Studies on pyruvate and carbohydrate metabolisms by lactic acid bacteria. IV. Analysis of dismutation by L. fermentum-香川大学学術情報リポジトリ

Loading.... (view fulltext now)

Loading....

Loading....

Loading....

Loading....

全文

(1)

Vol.8,No.2(1957) 221

STUDIES ON/PYRUVATE AND CARBOHYDRATE

METABOuSMS BY LACTIC ACID BACTERIA

ⅠⅤ.Analysis of Dismutation by L.fermentum

KeiYAMANAXA(LaboIatOryOfTechnicalMicrobiology)

(ReceivedJanuaIy8,1957)

TheapplicabilitytobacteriaoftheEMBDEN−MEYERHOF(EMl)schemeofcarbohydratedi早Simi1ation has beeninvestigated by many woIkeIS.,Since WARBURG(1)in1931demonstIated the occurrence of so−

Ca11ed“ゐi5‘henf‘r7rCnt,”namely glucose・6・Phosphate(G−6−P)dehydrogenasein yeast,itsimportancein

aerdbic dissimjlation of carbohydrate has beenincIeaSed greatly‖ Asit was elucidatedthat a number・Of bacteriahadtheabilitytooxidizeG−6−P・SOthenewschemeRforhexoseoxidationwere 且MscIleme

For example,glucose fermentation bylactic acid bacteria occurs by atleast two di茸erent routes Homolactic species,the streptococciand homolactobaci11i,COnVert hexose tolactate by the EM.pathwaY Incontrast,fdrmentationofhexosebyheterolacticoIganisms(LeuconoSiocandLaciobacilli)ischaracteTizeすby

theequimolaIfoImationoflactate,ethanolandcaIbondioxideqUNSALUSandG‡甲S(1952)(ヱ,予〉obtainedlthe

result ofisotopic analysis with C141abe11ed gh!COSe by Leuc,n2eSenterOides,by Z.eucde.Airanicum,Lfern;enii,

andエbentoateti(uSく小… Fe‡mentation ofglucose・・lHClよand glucose・3,4−C14reVealed thatcarbonユ.ofglucose

appears as carbon dioxide,and caIbon2as methylgIOup Of ethanol.Fuzthermore,it was free from al・

dolase(盲)butcontairledG−6−Pdehydrogenase(O・7)and6−phosphogluconate(6−PG)dehydrogenase(8)in Leuc

me・SenterOiders,From these results,itis proposed that glucose may be decomposed via so:calledanae’zdbic hexosc monophosphate pathway,Although the demonstration of enzymes and unknowninteImediates be・

longio such oxidative scheme by het畠主olactic acid bacteIia aIe aS yetincomplete

A new dismutation of gluconate(glucosc)and pyruvate by Lh fernlentum has been presentedin a

previous paper(9).In suchexpeIimcnts,metabo王ic gas changewas measuredwithaWARBURGmanOmeter,

While analyses of aliquoteⅥere Carriedoutinothermacroexperiments thusitwasdifEculttocomparethese

data fo工 di#erent manner

In this paper,gaSeOuS metabolism measu;ed with a WARBURG manOmeter andanalyzed metabolites inthis vesselof WARBURGin orde工tOde丘nefurthe:eXaCtly,and seveIalconditionsof colorimetricdeterm・

inations for pyruvate,1actate,diacetyl(DAC)and acetylmethylcarbinol(AMC)were descIibed.

二眼a乞erial$and恥乞et王10ds

伽血re L”feYmenium,ahetero・・iermente:,hasbeenusedthroughouttheexperimentslTheorgan子$P,

grownin3L of malt extract(4∼6%as sugar content)with4%of CaCO3.for40 hours at370C,WaS harvested by centrifuge,After washing the bacteria cells by distilled water for twice,thewetcellpe11ets

WeIe dIied over CaC12undeIreduced pressure

REanome七ric procedtlreS Manometric experiments were carried out by・COrlVentional− WARBURG

technique,aS describedin a previo11S paper(10)・0Ⅹygen uPtake and CO2eVOlution weremeasuredat3フOC

acco工dirlg tO the procedures descIibedbyUMBREIT,BuRRISandSTAUFFER(11).The reactionmiⅩtuIe WaS

incubated for600r9C)min,at Which period reaction was stopped by tipping ofO2mlof20 % trichlor・ acetic acid(TCA)fIOm2zld side arm,and then the amount ofliberatedCO2WaSreadh NoCO2retention WaS Observedin endogeneous andin the case of sugar added onlyl ReactionmiⅩtureS WereremOVedand

(2)

TecIlり Bl軋Fac.AgI・1.XagawalJniv.

