temperature was held at 25 ºC and UV detection at 254 nm was performed. An untreated fused-silica capillary of 75 µm I.D. x 375 µm O.D. was used and the effective length was 50 cm to the detector. The mobile phase consisted of 50 mM ammonium carbonate buffer (pH 9.5). Aliquots were taken at different times and injected into CE. Sample solutions were introduced by 5 s at 3.5 kPa and the separation voltage was 20 kV. Finally, biodegradation products from Xylidine were determined by capillary electrophoresis followed by MALDI-TOF mass spectra. A new degradation pathway was proposed and the final products were naphthalene sulfonate and xylene.   1. S. Rodríguez Couto, J. Hazard. Mater. 148 (2007) 768-770. 2. D. Wesenberg, I. Kyriakides, S.N. Agathos, Biotechnol. Adv. 22 (2003) 161-187. 3. X. Zhao, Y. Lu, D.R. Phillips, H. Hwang, I.R. Hardin, J. Chromatogr. A. 1159 (2007) 217–224. 4. S. Takeda, Y. Tanaka, Y. Nishimura, M. Yamane, Z. Siroma, S. Wakida, J. Chromatogr. A 853 (1999) 503-309.   Financial support from UBACyT IJ st1:*{behavior:url(#ieooui) }   1. S. Rodríguez Couto, J. Hazard. Mater. 148 (2007) 768-770. 2. D. Wesenberg, I. Kyriakides, S.N. Agathos, Biotechnol. Adv. 22 (2003) 161-187. 3. X. Zhao, Y. Lu, D.R. Phillips, H. Hwang, I.R. Hardin, J. Chromatogr. A. 1159 (2007) 217–224. 4. S. Takeda, Y. Tanaka, Y. Nishimura, M. Yamane, Z. Siroma, S. Wakida, J. Chromatogr. A 853 (1999) 503-309.   Financial support from UBACyT IJ st1:*{behavior:url(#ieooui) } In the present study the ability to decolourise Xylidine and Methyl Orange by crude culture filtrates from Trametes trogii with outstanding laccase (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) activity, was assessed. T. trogii is a worldwide distributed white-rot basidiomycete. It demonstrated to be a good producer of laccases and other ligninolytic enzymes including LiP and MnP. Our main objective was to evaluate degradation products of two azo dyes by ecofriendly techniques. This analysis was performed in a P/ACE MDQ (Beckman Coulter), equipped with a UV-vis photodiode array detector. For all experiments, the CE system temperature was held at 25 ºC and UV detection at 254 nm was performed. An untreated fused-silica capillary of 75 µm I.D. x 375 µm O.D. was used and the effective length was 50 cm to the detector. The mobile phase consisted of 50 mM ammonium carbonate buffer (pH 9.5). Aliquots were taken at different times and injected into CE. Sample solutions were introduced by 5 s at 3.5 kPa and the separation voltage was 20 kV. Finally, biodegradation products from Xylidine were determined by capillary electrophoresis followed by MALDI-TOF mass spectra. A new degradation pathway was proposed and the final products were naphthalene sulfonate and xylene.   1. S. Rodríguez Couto, J. Hazard. Mater. 148 (2007) 768-770. 2. D. Wesenberg, I. Kyriakides, S.N. Agathos, Biotechnol. Adv. 22 (2003) 161-187. 3. X. Zhao, Y. Lu, D.R. Phillips, H. Hwang, I.R. Hardin, J. Chromatogr. A. 1159 (2007) 217–224. 4. S. Takeda, Y. Tanaka, Y. Nishimura, M. Yamane, Z. Siroma, S. Wakida, J. Chromatogr. A 853 (1999) 503-309.   Financial support from UBACyT IJ st1:*{behavior:url(#ieooui) }   1. S. Rodríguez Couto, J. Hazard. Mater. 148 (2007) 768-770. 2. D. Wesenberg, I. Kyriakides, S.N. Agathos, Biotechnol. Adv. 22 (2003) 161-187. 3. X. Zhao, Y. Lu, D.R. Phillips, H. Hwang, I.R. Hardin, J. Chromatogr. A. 1159 (2007) 217–224. 4. S. Takeda, Y. Tanaka, Y. Nishimura, M. Yamane, Z. Siroma, S. Wakida, J. Chromatogr. A 853 (1999) 503-309.   Financial support from UBACyT IJ st1:*{behavior:url(#ieooui) }