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HATTORI, Takefumi

HAYASHI, Nobuya

SHIMADA, Mikio

TOKIMATSU, Toshiaki

Kyoto University Research Information Repository

Title<Preliminary>An Enzymatic Study of an Oxalate ProducingSystem in Relation to the Glyoxylate Cycle in White-rotFungus Phanerochaete chrysosporiumAuthor(s)HAYASHI, Nobuya; TOKIMATSU, Toshiaki; HATTORI,Takefumi; SHIMADA, MikioCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (2000), 87: 15-16Issue Date2000-09-30URL http://hdl.handle.net/2433/53158RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminaryAn Enzymatic Study of an Oxalate Producing Systemin -Relation to the Glyoxylate Cycle in White-rot FungusPhanerochaete chrysosporium*lNobuya HAYASHI*2, Toshiaki TOKIMATSU*2, Takefumi HATTORI*2and Mikio SHIMADA*2(Received May 31, 2000)Keywords: Oxalate, Glyoxylate dehydrogenase, Glyoxylate cycle, Phanerochaete chrysosporium, White-rot fungiIntroduction assayed spectrophotometrically.It has been well established that oxalate produced fromwhite-rot and brown-rot fungi plays several importantroles in wood decay processes!). Furthermore, it is acommonly known physiological trait that most brown-rotbasidiomycetes, including Fomitopsis (formerly calledTyromyces) palustris, accumulate oxalate in culture med~a,whereas white-rot ones do not. However, the enzymaticstudy of an oxalate producing system in white-rot fungi hasnot been fully studied, although glyoxylate oxidase fromCoriolus versicolor has been reported2). Thus, the purposeof this study was to investigate the changes in the activitiesof glyoxylate dehydrogenase (GDH), formate dehydro-genase (FDH), and glyoxylate cycle enzymes such asisocitrate lyase (ICL) and malate synthase (MS) in awhite-rot fungus Phanerochaete chrysosporium, as glyoxylatecycle has been reported to distribute among many woodrotting basidiomycetes3 ,4).Materials and MethodsPhanerochaete chrysosporium (ATCC 24725) was grownstatically in the Kirk's basal medium5 ) containing Glucoseas a sole carbon source and ammonium tartrate as anitrogen source. The crude enzymes including glyoxylate;dehydrogenase (GDH)6), formate dehydrogenase (FDH) 7),isocitrate lyase (ICL)8), malate synthase (MS)9) wereResults and DiscussionTotal activity ofGDH obtained at 33°C (lOS pkatlflask)was 3 times that obtained at 3SoC. Mycelia grew well at33°C than 3SoC, whereas the FDH activities at 33°C and3SoC were almost equal (500 p~at/ftask). The specificactivity of GDH in low nitrogen culture (LN, 2.4 mM ofNH4 +) was 17 pkat/mg protein, which was twice thatfound in high nitrogen culture (HN, 24 mM). However,almost equal total GDH activities (92 pkatlftask) wereobserved in the both cultures. Addition of Tween SO(I %, w/v) resulted in a 2-fold increase in specific activity ofGDH (33 pkat/mg protein). Thus, it was found that thehighest GDH activity was obtained form the culture with2.4 mM ammonium ion at 33°C in the presence of TweenSO.During the cultivation of the fungus, the accumulation ofoxalate reached the maximum level on day 4, butdecreased thereafter (Fig. I (A)). The highest GDHactivity was obtained on day 2, whereas the glyoxylatecycle enzymes, ICL and MS, showed the maximumactivity on day 4 and 2, respectively (Fig. I (B)). Thus,taking into consideration of the results, the reason whyhighest amounts of oxalate accumulated on day 4 is notclear but probably because large amounts of glyoxylatecould be supplied by ICL. The results also suggest thatis 1.5 it;:: t;::~ ~"" ""S2 1 S~ ~] ~Bi 0.5CZl....J:E S2____ MS........... ICL(A)i6~.Iod~4~§82I:500 13oiO.03~~ 4)..:..: ~20E:O.02 ]~ 'E10~O.Ol .§~~ "S.iIIII:::"_~;;"'''''''_...L._''F-''''''_~''''' 0 0 J O..'""-...-...L...._...L...._..&.-_.L-_..L..-_L-.I2 3 4 5 6 7 ...... 012 S 45670Incubation time (days) Incubation time (days)Fig. (A), (B). Changes in the accumulation of oxalate and activities of GDH, FDH, MS, and ICL duringcultivation of P. chrysosporium.*1 A part of this work was presented at the 49th Annual meetings of the Japan Wood Research Society in Tokyo, April, 1999.*2 Laboratory of Biochemical Control, Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan.-15-WOOD RESEARCH No. 87 (2000)Acetyl CoA~C;t,ate --lsocitrate ~':( Glyoxylate ~SUCCinate~~oxaloacetate (4) Oxalateoxaloace~tate cycle ,(~) f2)Glyoxylate (1) ~ Oxalate ~ Formate~Carbondioxide~ (2) (3)Malate- (6). '\Acetyl CoAFig. 2. Glyoxylate cycle and oxalate metabolism in white-rot fungo (I) Glyoxylate dehydrogenase,(2) Oxylate decarboxylase, (3) Formate dehydrogenase, (4) Oxaloacetase, (5) Isocitrate lyase,(6) Malate synthase.besides lignin peroxidase/veratryl alcohol and manganeseperoxidase/Mn(II)-mediated degradation of oxalate asreported 10;11), FDH together with oxalate decarboxylasemight also be involved in the complete decomposition ofoxalate. Because FDH activity increased around day 4 today 5 while the amounts of accumulated oxalate decreased.However, oxalate decarboxylase was not assayed at thistime. Alternatively, another enzyme, oxaloacetase mightbe involved' in . the production of oxalate, althoughoxaloacetase activity could not be detected from thisfungus.In conclusion, the glyoxylate cycle enzymes such a,s ICLand'MS, and GDH and FDH are suggested to beinterlinked with each other and function cooperatively inthe production and decomposition' of oxalate in the cultureof P. chrysosporium, as shown in Fig. 2. Further research isneeded' to elucidate the pathway for the production ofoxalate. .ReferencesI) M. SHIMADA et al.: J. Biotechnol., 53, 103-n3 (1997).2) K. MIl et al.: Wood Research, No. 83, 23-26 (1996).3) E. MUNIR et al. : Abstracts ofthe 50th annual meeting ofthejapan Wood Research Society, p.456 (2000).4) E. MUNIR et al. : Abstracts of the 50th annual meeting of thejapan Wood Research Society, p.503 (2000).5) T.K. KIRK et al. : Arch. Microbiol., 117, 277-285 (1998).6) T. TOKIMATSU et al.: FEBS Lett., 437, 117-121 (1998).7) T. OHYAMA and I. YAMASAKI: j. Biochem., 75, 1257-1263(1974).8) O.P. MAcHoTRA: Arch. Biochem. Biophys., 214, 164-171(1982) ..9) j.R. COOK: J. Protoz:,ool., 17, 232-235 (1970).10) Y. AKAMATSU et al.: FEBS Lett., 269,261~263 (1990).11) A. KHINDARIA and S. AusT: Environ. Sci. Technol., 29,719-725 (1995).- 16-

<Preliminary>An Enzymatic Study of an Oxalate Producing System in Relation to the Glyoxylate Cycle in White-rot Fungus Phanerochaete chrysosporium

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<Preliminary>An Enzymatic Study of an Oxalate Producing System in Relation to the Glyoxylate Cycle in White-rot Fungus Phanerochaete chrysosporium

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