The Lignin-Degrading Fungus, Phanerochaete Spectrophotometrically As the Decrease in Absorbance at Chrysosporium, Was Shown to Be Unique in 340Nm

Home , Lignin peroxidase, NADH peroxidase

Agric. Biol Chern., 50 (2), 525-529, 1986 525

Rapid Paper other than lignin-peroxidase is thought to be to supply H2O2to lignin-peroxidase by oxi- Oxidation of NADHby a Peroxidase dizing NADH. This report deals with the of a Lignin-degrading purification of a peroxidase (NADH-per- Basidiomycete, oxidase) which catalyzes the oxidation of NADHfrom a culture of P. chrysosporium, Phanerochaete chrysosporium, and also the involvement of this enzyme in and Its Involvement in the degradation of a lignin model dimer by the Degradation of lignin-peroxidase. a Lignin Model Compound MATERIALS AND METHODS Yasuhiko Asada, Miwako Miyabe, Makoto Kikkawa Culture of P. chrysosporium and purification of the and Masaaki Kuwahara enzyme. P. chrysosporium ME446 was cultured in 100ml of a mediumcontaining 1%glucose, 1.2him ammonium Department of Food Science, Faculty of Agriculture, tartrate (low nitrogen medium), 20mM Na-2,2- KagawaUniversity, Miki-cho, dimethylsuccinate buffer (pH 4.5) and salts as described by Kagawa 761-07, Japan Kirk et al.9) in a 500-ml Erlenmeyer flask. Two grams of polyurethane foam cubes of 5-mmside length was added Received September 19, 1985 to the flask to support the growth of the fungus. A suspension ofconidia was inoculated into the mediumand the culture was carried out at 37°C. The cultures were A lignin-degrading Basidiomycete, Phanerochaete purged with 100% O2 every 3 days. After 7 days, the chrysosporium, produced peroxidases other than lignin- cultures were filtrated through Miracloth (Calbiochem- peroxidase in a low nitrogen culture. One of the per- Behring, La Jolla), and then the filtrate was centrifuged at oxidases was purified by DEAE-Sepharose column chro- 9000xg for 30min to remove mycelia and conidia. The matography. The enzyme (NADH-peroxidase) oxidized filtrate (1 to 2 liters) obtained was concentrated with a NADHbesides phenol red and other substrates for per- Diaflo ultra filtration membrane YM10 (Amicon, Danvers) to 10 to 20ml. The concentrate was applied to a oxidases. The product on NADHoxidation by the enzyme column (1.0x2.7cm) of DEAE-Sepharose CL-6B was H2O2. The reaction was stimulated by Mn2+and inhibited by heme protein inhibitors. A diarylpropane (Pharmacia Fine Chemicals, Uppsala) and proteins were compound, a lignin model, was degraded in the presence eluted from the column with 0.02m succinate buffer (pH of both the NADH-and lignin-peroxidase with NADH. 5.5) containing a stepwide gradient of0 to 0.2m NaCl. The This reaction showed that NADH-peroxidase played a elution of enzymeswas monitored spectrophotometrically role as a H2O2donor in the lignin-peroxidase reaction. at 280and 407nm. Enzymeassay. NADHoxidation activity was assayed The lignin-degrading fungus, Phanerochaete spectrophotometrically as the decrease in absorbance at chrysosporium, was shown to be unique in 340nm. The reaction mixture (l ml) contained 0.3him secreting NAD+and NADP+, and their re- NADH,0.2mM MnSO4, 50mMsuccinate buffer (pH 4.5) duced forms,1} and the enzyme preparation and enzyme. Although the activity was maximal at pH 3.0 obtained from this fungus oxidized reduced to 4.5, the reaction was conducted routinely at pH 4.5 to minimize the non-enzymatic degradation of NADH.The pyridine nucleotides.2) This fungus was