Agric. Biol. Chern., 52 (2), 419-426, 1988 419

Characterization of D- Involved in D-Sorbitol Production of a Methanol Yeast, Candida boidinii (Kloeckera sp.) No. 2201 Vitchuporn Vongsuvanlert* and Yoshiki Tani Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received August 18, 1987

D-Sorbitol (sorbitol) production from D- could be achieved by initial isomerization of D-glucoseto D- via xylose , then reduction to sorbitol via sorbitol dehydrogenase coupled with NADHregeneration from the methanol oxidative system by an intact cell system of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. Sorbitol dehydrogenase was purified 351-fold from a cell-free extract of D-xylose-grown cells and characterized. It oxidized preferably D-mannitol besides sorbitol, and reduced preferably D-xylulose and D-ribulose, besides D-fructose. Kmvalues for sorbitol and D-fructose were 7.7 and 263mM,and Vmaxvalues were 301 and 384/imol/min/mg, respectively. The activity was strongly stimulated by Fe3+ and significantly inhibited by Pb2 + , EDTA, and o-phenanthroline.

Sorbitol dehydrogenase (SDH) (polyol and the characterization of SDHof C. boidinii dehydrogenase, L-iditol: NAD+-5-oxido- No. 2201, responsible for the reduction of d- reductase, EC 1.1.1.14) is an enzyme which fructose after isomerization of D-glucose. catalyzes the oxidation of sorbitol to D-fruc- tose.1} It is widely distributed among liver MATERIALS AND METHODS and brain of mammals,1 ~9) plant tissues10* and microorganisms, especially , n ~13) and Chemicals. DEAE-Sephacel, Phenyl-Sepharose CL-4B, Sephadex G-200, and Sephacryl S-300 were purchased that from mammalianliver was extensively from Pharmacia Co., Ltd. D-Xylulose, and D-ribulose purified and characterized. However, only the were obtained from Sigma Chemicals Co., Ltd. and l- metabolic pathway of the enzymes from yeasts iditol from Lab. Sarget Merignac. Other chemicals were were thoroughly investigated.14' 15) Moreover, usual commercial products of analytical grade and used SDHmay be clinically useful in the diagnosis without further purification. of liver diseases.5) We reported glucoamylase production to Microorganism and cultivation. C. boidinii No. 2201, which was selected as the strain for sorbitol production make D-glucose from raw cassava starch.16) In from D-glucose,17) was used. The basal medium was an attempt to further use the D-glucose, composed of 0.4g of NH4C1, 0.1g ofKH2PO4, 0.1g of the production of sorbitol, a sweet polyol, was K2HPO4, 0.05g of MgSO4-7H2O, 0.2g of yeast extract, studied with a system coupled with the oxida- and 0.3g of Polypepton (Daigo) in 100ml of deionized tion of methanol to regenerate NADHfor the water, pH 5.5. The inoculum was prepared by growing reduction of D-glucose by cells of a methanol cells on the basal medium containing 0.5 g of D-glucose in yeast, Candida boidinii {Kloeckera sp). No. a 16.5 x 165-mmtest tube for 24hr at 28°C under recipro- cal shaking at 200rpm, and added at a rate of 1%. 2201.17) In this paper, we describe the mech- Resting cells for sorbitol production was prepared as anism of sorbitol production from D-glucose described previously.17) In preparing cells for enzymatic

* On leave from Central Laboratory and Greenhouse Complexes, Kasetsart University, KamphaengseanCampus, Nakhon Pathom 73140, Thailand. 420 V. Vongsuvanlert and Y. Tani