222

anddeproteinized bycetrifugation”Aliquots wereassayed as describedbelowトRecoveIies ofmetabolites fromvesselofWARBURG,Were90and96.9∼98.3%withoutandwith washing three times by5%TCA,

respectively

Determinations For the deteImination of pyIuVate,the method of FRIEDEMANN and HAUGEN(12) wasused.Standard ofpymvatewasestimatedbythemethodofCLIFT−CooK(13)… HydIaZOneOfpyruvate formedwasextracted by8mlofxyleneinstead ofbytoluene,COloured with NaOH,5miofl5Nre・ placed by2mlof4N DAC and AMCwereidentified both by VoG‡S・PROSKAUER reaCtion(14)and by

iormationofnickel・dimethylglyoxime,deteIminedbythemethodasdescribedby WHITE and KRAMP‡IZ(Z5), and that of EGGLETON et alh(1(;)。The foImeImethod wasless sensitive than thelatter。Forstandard of

these methods,DAC from WakoCo.WaS disti11ed and estimated by the method of VAN NIEL(17)Distil・

1ate from one or twomiof aliquot was collectedin 負ve to ten ml,then one or two mlof this distillate WaSuSedfor determination小 AMCwasdetermined as DAC,distiユ】ed by oxidationwith additionof20mlof 20%FeCl3SOlution,amOuntS OfAMCvascalculated fromtotalamounts of DACandamounts of freeDAC

Res111tsfor recoveIy OfDACfor distillationwas sho・ Wnin Tablel.

Tablel Recovery of diacetylby distillation

lfDACfound慧㌫ど

dnt (鵬) (min) (ml) (%) 4 10 15−ユ6 500 12

I)AC was used245.2FLgin each cases・

DAC waseasily recoveredby distillation for10min completely.For thesetwomethods,pyruVatedid notinterferewithin2000pg,andglucose also did nota#ectwithin2500pg,eVen When added to each DACsolutionafterdisti11ation.ForVoGES・PROSXAUER(V.P”)reaction,aliquotswereusedwithoutTCA

Or With10%metaphosphoric acid,While this reaction wasinterfeIedby TCA to form a blue colour.For the methodofEGGLETONetal.,eだectof time forcoIouring,WaSteStedunder20∼25OC,The same colo11r densities we王e Obtainedbetween 10∼20minafter alkalineα・naphthoIsolution was added.Lactate was determinedbythe method modiRed by BARKER and SuMMER・ SON(ユ8〉.In this method,Visualization was performed by Oい1mi of p・hydroxydiphenyl and0.,05miof4%CuSO4・The opticaldensi− ties obtainedまⅠOm125JLg OflactateandOnO5 mlof4%CuSO4,With vaIied amo11ntS Ofp・ hydIOXydiphenyl:0.3フ5byO.05mlof♪HDP, 0.41by O.lmland OA(15by O.15 ml,and COlouring time at300C was25 min.This time was not eぽectedat20∼40min whereby Opticaldensities obtained O0フ3 at O min. 0り434at15min,0。439at2(〕min,043フat30 mi1】,and O.{436at40min.However,irlthe presence of pyruvate,reprOducible res111ts COuld not beachievedwiththisoriginaltech・ nlque,eSpeCiallyin thecaseofsmallamount Oflactic acid withlarge amount of pyruvic acid・・Thenseveralmodi負cationsofthispro・ Table 2 DAC determination as a function of time

Time(min) 5 10 20 30 40 60

Optical density “ 0 2 ︻h︶l C︺3 00 02 フl OC3 0〇 52 72 ︵03 00 1 82 0C3 〇 鵬20 3 0 AB

