この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました。Oxaloacetase, which catalyzes production of oxalic acid from oxaloacetic acid, was cell-free extracted for the first time from a white-rot fungus, Coriolus versicolor. Several other wood destroying fungi were grown on glucose and their oxaloacetase activities in cell-free extracts were compared. It was shown that the amounts of oxalic acid accumulated in the culture media reached the maximal value on day 7 of the cultivation, and decreased thereafter. The enzyme activity gradually increased until day 7 and more rapidly increased thereafter until day 10

AKAMATSU, Yasumi

SHIMADA, Mikio

TAKAHASHI, Munezoh

Kyoto University Research Information Repository

Title<Original>Cell-Free Extraction of Oxaloacetase from White-Rot Fungi, Including Coriolus versicolorAuthor(s)AKAMATSU, Yasumi; TAKAHASHI, Munezoh; SHIMADA,MikioCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (1993), 79: 1-6Issue Date1993-02-26URL http://hdl.handle.net/2433/53261RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityOriginalCell-Free Extraction of Oxaloacetasefrom White-Rot Fungi, IncludingCoriolus versicolorYasumi AKAMATSU*I, Munezoh TAKAHASHI*2and Mikio SHIMADA*3(Received October 31, 1992)Abstracts--Oxaloacetase, which catalyzes production of oxalic acid from oxaloacetic acid,was cell-free extracted for the first time from a white-rot fungus, Coriolus versicolor. Several otherwood destroying fungi were grown on glucose and their oxaloacetase activities in cell-free extractswere compared. It was shown that the amounts of oxalic acid accumulated in the culture mediareached the maximal value on day 7 of the cultivation, and decreased thereafter. The enzymeactivity gradually increased until day 7 and more rapidly increased thereafter until day 10.Keywords: Oxalic acid, oxaloacetase, white rot fungi, Phanerochaete chrysosporium, Coriolus versicolor,wood decay, malate dehydrogenase.I. IntroductionElucidation of biochemical mechanisms for wood decay is ofgreat importance to protectwoods against the fungal attack. Since it was postulated that oxalic acid which is ametabolite of wood-rot fungi might be involved as an electron donor in Fenton's reactioncausing oxidative degradation of cellulose l - 3) or as a proton donor in hydrolysis of woodcarbohydrates such as cellulose and hemicellulose4--6). Thus, oxalic acid produced bybrown-rot fungi has been receiving keen. attention.Although the enzymatic decomposition of oxalic acid catalyzed by oxalatedecarboxylase7,8) and lignin peroxidase system9 ,1O) was reported, biosynthesis of oxalic acidIII wood-destroying fungi have little been investigated.Recently, Akamatsu et al. 12) reported that oxalic acid is produced from oxaloacetic acidby the cell-free extracts from the brown-rot fungus Tyromyces palustris (Berkely et Curtis)Murrill. The experimental conditions for assay of oxaloacetase activities and variousinfluencing factors on the enzyme activities have been reported 13,14).As a chain of biochemical studies on oxalate metabolism in wood-destroying fungi 15),we attempted to survey the distribution of oxaloacetase among the wood-rot fungi, because*1 Present Address; The Fukui Prefectural General Green Center Maruoka, Fukui, Japan, 910-02.*2 Laboratory of Deterioration Control.*3 Laboratory of Biochemical Control.