study of SDH, yeast was grown on 500ml of the basal Methanol was measured by the method ofTani et al.l9) medium containing 2%(w/v) D-xylose in a 2-1 shaking flask. The cultivation was done at 28°C under reciprocal Purification ofSDH. All purification steps were done at shaking at 200rpm for 45hr. Cells were collected by 4°C and centrifugation was done at 14,000 rpm for 20 min. centrifugation and washed twice with 0.05 mpotassium The buffer was 0.05 mpotassium phosphate buffer, pH 7.0, phosphate buffer, pH 7.0. containing 0.25mMDTTunless otherwise stated. The enzymewas concentrated by ukrafiltration using Amicon Preparation of cell-free extract. The cell paste Was PM-10. suspended in 50 mMpotassium phosphate buffer, pH 7.0, Step 1. Ammoniumsulfate saturation. Solid ammonium containing 0.25 mMdithiothreitol (DTT), and disrupted by sulfate was added to the cell-free extract from 101 of an ultrasonic oscillator (Insonator Kubota Model200 m) culture medium to give 40% saturation (243g/1) with with a constant current at 2A for 45min. After centri- stirring, adjusting the pH to 7.0 with 10% NH4OH fugation the resultant supernatant solution was used as the solution. After this was left for 1 hr, the precipitates that cell-free extract. formed were removed by centrifugation and ammonium sulfate was added to the resultant supernatant up to 80% Assay of enzyme activities. The oxidative activity of saturation (285 g/1). After this was left standing overnight, SDH was measured by the method of Leissing and the precipitates that formed were collected by centrif- Guinness.3) The assay mixture contained 10/miol NAD+, ugation and dissolved in the buffer at a minimumvolume. 50mmol Tris-HCl buffer, pH 9.0, 8.69mmol sorbitol, The enzymesolution was dialyzed against the samebuffer and enzyme solution appropriately diluted with 50mM overnight. Insoluble materials formed during dialysis were potassium phosphate buffer, pH 7.4, containing 0.25 mM removed by centrifugation. DTT, in a total volume of 1.15ml. Initial velocity was Step 2. DEAE-cellulose column chromatography. The measured by the increase in absorbance at 340nm at dialyzed enzyme solution was put on a DEAE-cellulose 30°C due to the reduction of NAD+by monitoring for at column (4.0 x 40cm) equilibrated with 0.05m potassium least 1 min with a Hitachi Model 200 spectrophotometer. phosphate buffer, pH 7.0, containing 0.25 mMDTT. The The reductive activity was measured by the method of elution wasstepwise. The active fractions eluted by 0.1 m Gerlach and Hiby18) through the decrease of absorbance potassium phosphate buffer, pH 7.0, containing 0.1 m at 340mn due to the oxidation of NADH. The assay NaCl, were pooled and concentrated. mixture contained 107 mmolTris-maleate buffer, pH 5.6, Step 3. DEAE-Sephacel column chromatography. The 0.4mmol NADH,400mmol D-fructose, and enzyme so- concentrated enzyme from Step 2 was put on a DEAE- lution in a total volume of 1 ml. One unit of enzymewas Sephacel column (2.5 x 34cm) equilibrated with 0.05m defined as 1 /miol of NADHincreased or decreased per potassium phosphate buffer, pH 7.0, containing 0.25mM min for the oxidative and reductive activities, respec- DTT,and eluted like the DEAE-cellulose column except tively. for a flow rate of 40ml/hr. The active fractions were (AOD) activity was measured by the pooled and concentrated. ABTS/PODmethod as described by Tani et al.l9) The Step 4. Phenyl-Sepharose CL-4B column chromatog- color produced was measured spectrophotometrically at raphy. The concentrated enzyme from Step 3 was put on a 420nm.Oneunit of enzymewas defined as the amount Phenyl-Sepharose CL-4B column (2.5 x 6.0cm) equili- which oxidized 1 ^mol of ABTSper min at 28°C. brated with 3mKC1,and eluted stepwise at a flow rate Formaldehyde dehydrogenase (FAL-DH) activity was of 20ml/hr with 3.0, 1.5, 0.75, and 0m KC1. The enzyme assayed based on the reduction of NAD+as described by was eluted with goodseparation and the active fractions Schiitte et al.20) by following the increase in absorbance at eluted by 0 mKC1were pooled and concentrated. 340nm.Oneunit of enzymewas defined as the amount Step 5. Hydroxyapatite column chromatography. The which produced 1 /rniol of NADHper min at 30°C. concentrated enzyme from Step 4 was put on a hy- Xylose isomerase activity was measured by the method droxyapatite column (2.5 x6.0cm) equilibrated with of Yamanaka21) using D-glucose as substrate. The d- 0.01 Mpotassium phosphate buffer, pH 7.0, containing fructose formedwas measured spectrophotometrically at 0.25 mMDTTand eluted stepwise at a flow rate of20 ml/hr 540nm by the cysteine-carbazole reaction. One unit of with 0.01, 0.05, and 0.1 m potassium phosphate buffer, pH enzyme was defined as the amount which produced 1 7.0, containing 0.25mMDTT. The active fractions were /miol of D-fructose per min at 40°C. pooled and concentrated. Step 6. 1st Sephadex G-200 gel filtration. The con- Analytical methods. Protein was estimated from the centrated enzymefrom Step 5 wasput on a Sephadex G- absorbancy at 280 nm and by the method ofLowry et al.22) 200 column (1.5x85cm) equilibrated with 0.05m po- with bovine serum albumin as the standard. tassium phosphate buffer, pH 7.0, containing 0.25mM Sorbitol was measured enzymatically under the en- DTT.The enzymewas eluted with the same buffer and the dpoint system as described in our previous paper.17) active fractions were pooled and concentrated. D-Sorbitol Dehydrogenase of Methanol Yeast 421