By the method of EGGLETON eial

A,S530withIto electrophqtometer,DAC25.8JLg B,at530mp,withShimadzuspectrophotometer,mOdel

QB50,DAC14.O pg

Table3 The eぽect of successive coppeI・lime treat・ ments on the colour obtained with pyruVic acid samples

0√DけS70m〃・

acid (1)After (ⅠⅠ)After (IlI)After

(pg/鮎st Without one copper・tWO COpper−threecopper・

Smi) treatmentlime ljme lime

treatment tIeatment treatment

20 0.09フ 0.036

5U O.215 0..036 0023 0.018 1∞ 0..440 0.061 0.022 0.018 200 0.890 0い064 0.035 0、027 400 0い○フ4 0.038 0.032

(Ⅰ)4“フmlof standard solution(COntaining20up to4(刀 pg of pyIuVic acid)+0”3miof20%CuSO4+Ca(OH)2‖(ⅠⅠ) 3mi of supernatant sollユtion from(Ⅰ)+0・3 mi of 20% CuSO4+Ca(OH)2,Which volume make to5‖Oml,(lII)3mi Of supeL.natant SOlution fr’Om(ZI)another are same as王L

Cl.5mlof supernatant solution are taken for analysisin each case,and opiticaldensities areIead at570mp ac− COIding to the method ofBARKER and SuMMERSON.

(3)

Vol二8,No∴2(195L7) 223

Cedure were made accoIding to the technique described by HuLLIN and NoBLE(19)り As seenin Table3, COpPer・lime treatment was repeated壷or two or three times,Whereby the value of colour development due to pyruvate became smal1and constant which may be deduced fェom thefinalopticaldensity

The absorption spect川m Of the aldehyde・b−hydroxydiphenylcomplex shows a sharp maximum at570 mjL aSillustIated by the fo1lowing opticaldensities obtained from8pg oflactic acidundertheconditions described above at wavelengths between460to620mJL:460,OhO54;480,OhOフ2;500,01フフ;520,0,353;530,

0.4eO;540,0640;550,0.754;560,0,860;565,0。S95;5フ0,0.900;580,0835;590,0.678;600,0い471;610,0.302; 620,016l.The absorptjon curve Ofalkaline2,4・dinitIOphenylhydrazone of pyIuVate Shows also a sharp maximum at4E;O m〃′by the壬ollowing opticaldensitiesobtained壬Ibm2012pgofpyuvicacid at wavelengths

between3eOto580mIL:380,0165;400,0260;420,0422:430,0482;435,0,503;440,0520;450,0・528;455, 0”520;460,Oh508;4eO,0408;500h O”36フ;540,0,335;530,0235h Andthe absorption curveof DAC by the methodofEGGLETONetal.showsfollowingopticaldensitiesobtainedfIOm224pgofDACatwavelengthsbe・ tween320to600m〃:320,02∂フ;340,02e6;360,02∋5;380,○269;400,0り215;420,0192;440,0/190:460,0い236; 4eO,0367;500,○539;520,0・642;525,0・642;530,0642;535,0638:540,0・622;560,0448;5∈氾,0小ユ58;600,0.007

To deteImine colorimetric,byS480,at450mp for pyIuVate,byS530,at530mp forDAC and AMC, and by S580,at5フOmpforlactatewereusedwithelectIicphotometerandwithShimadzu spect【Ophotometer, m−odelQB50,reSpeCtively

Pentose was detected by the BIAL、’sIeaCtionintIOduced by ME・

JBAUM,aSgivenin UMBREIT,BuR− RIS and STAUFFER(11)”Nitrogen COntent Of cells was determined by the method of micro−KJEHLDAHL

ResⅦlts and D紬eussions I.DismuねtiomwitIlIn七aet

Cells

ltis evidentinFig,l.that glu− COSeandgluconate are dissimilated by intact celis grown in mait ex- tract,and the respiratory quotient values(RQ)foIglucose and glu・ conatearelh14andl89,reSpeCtive・ ly

Since the formation o土1actic acidisIeCOgmized both from glu・ COSe and gluconateandtheyieldof COz for gluconate is gIeater than that for glucose,SO the RQ Valuelor gluconateis as g工eat a− bout two til羊eSaSthatfor glucose, it is therefore suggested that the glucoseisfermentedbythisorgan・ ism via some scheme containlng gluconateoritsderivativesasinter−