-1-WOOD RESEARCH No. 79 (1993)the occurrence of this enzyme in white-rot fungi has not yet been reported. Here, we reportthe first example of the detection of oxaloacetase activity in the white-rot fungi such asCoriolus versicolor (Linnaeus ex Fries) Quele and Phanerochaete chrysosporium Burds.2. Materials and Methods2.1. Chemicals and reagentsAll chemicals, including oxaloacetic acid, were purchased from Nacalai Tesque andBiorad Laboratories. The commercial enzyme kit (Cat. No. 755,699) for the assay of oxalicacid was procured from Boehringer Mannheim Biochemicals.2.2 Culture conditionsThe following wood-rot fungi were grown at the temperatures shown on Fukinnbara'smedium l6) as previously reported I3); Coriolus versicolor (COV-1030) at 33°C, Phanerochaetechrysosporium (PHC BKM-1747) at 3rC, Lentinula edodes (Berkeley) Singer (L22) at 27°C,Gloeophyllum trabeum (Person ex Fries) Murrill (GLS-L4e), Serpula lacrymans (Wulfen exFries) Schroeter (SEL-8697) at 22°C, Lentinus lepideus Fries (LEL-8719) at 2rC. All fungiexcept for the fungus L22 have been maintained in this Institute, and the fungus L22 wasfrom the stock culture of Fukui Prefectural General Green Center.2.3 Assay of oxalic acidThe amounts ofoxalic acid enzymatically formed from oxaloacetic acid or accumulatedin the culture media of C. versicolor were determined by use of the enzyme kits as previouslyreported13).2.4 Enzyme preparationThe wood-rot fungi were grown for the different periods depending on their species untilthe mycelia reached a significant weight. The fungal mycelia were harvested andhomogenized as described previouslyI3). Since it was found that oxaloacetase activity wasconsiderably lost after the overnight dialysis, the cell-free extracts were used without dialysisfor the assay of the oxaloacetase activities.2.5 Assay of oxaloacetaseOxaloacetase activities were assayed by use of the commercial assay method throughoutthis experiment as previously reported 13). However the procedure was slightly modified asfollows. After the incubation of the oxaloacetase system, the mixtures were heated at 70°Cfor 10 min in order to stop the reaction as well as to inactivate malate dehydrogenase, whichsubsequently interferes the measurement of formate dehydrogenase. For this heattreatment, we also confirmed that oxalic acid is not decomposed by this heat treatment andthat oxaloacetic acid is not decomposed either to form oxalic acid.2.6 Assay of malate dehydrogenaseThe reaction mixture (3.0 ml) contained 0.1 ml of the enzyme solution, 0.05 ml of 0.6M oxaloacetate, 0.06 ml of 0.1 M NADH and 2.8 ml of 0.1 M phosphate buffer (pH 7.5).AKAMATSU et al: Cell-Free Extraction ofOxaloacetase from White-Rot FungiThe mixture was incubated at 30°C and the decrease in the absorbance at 340 nm wasfollowed by Hitachi UV-Visible spectrophotometer Model U-3210.3. Results3.1 Assay of oxaloacetase in cell-free extracts of C. versicolorWe chose C. versicolor as a model fungus for this investigation, since it has been used asthe test fugus ofJIS Oapanese Industrial Standard) for wood preservation tests. As shownin Table I, the cell-free homogenate of the mycelia of C. versicolor exhibited a considerableactivity in the complete system. The results clearly show that production of oxalic acidfrom oxaloacetic acid substrate completely depends on the presence of both the enzymepreparation and the substrate, since neither of the reaction systems lacking the enzyme orthe substrate yielded any significant amounts of oxalic acid. The heated enzyme gave only7% of the amount of oxalic acid formed in the complete system. It is noteworthy that thecrude enzyme preparation also contained strong activity of malate dehydrogenase whichinterfered the assay of formate dehydrogenase.Table 1. Occurrence of oxaloacetase and malate dehydrogenase