Step 7. 2nd Sephacryl S-300 gel filtration. The con- centrated enzyme from Step 6 was put on a Sephacryl S- 300 column (1.0x85cm) equilibrated with 0.05m pot- assium phosphate buffer, pH 7.0, containing 0.25mM DTT.The enzymewas eluted with the same buffer and the active fractions were pooled.

RESULTS

EnzymesFigure for1 showssorbitolenzymeproductionactivities fromrelatingglucoseto sorbitol production from D-glucose in cell-free Fig. 1. Enzyme Activities Relating to Sorbitol extracts from different growth phases of cells grown on a mediumcontaining 1%methanol Production in Cells. and 0.3% D-xylose. The enzyme activities pos- Cell-free extracts from different growth phases were pre- sibly involved in sorbitol production, xylose pared from cells grownon the basal mediumsupplement- ed with 1% (v/v) methanol and 0.3% (w/v) xylose. The isomerase, SDH, AOD, and FAL-DH, ap- sorbitol produced (#) was measured and growth (O) was peared during the sorbitol production. The measured spectrophotometrically at OD610, as shown in maximumsorbitol production was obtained Fig. 1A. Enzyme activities for the oxidative activity,of by cells from the late logarithmic phase at SDH (A), xylose isomerase (A), AOD (å¡) and FAL-DH (å ) were assayed, as shown in Fig. IB. 45 hr of cultivation. Moreover, enzyme activ- ities relating to sorbitol production of cell- free extracts from cells grown on different carbon sources and of growth phases were also investigated. We confirmed that cells having high sorbitol productivity were obtained by growth on methanol and D-xylose. In this condition, cells having higher activities of xy- lose isomerase, SDH,and methanol oxidative enzymes produced the large amount of sor- bitol. On the other hand, cells grown on other carbon sources, which lacked the activities of methanol oxidative enzymes, could also pro- duce sorbitol by endogenous NADH in the Fig. 2. EnzymeActivities Relating to Sorbitol cells. However, the sorbitol productivity was Production in Resting Cells System. lower than that of the cell reaction coupled Cell-free extracts were prepared from resting cells of with NADH regeneration by the methanol various reaction times. The methanol remaining (©) and oxidative enzymes. It was suggested that the sorbitol produced (#) were measured as shown in Fig. 2A. Enzymeactivities for the oxidative activity of SDH(A), methanol oxidative enzymes enhanced the xylose isomerase (A), AOD(å¡) and FAL-DH(å ) were sorbitol production with regeneration of assayed, as shown in Fig. 2B. NADHfor the reduction of D-fructose after isomerization from D-glucose by xylose duction in the early period of cell reaction that isomerase. decreased at the late period. However, the Furthermore, enzyme activities relating to accumulation of sorbitol reached the max- sorbitol production during the reaction with imum as all enzyme activities declined. The resting cell system were also measured. As decrease of these enzyme activities and the low shown in Fig. 2, the rapid rate of sorbitol activity of xylose isomerase could limit the production was obtained with cells having sorbitol production from D-glucose. high enzyme activities relating to sorbitol pro- From these results, sorbitol may be pro- 422 V. Vongsuvanlert and Y. Tani duced from D-glucose using mainly the system Effects of carbon source on SDHproduction including the isomerization of D-glucose to d- Table I shows the activity of SDHin cell- fructose by xylose isomerase followed by the free extracts from cells grownon various car- reduction of D-fructose to sorbitol by SDH bon sources. It was found that D-xylose was coupling with NADHregeneration from the the most favorable carbon source. The highest oxidation of methanol by methanol oxidative SDH activity was obtained from a cell-free enzymes, AODand FAL-DH. extract of cells grown on the mediumcontain- ing 2% D-xylose. It was suggested that d- xylose was an inducer for the synthesis of Table I. Effects of Carbon Sources on Sorbitol NAD-linked dehydrogenase, especially in Dehydrogenase Activity Candida utilis}^ The cultivation was done in a 500-ml shaking flask containing 100 ml of the basal medium with each carbon source indicated. Culture conditions and the measure- Purification of SDH ment of the oxidative activity of SDHof each cell-free Asummaryof the purification procedure is extract were described in Materials and Methods. given in Table II. The elution patterns of the Specific DEAE-cellulose column chromatography and Growth activity the second gel filtration with Sephacryl S-300 Carbon source (OD610) (units/mg are shown in Fig. 3 and Fig. 4, respectively. protein) One symmetrical protein peak was eluted by Sephacryl S-300gel filtration. The yield of D-Glucose 1% ll.1 <0.01 Sorbitol 1% 9.5 0.06 4.9% of the total activity was obtained from Methanol 1% +D-glucose 1% 5.8 <0.01 overall purification steps, the final preparation Methanol 1%+sorbitol 1% 8.5 0.03 having 351-fold the specific activity of the Methanol 1% +D-xylose 0.5% 7.8 0.03 Methanol 1%+L-sorbose 0.5% 3.4 0.03 original extract. It was almost completely pu- Methanol 1% 6.1 0.01 rified but it still had a small amount of another Methanql 2% 5.9 0.02 protein, one minor protein band on SDS- D-Xylose 0.25% 2.9 0.05 D-Xylose 0.5% 5.9 0.05 polyacrylamide gel electrophoresis.23) D-Xylose 1.0% 7.6 0.05 D-Xylose 1.5% 9.4 0.07 Effects of pH and temperature on SDHactivity D-Xylose 2% 12.1 0.15 The influence of pH on the oxidative and D-Xylose 5% 10.3 0.13 reductive activities wasmeasuredwith Tris-