Fig‖1Gaseous metabolism ofdismutation byintact ce11s ⑳−−⑳CO2eVOIved,○−00ゴCOnSumed

l・Pyruvate50FLM+glucose40pM,2”Pyruvate50pMエ gluconate40p〃,3Pyruvate50FLM:4.Glucose40jLM1

(4)

224 Tech.Bu11.,Fac.Agr.,:Kagawa Univ. mediates oxidatively. Pyruvateis

also王eadily oxidizedtoacetateinae・ robically by pyruvic oxidase of this OIganism as alIeadyshownin a pre・ vious pape工(10).As seeninTable4, smallamountsofDACandAMCfrom

pyIuVate are Obtained,identi丘ed by theformatior)OfNi・dimethylglyoxime, Table4Analysis of dismutation of pyruvate and glucose by

intact cells. 1ⅠⅠ ⅠⅤ Ⅰ (witb glucose) (浣雲3別品三豊)(霊∑芝;) Substrate PyIuVate(pM) 50”85Eぼect of 5085 Glucose(pM) 40 glucose O PyI・uVate IemOVed(〝朗) 48.6 十35..0 1356 CO2eVOlved(〝凡才) 42,2 +28・2 14小0 02COnSumed(〝朗)13.2」−11.1 2.1 9.25 01 8.25 0.1 ーーーー「− 0 and by theVoGES−PROSKAUERreaC・  ̄ J二ユ5 formed(〝〟) 0‖J5+ 0 0.13

AMC formed(JLM) 0”96+0.83 tion,afterdistillationwithandwithout

20%FeCl$

In addition,Fig”lalsoindicated the remarkable increase of carbon dioxideevolutionfromthe mixture of pyIuVate and glucose or gluconate, whereas O2COnSumption was alittle decreased.A result of this reaction byintact cellsis shownin Table 4. Lactic acid which occurein this

0 0 3.3フ 0 Lactateformed(pM)2l.0 +2l.0 0 BIAL’s test + + RいQい 3..20 6い67 1小12 1.0 C・RecoveIy鴇 122 33.O

Reactionperiod,90min,andNcontentofcel王per cup wasO小/6mg 場C−Recorveryis expressed by following equation

2×(DAC・AMC+02)+1actate

C・RecoveIy(%)=

pyruvate removed

reactionwasidentiRed by papeIChromatography,the sample were developped byascendingchromatogIaph

using BuOH:aCetic:H20(4:ユ:2)as soIvent,and visuali2:ing the spots with BPB−ethanoIsolutionFrom Table4,theincreasedamountsofCO2p工Oduction from glucose and pyruvateis 28」2pM,that of pyruvate removedis35OILM.and the formation oflactateis2l10pM,While the formation of both DAC andAMC areincIeaSed sユightly.In generaいt js unable toexpect to obtain a typicalresult byintact cells,While

glucoseaddedasasole substate is degraded byintact cells,SO de・

tails of this reaction

willbe elucidated by 山ied ceユユs. 2.DismlltatioIIWith Dl・iedCeils ThesimilaIgaSeOuS metabolismis observ− ed with dried cells as glVeninFig2,andthe e仔ect of adding variq oussugaISO点CO2prO・ duction from pyruvate and sugaI:Sislistedin Table5 As notedpIeVious− 1y:9)the additions of gttlCOSe,fructose,man− nose,maltose,andglu−

Fig‖2 Gaseous metabolisms of dismutation by dried cells −O COゴeVOIved,−▲Oi2COnSumed

A.lPyruvate50pM+glucose20lLM, 2・Pyr 50〃M+fructose20pM,

3。Pyrl5〔)FLM十galactose20JJ・M

B.l.Pyruvate5C)pM+・maltose20pM,2h Pyr”50FLM+sucrose20pM,

3・Pyr.50神灯+ユactose20/J肱

C‖ ⊥い PyIuVate50FLM+gluconale2C)JLM,2.Pyr50pM十mannOSe20pM, 3Pyr‖50ILM+Ⅹylose2C)FLM,4PyruVate5C FLM

(5)

Vol.8,No.2(195フ)

Table5 E仔ectofsugarsonCO2prOductionindismu− tation system.

