BIOTECHNOLOGY TECHNIQUES Volume 10 No.4 (April 1996) p-273-276 Received as revised 14th February.

COLORIMETRIC ASSAY FOR LIGNIN DETERMINATION USING METHYLENE

PEROXUlASE ACTIVITY BLUE AS SUBSTRATE

Denise Bello Magalhks, Maria Eleonora Andrade de Carvalho2, Elba Bon, Julio Silva Araujo Neto3 (in memoriam), Sergio HClio Klin$*
Institute de Quimica, UniversidadeFederaldo Rio de Janeiro,RJ,Faculdadede Engenharia Quimica de Lorena, SP, Escola de Quimica, UniversidadeFederaldo Rio de Janeiro, RJ, 41nstituto de Quimica, Universidadedo Estado do Rio de Janeiro, Rua S%oFrancisco Xavier, 524, MaracanB,CEP 20550-000,Rio de Janeiro,RI, Brasil.

SUMMARY A calorimetric method for determination of lignin peroxidase activity has been developed which is based on the demethylation of methylene blue. The use of this dye shows the practical advantage of using a wavelength in the visible region of spectrum. The method can be efficiently used for the enzyme quantification as its sensitivity is close to the veratryl alcohoi assay. INTRODUCTION The veratryl alcohol has been frequently reported as substrate for the assay of lignin peroxidase activity. The method is based on the rate of oxidation of veratryl (3,5dimethoxybenzyl) alcohol to veratraldehyde (Tien and Kirk, 1984). Other methods which use the 2-keto-4 methiolbutyric acid (KMB), lignins and Azure B as substrates for oxidations (Umezawa et al., 1986, Perez and Jeffries, 1990, Archibald, 1992) have also been reported. This study presents a method for lignin peroxidase activity assay using the dye methylene blue, as substrate. It is based on previous findings by KIing and Neto ( 199 1) who observed the ability of the cultures of Phanerochaete chrysosporium to demethylate methylene blue in the presence of H202. The final produce was Azure C, a tri-demethylated methylene blue derivative. The use of this dye for lignin peroxidase assay has the advantage of being a readily available reagent and has its absorbancy in the visible region of the spectrum, i.e. 664 run. It is also worth mentioning that the reaction occurs at pH 4.0, which is adequate for the enzyme stability and matches the physiological pH for the enzyme production by the fungus. Also, considering the potential use of lignin peroxidase in the

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wastewater treatment this method is more advantageous as the use of the veratryl alcohol is hindered by it and other wavelength. MATERIALS AND METHODS aromatic pollutants absorbing at the same

Enzyme source Immobilized mycelium of the fungus Phanerochaete chrysosporium ATCC 24725 was used for enzyme production. For the immobilization step, shake-flask fermentations using a chemically defined carbon-limited medium (Link0 and Zhong, 1987) were carried out in the presence of nylon-web cubs (Linko, 1988). The cultures were flushed with pure Oz. After the growth phase the immobilized mycelium was transferred to a packed-bed reactor, which was operated on a semicontinuous mode, by the supply of the production medium: 5.5 mM glucose and 2.5 mM veratryl alcohol. Tween 80, to a final concentration of 1.3 g/L, was also added. Unless otherwise stated the enzyme activity assays were performed using the culture supernatant (crude enzyme) which was dialysed against 20 mM sodium tartrate buffer, pH 4.5 at 8.0 C. Dialysis were carried out for 72 hours with three buffer replacements. Enzyme Activity Assays Quantitative Lignin Peroxidase Assays Methvlene Blue Assay. The assay mixture contained in 3.0 ml: 2.2 ml of the diluted supematant, 0.1 ml of 1.2 mM methylene blue and 0.6 ml of 0.5 M sodium tartrate buffer (pH 4.0). The reaction was started by the addition of 0.1 ml of 2.7 mM H,Oz The conversion of the dye to Azure C was monitored by the measurement of the decrease in absorbance at 664 mn. The results were expressed as the change of absorbance per minute. Veratrvl Alcohol Assav. For comparison, lignin peroxidase activity was measured by the veratryl alcohol method: the initial rate of oxidation of veratryl alcohol to veratraldehyde was followed by absorption at 310 nm. The assay mixture contained in a final volume of 2.5 ml: 1.8 ml of the diluted supematant, 0.1 ml of 50 mM veratryl alcohol, 0.5 ml of 0.5 M sodium tartrate buffer (pH 3.0) and 0.1 ml of 10 mM H20?. One unit of enzyme activity was considered as the amount of enzyme which oxidizes one micromole of veratryl alcohol per minute. Qualitative Lignin Peroxidase Assay The methylene blue reaction can also be used for a visual inspection for the enzyme presence in the culture supematant. For this purpose the assay mixture contained in 2.7 ml: 2.2 ml of the supematant (without dialysis), 0.1 ml of 1 mM methylene blue, 0.3 ml of 0.5 M sodium tartrate buffer (pH 4.0). The oxidative reaction was started by the addition of 0.1 ml of 4.5 mM H202 The color that develops in the presence of lignin peroxidase was compared to a blank assay where twice distilled water was used to replace the supematant. Protective Role of Methylene Blue against Lignin Peroxidase Inactivation by Hydrogen Peroxide According to the literature (Haemmerli et al., 1986, Aitken and Irvine, 1989, Neto and Kling, 1993) lignin peroxidase is susceptible to H202, It has been also shown that veratryl alcohol protects the enzyme against inactivation. In order to identify a similar role for the methylene blue, 2.3 ml of the dialysed supematant was incubated with 0.1 ml of 1.5 mM H202 in the presence of either 0.1 ml of 50 mM veratryl alcohol or 0.1 ml of 4.68 mM methylene blue at 38 OC, during 10 minutes. After the treatment the

