Biosci. Biotechnol. Biochem., 66 (4), 717–721, 2002 NADH Oxidation by Manganese Peroxidase with or without a-Hydroxy Acid Tetsuya DEGUCHI, Masaaki MATSUBARA,andTomoakiNISHIDA* Chemical and Environmental Technology Laboratory, KOBE STEEL, LTD., Kobe 651-2271, Japan *Department of Forest Resources Science, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan Received August 3, 2001; Accepted November 28, 2001 NADH oxidation by manganese peroxidase (MnP) organic acids chelate the generated Mn(III) and was done in a reaction mixture including either a- release the Mn(III) from the enzyme-manganese hydroxy acid or acetate. The oxidation in the former complex. The released Mn(III)-organic acid, in turn, reaction mixture was inhibited by a catalase and was ac- oxidizes various substrates.7,8) This oxidizing system celerated by exogenous H2 O2, while the oxidation in the in which the low molecular weight compound, latter reaction mixture was inhibited by a superoxide Mn(III)-organic acid, acts as the direct oxidizing dismutase and was not accelerated by the exogenous agent is believe to be e‹cient for oxidation of poly- H2O2. These results indicated that there are signiˆcant meric substrates such as lignin because the low diŠerences between the two reaction systems, particu- molecular weight compound is mobile in polymeric larly, in the active oxygen species involved in the reac- substrates which may be inaccessible to polymeric en- tions. Additionally, the experiment of MnP reduction zymes. with Mn(II) suggests that MnP has a separate catalytic On the other hand, we have reported that the white activity other than an oxidation of Mn(II) to Mn(III) in rot fungi were able to oxidatively degrade nylon un- the reaction mixture including acetate. der ligninolytic conditions9) and that puriˆed MnP was able to degrade nylon without the exogenous 10,11) Key words: manganese peroxidase; NADH; nylon H2O2. In addition, surprisingly, the a-hydroxy degradation acid was a strong inhibitor of the nylon degradation. These results cannot be explained by the known fea- White rot fungi are the best-known and most eŠec- tures of the MnP catalytic cycle. tive lignin-degrading microorganisms. These fungi Recently we recognized that MnP could oxidize have recently received worldwide attention because NADH either with or without a-hydroxy acid. The of their detoxiˆcation of recalcitrant environmental NADH oxidation by MnP is very interesting and may pollutants such as dioxins,1–3) DDT,4) and PCB,5,6) un- be useful to increase the understanding of the MnP der ligninolytic conditions. The process of their lig- reaction without a-hydroxy acid. In this paper, we in- nin degradation is nonspeciˆc and nonstereoselec- vestigate the NADH oxidation by MnP and reveal tive, which explains why these fungi can degrade the diŠerences in the NADH oxidation between with various organic materials. and without a-hydroxy acid. Under ligninolytic conditions, many white rot fungi secrete extracellular peroxidases. Manganese Materials and Methods peroxidase (MnP) is one of the most frequently en- countered peroxidases among white rot fungi. MnP Chemicals. NADH and superoxide dismutase has the same catalytic cycle as other peroxidases but (SOD) were purchased from Wako Pure Chemical MnP is unique in its ability to catalyze the oxidation Industries (Osaka, Japan). Catalase was purchased of Mn(II) to Mn(III). Resting MnP is oxidized by fromSigmaChemicalCo.(St.Levis,Mo.). H2 O2 in a single two-electron step to form MnP com- pound I and the latter is reduced by Mn(II) back to MnP preparation. The white rot fungus strain the resting MnP. In this reduction step, a total of two IZU-154, which had been isolated in our laborato- equivalents of Mn(III) is formed. In this catalytic ry,12) was used for the production of MnP. MnP was cycle, a-hydroxy acids such as lactate, malate, and puriˆed from the extracellular ‰uid of the IZU-154 tartrate are known to have an important role. These cultures (6 days incubation) as described previous- To whom correspondence should be addressed. Tetsuya DEGUCHI, Phone: +81-78-992-5618; Fax: +81-78-992-5604; E-mail: te-deguchi＠ rd.kcrl.kobelco.co.jp Abbreviations: MnP, manganese peroxidase; 2,6-DMP, 2,6-dimethoxyphenol; SOD, superoxide dismutase 718 T‚ DEGUCHI et al. ly.10) NADH oxidation. The reaction mixture, contained 50 mM organic acid and 1 mM MnSO4,wasadjusted to pH 4.5 by sodium hydroxide. The organic acid was selected from lactate, tartrate, malate, and acetate. The MnP was added to the reaction mixture and reaction was initiated by the addition of NADH (ˆnal conc. of 0.17 mM). After mixing them, NADH oxida- tion was followed by monitoring the absorbance at 340 nm. In experiments investigating the eŠects on H2 O2,SOD,andcatalase,theywereaddedtothe reaction mixture before the addition of NADH. In order to investigate whether the reaction involved the production of low