225

COnate tO pyruVate rise the accelerativeevo・ 1ution of CO2,although no e鱈ectiveIeSults aエe eVer Obtained with galactose,SuCrOSe, lactose,and xylose.The analyticaldetails of these Ieactions are presented in Tables

6a−6c

In experiment with pyIuVate and gl11COSe in Table6a,the amo11ntSOf pyruvateIemOVed, CO2prOduced,andlactate foImedin the pre・ SenCe Ofglucose were386pM,33。O pプ吼and 30r18 p爪先IeSpeCtively;the amountsin the COrreSPOnding contIOlrun,in which glucose plin60min increased ratio(%)

CO2 02 CO2 02 ︵∪80∪︵つ1﹂︵〇9︵○︻b︻ノ 091つん00909∩︶ ﹁l l l l ユ l 1 0237︵U180︵D3 ∩︶335∩︶24196 11⊥ll l11⊥⊥ ﹁⊥ PyIuVate 5【】0−640 260−295 +gl11COSe 840 290 +fIuCtOSe 850 350 十mannose 910 370 +galactose 640 30〇 +Ⅹylose 700 311; 十maltose 860 255 +SuCrOSe 640 275 +1actose 56〔) 245 +・gluconate 945 28〕

Table6 Dismutation of pyruVate and various sugars by dIied cells (Reaction’period,60min,Ce11used,15mg per cups) a。With glucose,fructose,and galactose

1V (witbo扇sugar)

Ⅰ(+glucose) ⅠⅠ(+fructose) lII(+galactose)

E庁ect 45.2 0f 20 glucose Effect E償ect 45.2 0f 45.2 0f 20 frutose 20 galactose Stlbstrate Pyruvate SugaI 38.6 ・+13りユ. 35,8 十10.4 23=2 −2..3 ⅠemOVed CO2eVOlved 33・0 + 7.9

334 十e3

25・・2 ・+0.1 02COmSumed 11..3 −0.3 13”フ +2‖L ll.フ +0.ユ

DAC foImed Ol.20

AMC formed O”47

Lactate foImed 30.18 0.12 − 005 0い33 − 0,.11 1フ“94 +1Lフ.94 十 0.03 十0い03 十3018 0.1フ 0 0り24 ・− 020 L49 +1“49 BIAL’s test 寸十 + ± RQい 2.92 2.44 2.15 C・Recoveェy(%) ユ01 b小 With maltose,SuCrOSe,andlactose ⅠⅤ (Wit!10ut S11ga!) Ⅰ(+maltose) Ⅰ王(+SuCrOSe) ⅠⅠⅠ(+1actose)

Eぽect Eぽect 45い9 0f 45.9 0f 20 maltose 2つ sucrose SubstIate Pyruvate SugaI E仔ect 459 0f 20 1actose 395 +18.2 22‖9 +1.6 185 −2..7 Ⅰ・emOVed CO2eVOIved 34.0 +lL1 25rl +・21−2 22‖0 −0.9 02COnSumed lO.1 −0・、1

108 十06

9小フ ー0.5 0.12 − 0.C2 0,32 − 010 0 0

DAC formed O.24 AMC for・med へ70 Lactate formed 33..9 + ○。.10 + 0.28 + 33..9 013 −001 0.29 − 0..13 0 0 BIAL’s test 十1− R−Q.. 336 2.25 C・RecoveIy 86.4 98。3 109 110い8

(6)

TechBull.Fac.Agr”Kagaw∂Univ 226

C一With gluconate,mannOSe;and xylose

Ⅰ(+gluconate) lI(+mannOSe) ⅠⅠⅠ(+Ⅹylose) (without S11gaI)

Substrate Effect E任ect E庁ect

ate ”9 ・9 ”9 49

glate ma。Se Ⅹse

Pyruvate 3フ.フ +109 39.1ユ. +12け3 30.2 +33 26 9 IemOVed + 4り6 22.8 十 35 9.0 358 ・+13.0 2フ4 14.1 + 56 〕2。5 CO2eVOIved 37ll +14l3 02COnSumed ll0 +20 0。25 ・十 008 0.76 十 0.11 21フ0 + 2,フ○ 007 ・− 0け10 039 − 0…26 1387 +ユ.3…87 DAC foI・med O.24