shown reaction was also followed by using an oxygen electrode to produce extracellular peroxidases3A) includ- (Sensonix, Tokyo). Peroxidase activity was assayed using ing the enzyme, temporarily named lignin- phenol red as a substrate as described previoyusly3) with a peroxidase or ligninase, which catalyzes the modification. The reaction mixture (l ml) contained oxidative degradation of the Coc-Cfi propyl 0.27mM phenol red, 0.1 mMH2O2, 0.1 mMMnSO4, 20mM succinate buffer (pH 4.5) and enzyme. The reaction was side chain of lignin and lignin model com- stopped by the addition of 40fil of 2m NaOHand the pounds in the presence of H2O23'5~8) One of absorbance was read at 610nm. The activity of the lignin- the possible physiological roles of peroxidases peroxidase was assayed after oxidation of veratryl alcohol Abbreviation: DAP, l (3 /,4'-dimethoxyphenyl)- l ,3-dihydroxy(4//-methoxyphenyl)propane. 526 Y. Asada et al. to veratryl aldehyde according to the methodof Tien and RESULTS Kirk6) with a slight modification. The reaction mixture (1 ml) contained 0.4mM veratryl alcohol, 0.27mM H2O2, As shown in Fig. 1, DEAE-Sepharose col- 100mM tartrate buffer (pH 3.0) and enzyme. The increase in absorbance at 310nmdue to the formation of veratryl umnchromatography gave three enzyme frac- aldehyde was measured. The formation of veratryl al- tions, Fr-I (NADH-peroxidase), Fr-II and Fr- dehyde on the degradation of a diarylpropane model III (lignin-peroxidase), which oxidized NADH (DAP, 1 1 -(3 '^'-dimethoxyphenyl)- 1 ,3-dihydroxy(4/ '-meth- and phenol red. The activities were only de- oxyphenyl)propane), was assayed on the basis of the tected in fractions which showed absorbance absorbance at 310nm. The reaction mixture (l ml) contained l mMDAP, 0.27mM H2O2, 100mMtartrate buffer at 407nm, suggesting that the proteins con- (pH 3.0) and enzyme. For the coupled degradation of cerned were hemeproteins. The first enzyme DAP by peroxidases, the reaction mixture (0.5ml) con- fraction (NADH-peroxidase), eluted with tained l mMDAP, 0.6mM NADH, 50mMtartrate buffer 0.05m NaCl, showed the highest activity to- (pH 3.0) and enzyme (12.5 fig of NADH-peroxidase and ward both NADHand phenol red. The highest 10/ig of ligninperoxidase). The reaction was followed on peak fraction (fraction number 25) was dia- the basis of the absorbance at 310nm. The reaction products were also detected on a thin layer of Kieselgel 60 lyzed against 0.02m succinate buffer (pH 5.5) F254 (Merck, Darmstadt) with dichloromethane-methanol and used for further characterization of the (20 : 1, v/v) as a solvent system. Protein concentration was enzyme. This fraction gave a single band on determined by the method of Lowry et al.10) with bovine polyacrylamide gel electrophoresis, and the serum albumin (Sigma, St Louis) as a standard protein. molecular weight was found to be around 46,000. The third enzyme fraction was lignin- Chemicals and enzymes. The DAPmodel compound was synthesized by Dr. A. Enoki, Kinki University. Unless peroxidase.3) Although this enzyme fraction otherwise mentioned, all chemicals were of reagent grade. showed the ability to oxidize veratryl alcohol Bovine liver catalase and bovine erythrocyte superoxide in the presence ofH2O2, the specific activity of dismutase were the products of Sigma. oxidation ofNADHand phenol red was less in the case of NADH-peroxidase. The lignin-