in thecell-free extracts of C. versicolor.Reaction systemComplete systemComplete substrateComplete enzymeHeated enzyme systemc)Malate dehydrogenaseactivitya)1.41oo-0Oxaloacetaseactivityb)3.34tracetrace -0.24a) The assay conditions are described in the Material and Method.The activity are expressed the amount (,umole) of NADHconsumed/min/O.l ml of the enzyme used.b) The enzyme activity was assayed as described in the text; the valuesare the amounts ofoxalic acid (,umole) produced/hr/0.5 ml ofthe enzymeused.c) The heated enzyme was used instead of the untreated enzyme for thereaction.3.2 Occurrence of oxaloacetase in wood-destroying fungiTable 2 shows oxaloacetase activities of three white-rot fungi and three brown-rot fungi.The result shows that the homogenate of C. versicolor gave the greatest activity ofoxaloacetase and that P. chrysosporium also exhibited a significant activity of this enzyme.However, GLS, SEL, LEL, and L22 gave no significant amounts of oxaloacetase activities.3.3 Changes in oxaloacetase activities during the culture of Coriolus versicolorFig. I shows changes in oxaloacetase activities, pHs, and oxalic acid accumulatedduring the cultivation of C. versicolor. The results clearly show that activities ofoxaloacetase-3-WOOD RESEARCH No. 79 (1993)Table 2. Distribution of oxaloacetase among white-rot and brown-rot fungitested.Fungi testedWhite-rot fungiC. versicolorP. chrysosporiumL. edodesBrown.-rot fungiT. palustrish )G. tarabeumS. lacrymansL. lepideusCultureday118303113121Total actIvIty(nkat/culture)26.51.5n.da )5.5n.d.n.d.n.d.Specific activity(pkat/mg protein)603260a) n.d.; not detected.b) Data for T. palustrisH ).0c:&0~•a.C)E .i-<Il..... 1'01'0 ;:"! ::30........Cll 1'0~<Il.s1'0860 Cll<Il1'0 1'0~50 820~ 1'001540 iij)(>. 0~ 30 -0~ ~101'020 .:;(J ~;;:'g 10 !a.(/) 0 {}. 00 5Z'.~<Il5 0.5~-4 0.4~3· E0.3-"tJ2::r: 0.2·~a.1 0.1 ~)(0 0 010 15Culture da.yFig. 1. Changes in activities of oxa1oacetase during the cultivation ofCoriolus versicolor.appeared as a constitutive enzyme, which was also observed for the culture of T. palustris.The total activity/culture reached the maximum on day 11, and decreased gradualythereafter. The specific activity also increased, reaching the maximum on the same day.This means that the proportion of this enzyme in total proteins is increasing with thecultivation period. It is noteworthy, however, that the moderate increase in the totalactivity found until day 8 and more intensively thereafter until day 11 correlated with theincrease in amounts of oxalic acid produced, reaching the maximum (6 mM) on day 8.- 4AKAMATSU et at: Cell-Free Extraction ofOxaloacetase from White-Rot FungiThe reason for decrease in the amounts of oxalic acid accumulated may be due to activationof oxalate decarboxylase and LiP systems.It is interesting to note, however, that significant amounts of oxalic acid wasaccumulated in the culture of C. versicolor in contrast to the report that oxalic acid is notaccumulated in the culture medial}.4. DiscussionOxaloacetic acid is a very important member of organic acids involved in TCA cyclewhich functions as the energy-producing device accompanied by production of CO2 and alsoas the biochemical device to supply several aliphatic amino acids through the enzymatictransaminations of the keto-acids.This investigation has shown that oxaloacetate, which is a precursor of aspartic acid, isalternatively converted to oxalate in white-rot fungi and consequently oxalate is decomposedto CO2 by either the system of oxalate decarboxylase/formate dehydrogenase or the systemof LiP/veratryl