AMC formed O.81

Lactate f0Ⅰmed 19い33 + 0‖07 十 0.16 +・1.9い33 BIAL’s test l十 2.45 221 RいQ、 3.38 94い8 93 8 C−Recovery 89小4

was omitted,WeIe255pM,251FLM,andOILM・SOtheIeSpeCtiveincreasedamountsof pyruvateIemOVed, CO2prOduced,andlactateformedcausedbytheadditionofglucosewe工e13・lpM・7・9lLM・and3018〃・M・

respectively・Asis showninTables6a−6c,thereactionproceededrapidly to astable end−POint,SO the

postulatedunknownintermediateinthedismutationsystemwasrapidlydegIadedbelowthestageofacetate

andlactate undeIthe condition used.In agreement with the mac工O eXPerimentsin the previous paper(9〉,

detai1s of the dismutation weIe elucidated more exclusively壬rom the fact thattheratioofincreasesofCO2 evoIved,pyruVateIemOVed,andlactatefollmedwasnearlyl:l;2

One maythen suggestlogically thefollowingover−allsequencefor this dismutationbyLfermenlum

This sequence of reaction wouldyield thatabovemen・ tioned ratioisl:l:2。SincetheobseIVed工atiosarel.0

:06:2.3with giucose,ユ.。0:1、3:l.78withgluconate,itcan be concluded that thisdata危tthissequence。Thepze・

Glucose

Y Gluconate肌−>(6・Phosphogluconate) PyIuVate

1r

CO2+(Pentose・phosphate)/、\Lactate

〆′ \

C2(Acetate) C8(Lactate) sentdatadonotpermittodecide thissequence more

de丘nitely,however,HoRECKER andSMYRNIOIIS(2〇−22)have reported the董01lowing equations using6PG

andlactic dehydorogenases from yeast

6PG+TPN+→ ribulose−5−phosphate+TPNH+H++CO2

TPNH+H十−l−pyruVate一寸1actate

Sum(l)+(2)6−Phosphogluconate+・pyruVate→工ibulose・5・phosphate+lactatc+CO2

⊥ 2

Sincethe fermentation did not go to completion,itis not unlikely that appreciable quantitiesofinter・

mediates accumulate,SO tO eStablish the nature of theintermediate productsandultimatelytoevaluatethe

signi丘cance of the pathwayin carbohydratemetabolism by L・/’ermentum,detectionofpentoseexpectedas

anendproductofthisdismutationIeaCtion was undertakenin these aliqo11tS,SOitwasequally positivein

whichdismutationwasproceededlIncontract,in other aliquotsin whichdismutationwasneverobserved,it wasscaIeCely detectedby BIAL・sreactionEvidencehasbeenpIeSented,hoⅣeVer,thata ketopentose・Phos− phateisthesubstanceactuauy splitbyL♪enioISuSintoC3andC2units(23),SOaketopentosephosphateisp王0−

bablyexpectedastheendproduct for this dismutation system from phosphorylated gluconate,also as

comparedwiththe data of yeastenzymes by HoRECI(ER andSMYRNIOrISAsalready mentioned,this unknownintermediate willbe rapidly degraded to a stable end products,namely acetateandlactate,SOthe

fomationof ketopentose phosphate whichis expected to accumulatewi11bethe subjectof the succeeding

(7)

Voll8,Noり2(195フ) 22フ On the basis of these considerations,itis suggested that ketopentose phosphateis a product of this

dismutation system by L,fermenium,and thatits formation maybeinitialstepinthedegradationsequent色 Of6PG

Stlmmary

The new dismutationofpyruVate and gluconate by L。f■eYmenlum,a hetero・type Oflactic acid bacteIia, WaS mentionedinapreviouspaper(9)小Inthatpaper,inordertoel11Cidatethisreaction,tWOdi#erentmethods Were employed,that of the manometry for gaseous metabolism,and of tbe macro oscillationforanalyzing metabolites except gases

This paper dealed a quantitative analysis foraliquots of vessels of WARBURG,and seveIalconsider・

ations for the conditions of colorimetric determinations of DAC,AMC,andlactate,eSPeCially forlactate deteImination of sample contaminated with alarge amount of pyIuVate(Seein Table3).