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10 20 30 A0 50 60 ° Fraction number (2ml/fraction) Fig. 1. Chromatography of the Peroxidases on DEAE-Sepharose. The concentrated filtrate (20 ml) was applied to a column and the column was eluted with 0.02 mNa-succinate buffer (pH 5.5) containing a stepwise gradient ofNaCl; I, enzyme; II, without NaCl; III, 0.05 m NaCl; VI, 0.1 m NaCl; V, 0.15m NaCl; VI, 0.2m NaCl. Absorbance at 280 ( ) and 407nm (-#-); NADHoxidation measured at 340 nm (-O-); phenol red oxidation measured at 610 nm (-A-); veratryl alcohol oxidation measured at 310nm (-D-). Peroxidase of a Lignin-degrading Fungus 527 peroxidase fraction was concentrated with an ultra filtration membraneand then used for the coupling and other reactions. The properties of NADH-peroxidasewere studied. Its pH optimum was 3.0 to 4.5. On Lineveaver-Burk plotting, the Km value for NADHwas calculated to be 0.25him. The effects of various compounds on NADHoxi- dation were examined. Addition of a large reactionamount (1500completely.units) of Thecatalaseinhibitoryinhibited effectthe (81 % inhibition) of superoxide dismutase (650 units) is assumed to be due to dismutation of 25nmoles the superoxide radicals for the NADHoxida- tion. Inhibitors of heme proteins, azide, cya- Catalase nide and thiourea, caused 86, 73 and 57% inhibition, respectively. Amongthe metal ions 0 A 8 3.4-fold,tested, Mn2+suggesting(0.1 him) thatstimulatedthis enzymethe reactionwas a Mn-dependent peroxidase, as shown pre- Time (min) viously.^ The enzyme also oxidized guaiacol, Fig. 2. NADHOxidation Activity of the Fr-I (NADH) syringaldazine and other conventional sub- Peroxidase. strates for peroxidases. Figure 2 shows that this enzyme consumed The reaction was initiated by the addition of NADH. oxygen in the reaction mixture containing Catalase (1000units) was added at the arrow. No oxygen NADH.The addition of catalase to the mix- consumption was observed in the absence of the enzyme ofturetheledoxygento theconsumed.recovery Thisof approximatelyshows that this50% peroxidase produces H2O2 by oxidizing NADH as in the case of horseradish peroxidase.ll ~13) The formation of H2O2 by the NADH-peroxidase is thought to provide H2O2 for the ligninolytic reaction system catalyzed by lignin-peroxidase. Figure 3 shows time course of the degradation of DAPby enzyme veratrylpreparations,aldehydedeterminedproduced bythroughmeasurementCa-C/?of cleavage of the model. Only lignin-peroxidase degraded the model in the presence of H2O2 readily. In the coupling reaction system which contained both the NADH- and lignin- gradedperoxidasesin theandabsenceNADH,DAPof wasaddedalsoH2O2.de- Formation of veratryl aldehyde was also detected on thin layer chromatography. This indicates that the NADH-peroxidaseproduces i-.»-å »... å H2O2through oxidation of NADH,and the 1 2 3 lignin-peroxidase uses the generated H2O2to Time (h) Fig. 3. Degradation of a Diarylpropane (DAP) Model Compoundthrough a Coupled Reaction Involving the Fr- I (NADH) and Fr-III (lignin) Peroxidases. The reaction systems are described in the text; -#-, Fr-I and Fr-III peroxidases+DAP+NADH; ~O-, Fr-I and Fr-III peroxidases+NADH; -A-, Fr-I and Fr-III peroxidases+DAP; -A-, Fr-III peroxidase+DAP+ NADH; -O-, Fr-III peroxidase+DAP+H2O2

degrade DAP. Although the lignin-peroxidase oxidized NADH,the specific activity was low (Fig. 2), degradation of DAPin the presence and absence of NADH and H2O2, respectively, was not detected under the conditions used in this experiment. However, in the re- 528 Y. Asada et al.