alcohollH20 29-11). Now that oxaloacetase was found to occur in white-rotfungi, this enzyme is commonly important for oxalate metabolism among the major threegroups of wood-destroying fungi such as white-rot, brown-rot and soft-rot fungi 17).Although accumulation of oxalic acid might be very important for degradation of woodcarbohydrates in brown-rot decay1,2), the role of oxalate metabolism in white-rot decay isnot clear yet. We suspect one of the biochemical advantages of formation anddecomposition of oxalate for white-rot fungi is that surplus organic acid metabolites fromglucose are alternatively decomosed to CO2 via the oxalate pathway, which may function asa regulatory valve for controlling the metabolic pool sizes of the overflooded organic acidswhose metabolisms are beyond capacity of the TCA cycle in the manner similar to aceticAcetate--GlucoseHCOOH- CO2~d)(b) (c)Oxalate --- 2C02TCAe.ele J-CitrateIsocitrate /'OxoglutarateC02~~~Imoro.e ..010,\Succinate OxaloacetateC~Fig. 2. A metabolic linkage of oxalate pathway with TCA cycle.The reaction steps (a), (b), (c) and (d) are catalyzed byoxaloacetase, oxalate decarboxylase, LiP/veratryl alcohol!H 20 2 system, and formate dehydrogenase, respectively.-5-WOOD RESEARCH No. 79 (1993)acid accumulation by Escherichia coli 18). Thus, as shown in Fig. 2, oxalate pathway mayplaya role in maintaining rather low pool sizes oforganic acids to keep the operation ofTeAcycle normal even under the ammonium nitrogen limited condition in wood decay process.On the other hand, probably, under the ammonium-nitrogen-rich condition organic acidpool sizes are reduced since these acids flow into the amino acid pools required for proteinsynthesis. However, further research is required to explain the proposed role of the oxalatepathway in white-rot fungi.References1) G. HALLIWELL: Biochem. j., 95, 35-40 (1965).2) ].W. KOENIG: Wood and Fiber, 6, 66-80 (1974).3) C.J. SCHMIDT, B.K. WHITTEN and D.D NICHOLAS: Am. Wood Preserv. Assoc., 77, 157-164 (1981).4) M. SHIMADA, Y. AKAMATSU, A. OHTA and M. TAKAHASHI: Intern. Res. Gr. Wood Preser., Document, No.IRG/WP/1472 (1991).5) F. GREEN, M.J. LARSEN, ].E. WINANDRY and T.L. Highley: Material und Organismen, 26, 191-213 (1991).6) ]. BECH-ANDERSON: Intern. Res. Group Wood Preserv., Document, No. IRG/WP/1130 (1987).7) H. SHIMAZONO: Biochem. j., 42, 321-340 (1955).8) H. SHIMAZONO and O. HAYAISHI: j. BioI. Chem., 227, 151-159 (1957).9) Y. AKAMASTU, D.B. MA, T. HIGUCHI and M. SHIMADA: FEBS Lett., 269, 261-263 (1990).10) ].L. POPP, B. KALYANARAMAN and T.K. KIRK: Biochemstry, 29, 10475-10480 (1990).11) D.B. MA, T. HATTORI, Y. AKAMATSU, M. ADACHI and M. SHIMADA: Biosci. Biotech. Biochem., 56(9),1378-1381 (1992).12) Y. AKAMATSU, A. OHTA, M. TAKAHASHI and M. SHIMADA: Mokuzai Gakkaishi, 37, 575-577 (1991).13) Y. AKAMATSU, M. TAKAHASHI and M. SHIMADA: ibid., 38,495-500 (1992).14) Y. AKAMATSU, M. TAKAHASHI and M. SHIMADA: ibid., (1993, in press).15) M. SHIMADA, Y. AKAMATSU, D.B. MA and M. TAKAHASHI: In "Biotech. in Pulp and Paper Industry"(Eds. M. KUWAHARA and M. SHIMADA) Uni-Publishers, pp. 273-278 (1992).17) H. LENZ, P. WUNDERWALD and H. EGGERER: Eur. j. Biochem., 65, 225-236 (1976).18) K. HAN, H. C. LIM and ]. HONG: Biotechnol. Bioengineer., 30, 663-671 (1992).-6-

<Original>Cell-Free Extraction of Oxaloacetase from White-Rot Fungi, Including Coriolus versicolor

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<Original>Cell-Free Extraction of Oxaloacetase from White-Rot Fungi, Including Coriolus versicolor

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