The similar results for dismutation of pyruvate was obtained withglucose,fructose,mannOSe,maltose, andgluconate,SO the ratio ofincreases of pyruvate removed,CO2eVOIved,andlactateformedwasl:l:2・ In these cases,DAC anc AMC werelittleincreased.In contIaSt,the cells did not catalyze similar dis− mutationwithgalactose,SuCrOSe,lactose,and xylose‖ Moreover,by using the BIAL,s reaction,PentOSe was detected onlyin the cases whendismutations wereobserved」・ThissubstancewhichIeaCtWith orcinol might be a pentose phosphate(unpublished)

The signi丘cance of this dismutation and the formation of pentose phosphatein the fermentation of hexose by L.fermenium was discussed

AckItOWledgement

The a11thor wishes to express his gratitude to Prof.・HIKATAGIRI,Ky6to Umiversityforhis contin11ed interest and encouragementい He also wouldlike to thank ProfりTu KuROXAMIfor his helpfuladvise,and

Prof.,K…KATAKURA and Prof.S”KAWAMURA for many valuable suggestions and for use of technical equipments

BitIliography

(6)DEMoss,R.D一,GuNSALUS。Ⅰ.C,BARD,RC、:

Newdiphosphopyridine nucleotide・1inked dehydr−

OgenaSeSinエβ〝CO〃0∫わC桝β。ざβ乃gβγクよ■滋ざ.βαC′u 丹¢‘., 125(1951)

(フ)DEMoss,RD。,GuNSALUS,Ⅰ.C.,BARD,R C:

Aglucose・6・phosphate dehydrogenasein Leucono−

5わc沼♂ざβ搾ね′〃よゐざl./・励頭減れ66,10−16(1953) (8)DEMoss,R Dl,GIB73S,M:6−Phosphogluco・ natedehydrogenasefromLeuconosiocmesenieroides. JβαCgβγ∠クJ・,70,フ30−フ34(1955) (9)YAMANAKA,K”:StudiesonpyruVatemetabol− ismbylactic acidbacteriaⅠIlDismutationsys−

temof sugars and pyruvate by LfeYmentum

(inJapanese)”7Agr・ChemSoc・.Pb.2n,(遷化),

30,269・2フ4(1956)

(10)YAMA上qAKAK:On pyruvic oxidase.(in

Japanese)This Bulleiin,(香川盛大学術報告),7, (l)WARBRUG,0・,CHRISTIAN,W”:BiochemZ, 238,131(1931);242,206(1931). (2)GIBBS,M,DEMoss,R Dl:Anewmechanism of ethanolformationintheheterolacticfermenta・ tion釣dβ7■α如邦乃OC,10,189(1951). (3)GuNSALUS,Ⅰ‖C‖,GIBBS,M”:TheheteIOlactic fermentation。n Position of Cllin the products Of glucose dissimilation by Leucono.sioc mesenier・

0査♂βゞ一J・戯oJ・C蝕明,194,871−Sフ5(1952). (4)GIBBS,M,SoKATCH,JT.,GuNSALUS,Ⅰ.C: Productlabe11ing of glucose−l−C14fermentation by homofeImentative andheterofermentativelac. ticacidbacteriafBa(ieYiol,70,572−5フ6(1955) (5)I)EMoss,RI},BARD,RC,GuNSALUS,LC:

The mechanism of the heterolacticfermentation Anewrouteofethanolformation.IBacterio[,62, 499・511(195け

(8)

223

フl−30(1955)

(11)UMBREIT,WWい,BuRRIS,R・Hh,STAUFFER, JlFr:ManometIic Techniques and Tissue Me・ tabolism,(1951)

(12)FRIEDEMANN,T.E,HAUGEN,GE.:Pyru¢,

ic acid。ⅠⅠ。The determination of keto acidin

bloodanduIine・J・点わムC血糊/,147,415・・442(194外 (13)CLIFT,ElP・,CooⅨ,RP∴」財鉱如呪/,28,

1フ88(1932)