O2 NADH-peroxidase According to Forney et al.,14) H2O2 pro- v (FrI peroxidase) NADH- ^^ -NAD* duction activity in a cell extract of P. chryso- HjO2 sporium was observed to coincide with the CH2OH I CH2OH appearance of ligninolytic activity. They sug- HC-^-OCH3 } ^ X +H9°H gested that the production of H2O2 was at- HCOH Lignin-peroxidase ^^OCH3 ^J tributable to the glucose oxidase activity of ^Si (FrIII peroxidase) OCH3 T^H this fungus.14) Although they demonstrated T OCH3 3 OCH3 n that H2O2production and catalase were local- I ized in the periplasmic structure of this fun- Fig. 4. A Possible Coupling Reaction Involving the gus,15) no evidence has been obtained show- Peroxidases for the Degradation of a Diarylpropane ing the presence of a peroxidase in this sub- Model Compound (DAP). cellular structure. I, DAP; II, veratryl aldehyde. Involvement of NADHin the degradation of a lignin model compoundwas first reported action stystem to whichan excess amountof by Fukuzumi et al.16) They reported that a the enzyme (60fig or more) had been added, partially purified enzyme preparation from veratryl aldehyde was detected on thin layer Poria subasida degraded the arylalkylether chromatography after 3 hr reaction. The pos- bond in veratrylglycerol-j8-guaiacylether in the sible coupling reaction catalyzed by the two presence of both NADHand molecular oxy- peroxidases is illustrated in Fig. 4. gen with concomitant demethylation of the p- methoxy group of the veratryl moiety of this DISCUSSION model dimer. The mode of involvement of NADHin this unique reaction is obviously Lignin-peroxidase has been shown to de- different from that reported by us. grade lignin and lignin model compounds in the presence of H2O2.3'5~8) The question of Acknowledgment. We wish to thank Dr. A. Enoki, how H2O2 is provided for the ligninolytic Kinki University, Osaka, Japan, for providing the DAP model compound. Wealso thank Professor M. H. Gold, system has been raised. In this study, we found Oregon Graduate Center, Beaverton, Oregon, U.S.A, for that a peroxidase (NADH-peroxidase) plays a his useful suggestion. This study was supported in part by role as a H2O2donor in vitro. Formation of a Grant-in-Aid for Energy Research from the Ministry of H2O2 at the expense ofNADH (NADPH) has Education, Science and Culture of Japan. been demonstrated for horseradish peroxidase.11~13) This reaction was found to re- REFERENCES quire Mn2+and to be stimulated by various 1) M. Kuwahara, Y. Ishida, Y. Miyagawa and C. monophenols. The mechanism of NADH Kawakami, J. Ferment. TechnoL, 62, 237 (1984). oxidation has been well discussed by Gross et 2) M. Kuwahara and Y. Asada, "Recent Advances in ail2) and Halliwell.13) According to them, Lignin Biodegradation Research," ed. by T. Higuchi, NADHis oxidized through two different mech- H-m. Chang and T. K. Kirk, Uni Publisher, Tokyo, anisms; one involves Mn2+ and the super- 1983, p. 164. 3) M. Kuwahara, J. K. Glenn, M. A. Morgan and M. oxide radical, O2~, and the other depends on H. Gold, FEBS Lett., 169, 247 (1984). the presence of free O2~ and probably an 4) V-B. Huynh and R. L. Crawford, FEMS Lett., 28, enzyme-NADHcomplex. In either reaction, 119 (1985). oxidation of NADHgives equimolar H2O2. 5) M. Tien and T. K. Kirk, Science, 111, 661 (1983). 6) M. Tein and T. K. Kirk, Proc. Natl. Acad. Sci. The NADH-peroxidase of P. chrysosporium is U.S.A., 81, 2280 (1984). thought to have similar characteristics to the 7) M. H. Gold, M. Kuwahara, A. A. Chiu and J. K. horseradish enzyme, and to supply H2O2to Glenn, Arch. Biochem. Biophys., 234, 353 (1984). the lignin-peroxidase by oxidizing NADH 8) P. J. Kersten, M. Tein, B. Kalyanaraman and T. K. which is secreted in the culture medium.1 2) Kirk, J. Biol. Chem., 260, 2609 (1985). Peroxidase of a Lignin-degrading Fungus 529 9) T. K. Kirk, E. Schultz, W. J. Connors, L. F. Lorenz and J. G. Zeikus, Arch. Microbiol, 117, 227 (1978). 13) B. Halliwell, Planta, 140, 81 (1978). 10) O. H. Lowry,N.J. Rosebrough,A. L. Farrand R. J. 14) L. J. Forney, C. A. Reddy, M. Tien and S. D. Aust, Randall, J. Biol. Chem., 193, 265 (1951). J. Biol. Chem., 257, 11455 (1982). ll) E. F. Elstner and A. Heupel, Planta, 130, 175 (1976). 15) L. J. Forney, C. A. Reddyand H. S. Pankratz, Appl. 12) G. G. Gross, C. Janse and E. F. Elstner, Planta, 136, 271 (1977). Environ. Microbiol., 44, 732 (1982). 16) T. Fukuzumi, H. Takatuka and K. Minami, Arch. Biochem. Biophys., 129, 396 (1969).