molecular weight compounds which were able to oxidize NADH or not, an experiment using an ultra-ˆltration mem- brane, Ultrafree-PFL (10,000-molecular-weight Fig. 1. NADH Oxidations in Various Reaction Mixtures. Symbols: , acetate 50 mM; , lactate 50 mM; $, tartrate 50 cutoŠ; Millipore), was done. The ˆrst reaction mix- mM; ,malate50mM. ture and MnP were placed in the Ultrafree-PFL (total vol. of 0.5 ml). Then the ˆrst reaction was started by the addition of NADH (ˆnal conc. of 0.17 mM). After 5 minutes of reaction, the Ultrafree-PFL was pressurized and a solution containing low molecular weight compounds but no MnP was collected in a receiver. One-tenth of a ml of the solution was then added to the second reaction mixture (0.5 ml) con- taining 0.17 mM NADH and no MnP. The absor- bance at 340 nm of the second reaction mixture was monitored. Reduction of MnP with manganese. In order to in- vestigate the MnP reduction rate with manganese, the absorbance at 406 nm, which diŠers in extinction coe‹cient between resting and oxidized MnP,8) was monitored. Puriˆed MnP in the reaction mixture in- cluding either malate or acetate was oxidized by H2O2 (ˆnal conc. of 0.1 mM) and then MnSO4 was added to the reaction mixture (ˆnal conc. of 1 mM). The absor- Fig. 2. NADH Oxidation Rates in Various Reaction Mixtures. bance at 406 nm of the reaction mixture was immedi- Symbols: , acetate 50 mM; , lactate 50 mM; $, tartrate 50 ately monitored after the addition of MnSO . mM; ,malate50mM. The decreasing rates of absorbance at 4 340 nm were calculated as the average for 0.1 min. Results trapolation to the intercept of Fig. 2, was NADH oxidation proˆle 0.18 Abs.Wmin. and maximum. The oxidation rate Figure 1 shows the courses of absorbance at gradually decreased. 340 nm, indicating a NADH concentration. In the Figure 3 shows the course of absorbance in the reaction mixture including a-hydroxy acid, i.e. lac- presence of both acetate and malate. The proˆle of tate, tartrate, or malate, the initial oxidation rate, the reaction course resembled that of malate very whichwasestimatedbyextrapolationtotheintercept closely, indicating that malate is predominant in of Fig. 2, was close to zero and the oxidation rate deciding the reaction type. increased. The oxidation rate reached maxima, 1.72, 0.35, and 0.50 Abs.Wmin., after acceleration periods EŠectsofH2O2 of 0.3, 2.7, and 2.1 minutes in the reaction mixture The eŠects of exogenous H2 O2 on NADH oxida- including lactate, tartrate, and malate, respectively. tion in the reaction mixture including malate or On the other hand, in the reaction mixture including acetate are shown in Figs. 4(a) and 4(b), respectively. acetate, this acceleration period was not observed. In the former reaction mixture, the additional H2O2 The initial oxidation rate, which was estimated by ex- increased the initial oxidation rate and reduced the NADH Oxidation by Manganese Peroxidase with or without a-Hydroxy Acid 719 Fig. 5. EŠects of SOD and Catalase on NADH Oxidation in the Reaction Mixture Including Malate (a) and Acetate (b). Symbols: , no addition; , 340 uWml of SOD (ˆnal concen- tration); #, 2600 uWml of catalase (ˆnal concentration). Fig. 3. NADH Oxidation in the Reaction Mixture Including both Malate and Acetate. Symbols: , acetate 50 mM; #, malate 50 mM; ; acetate 50 mM+malate 50 mM. Fig. 4. EŠects of Exogenous H2 O2 onNADHOxidationinthe Fig. 6. NADH Oxidation by Low Molecular Weight Compounds Reaction Mixture Including Malate (a) and Acetate (b). Obtained from the Reaction Mixture Including Malate or Symbols: , no addition; , 100 mM of H2 O2 (ˆnal concen- Acetate. tration); $,10mM; ,1mM. Symbols: , malate; , acetate; #, control. The control was done by low molecular weight compounds obtained from the reaction mixture including malate but not enzyme. acceleration period. Particularly the 100 mM H2O2 completely extinguished the acceleration period. These results suggest that the NADH oxidation in the lase, on the whole, slightly accelerated the NADH reaction mixture including malate requires H2O2, oxidation (Fig. 5(b)). These results indicate that the especially in the initiation, and the acceleration two reaction mixtures diŠer in the active oxygen spe- period is caused by an insu‹ciency of H2O2. cies involved in the reactions. On the other hand, in the latter reaction mixture, the acceleration resulting from the addition of H2O2 NADH oxidation with low molecular weight com- was not observed. Besides, the inhibition of the reac- pounds tion was observed by the 100 mM H2O2, perhaps due Figure 6 shows the courses of absorbance at to the oxidative stress of H2 O2. 340 nm of the second reaction mixtures after the addition of the low molecular weight compounds EŠects of SOD and catalase obtained in the ˆrst reaction mixtures. Only low Figure 5 shows the eŠects of SOD and catalase on molecular weight compounds, obtained from the NADH oxidation.