(14)HELPRIN,J1。,SuLLrIVAN,MX.:The pIO・ duction of acetylmethylcarbinolby Aerobacier from a variety of ta工bon sourcesin asynthetic

medium.。′.βαCね′∠∂/・,67,90・93(1954) (15)WHITE;A.・G‖C.,KRAMPITZ,L・・0・,WERK・ MAN,C∴H..:Method′forthedirectdetermination Of diacetylintiss豆e andbacteIial別tratesりArck βよocお朋∴,9,229・234(1946) (16)EGGLETON,PN,ELSDEN,S・・R,GouGI子,N・・: The estimationof creatineanddiacetyl.Biochem J,37,526・529(1943)

(17)VAN二NJEL C”B:Notiziiber die quautita−

tiveBestimmung von DiacetylundAcetylmethyl・

Tech.Buu.FacAgrKagawa Univ CaIbino王戯鋸勉励Z,187,472・478(ユ927)

(18)BARKER,S・B.,SuMMERSON,W.H.:The

COlo!imetric deter・mination oflactic acidin bio・

logicalmaterialJBiotChem,138,535−554(1941) (19)HuLLIN,RH,,NoBLE,RL”:Thedetermina・

tion oflactic acidin microgram quantities小Bio・ Cゐ♂刑J.,55ぅ289・291(1953). (20)HoRECI(ER,B巾L,SMYRNIOTIS,P.Z.:The enzymatic productionofribose・5・phosphatefrom 6−Phosphogluconate”ArckBio:hemい,29,232q233 (1950) (21)HoRECRER,BL・,SMYRNIOrIS,PhZ:Phos− Phogluconic dehydrogenase fzom yeastJBiol

C蝕澗,193,3フ1−381(195け

(22)HoRECKER,BlL・,SMYRNIOIIS,Pl・Z,,S瓦EG− MILLER,J.E。:The enzymatic conversion of6− phosphogluconate to ribulose・5・phosphate and Iibose・5・phosphate小ibidい,193,383,396(i951). (23)GESr,H,LAMPEN,J‖0”:Fermentation of トC14・D−Ⅹylose如上融励ぬ摘町卸ぬ皿,左鮎d,1‡払 555−562(1952) 乳酸菌によるピルビン酸及び糖類の代謝に関する研究 ⅠⅤ エ,/−βグ’∽β紹わ‘∽に、よる共拒反応の分析

山 中

既に贋2報(9)にて,エ./βγ〃7¢花fα別によるグルコン酸とピルピン放との共玩反応を見出し,その結果を報告した. そのさい,アセトイン及び汐アセチルを蚤鼻法で定量したために・,検圧封の反応液を分析すること.が不可能であっ た.そのためにCOゴ及び0ゴについてのみ検圧計で測定し,反応の分析Lr3:別に.2〕倍のSCaleで振忽実験を行っ たものについて,ビルピン酸,酢酸,乳酸,アセトイン,汐アセチルを走還し,両者の結果を比較した・今回この 不備を補足するために,アセトイン,ジアセチルの比色定遠法を行い,検圧計についての分析が可能になったぃ 更 をこ他の比色定員億の条件も併せて検討し,特に多崖−のピルピソ酸の残存する場合の乳酸の走毘値な正確をこ求める方 法を検討した・又酪酸はこれでは建屈出来ないが,既にこの菌のpyruvicoxidase(10)について述べているので,吸 収0ゴをもって算出した その結果,第2報の結果と−・致し,グルコ−ス,プラクトース,マンノ−ス.マルトース,グルコソ酸ほピルピ ン放と.共妬反応を行い,いずれの場合にも,消費ピルビン酸:COJ:生或乳酸=1‥1:2となった アセトイン,ジア セチルの変化ほ僅少であった叉ガラグトース,レ.フ_−クロ−メ,ラクトー・ス,キンロ−スでほこの結果が得られ なかった更にこれら共甑反応庖示した場合にBIA・L反応が陽性匿なり,これほぺソトースである(未発表)こと から,この薗の研舷について岩1二の考察を行った

Updating...

参照

Updating...

関連した話題 :