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samples were dialysed against 20 mM sodium tartrate buffer (pH 4.5) and the enzyme activity was measured as usual. In order to avoid the denaturation of lignin peroxidase by the H202, the initial buffer used in the dialysis procedure contained the relevant substrate (except controls). In control experiments, Hz02 was replaced by twice distilled water and the inactivation treatment was carried out in the absence of veratryl alcohol and methylene blue. RESULTS AND DISCUSSION 1. Absorption Spectra Studies of absorption spectra for methylene blue and its demethylated derivative are presented in Fig. 1. Accordingly, the
1.2

wavelength

664 the

nm

was higher

choosen due to absorbance of

methylene blue at gives the

06

this wavelength which to the difference

best sensitivity to the method and in absorbance blue and between Azure C. methylene

Figure 1. Visible light absorption spectra for Methylene blue (B) and Azure C (P). 2. Methylene Blue Quantitative Assay

Comparative data for the methylene blue and veratryl alcohol assays are shown in Fig. 2. The results which were obtained indicated that the methylene blue method has a sensitivity close to the veratryl alcohol assay. In order to obtain linear response in the methylene blue assay a minimal enzyme amount of 65 U/L (in terms of veratryl alcohol) was used. In the veratryl
ml Crude enzyme/ml Assay mixture

alcohol

method

this

amount was of 44 U2. Figure 2. Effect of enzyme amount on the veratryl alcohol (D) and methylene blue (0) assay.
Veratryl alcohol activity of crude enzyme: 284 UiL

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3. Protective Role of Methylene Blue against Hz02 Inactivation The results concerning the protective role of methylene blue against lignin peroxidase inactivation by H202 are shown in Table I. The data indicate that, similarly to veratryl alcohol, methylene blue protects the enzyme against H202 inactivation. This set of data also confirm that the demethylation reaction of methylene blue was carried out by lignin peroxidase: the results of veratryl alcohol activity when methyiene blue was used as the protective substrate were equivalent to the data observed for veratryl alcohol. Table L Protective Effect of Methylene Blue against H202 denaturation. Veratryl Alcohol Activity Control 1 Control 2 Veratryl Alcohol Methylene Blue* 806.4 341.1 641.9 702.1 (U/L)

Control 1 - no HzOz treatment (twice distilled water addition in place of H,Oz solution) Control 2 - H202 treatmentwith no substrateaddition. Denaturation assayin presenceof Veratryl Alcohol Denaturation assayin presenceof Methylene Blue

4. Methylene Blue Qualitative Assay The methylene blue method can be used as a fast qualitative assay for lignin peroxidase in cell cultures. Supematants with enzyme concentration above 50 U/L (in terms of veratryl alcohol) show a sharp change in the blueness from a greenish blue to purple blue. This color may be developed immediately, depending on the enzyme concentration, REFERENCES Aitken, M.D. and Irvine, R.L. (1989). Biotechnol. Bioeng. 34, 1251-1260. Archibald, F.S. (1992). Appl. Environ. Microb. 58,3110-3 116. Haemmerli, S.D., Leisola, M.S.A. and Fiechter, A. (1986). FEMS Microbial. Lett. 35, 33-36. Kling, S.H. and Neto, J.S.A. (1991). J. Biotechnol. 21,295-300. Linko, S. and Zhong, L-C. (1987). Biotech. Techn. 1,249-254. Linko, S. (1988). Em. Microb. Technol. 10,410-417. Neto, J.S.A. and Kling, S.H. (1993). Biotech Techn. 7, 81-84. Perez, J. and Jeffiies, T.N. (1990). Appl. Environ. Microb. 56, 1806-1812. Tien, M. and Kirk, T.K. (1984). Proc. Natl. Acad. Sci. USA 81,2280-2284. Umezawa, T., Shimada, M., Higushi, T. and Kusai, K. (1986). FEBS Lett. 205, 287292

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