Table II. Summaryof Purification of Sorbitol Dehydrogenase

Total Total Total Specific activity Purification Recovery volume protein activity" (units/mg Step (fold) (%) (ml) (mg) (units) protein)

Cell-free extract 950 36,420 3,209 0.09 1 Ammonium sulfate 140 6,568 2,900 0.45 5. 1 (40 ~ 80%) saturation DEAE-Cellulose ll 1,255 1,017 0.83 9.4 31.7 DEAE-Sephacel no 373 377 1.01 ll.4 ll.7 Phenyl-Sepharose CL-4B 170 116 273 3.67 41.6 8.5 Hydroxyapatite 4 14.2 204 19.19 218 6.4 1st gel filtration 1.4 13.1 193 20.99 238 6.0 (Sephadex G-200) 2nd gel filtration 15 158 (Sephacryl S-300)

The activity was defined as the oxidative activity. D-Sorbitol Dehydrogenase of Methanol Yeast 423

Fig. 3. Chromatography of Sorbitol Dehydrogenase on DEAE-Cellulose. The elution was stepwise at a flow rate of 60ml/hr with potassium phosphate buffer, pH 7.0, containing 0.25 mM Fig. 5. pH Optima for Sorbitol Dehydrogenase DTT; A, 0.05m buffer; B, 0.1m buffer; C, 0.1m Activity. buffer+0.1 m NaCl and D, 0.1 m buffer+0.2m NaCl. The A reaction mixture with 1.75jug of the purified enzyme amount of protein and the oxidative activity of SDHwere from Sephadex G-200 column chromatography was used i to examine the optimal pH on SDH activity for the oxidative activity (O) and the reductive activity (#). The assay for both activities was done by variation of pH values, as indicated. examined with Tris-maleate buffer from pH 5.0~8.6. The purified enzyme was kept in Tris-maleate buffer at different pHs at 4°C overnight and the remaining activity was mea- sured. The enzymeactivity was stable at pH 7.0 ~8.0, but it was slightly inactivated during the incubation.

Coenzyme of SDH Fig. 4. Chromatography of Sorbitol Dehydrogenase on Sephacryl S-300. NAD(H)and NADP(H)were examined as the coenzymefor the oxidative and reductive The elution was by 0.05 m potassium phosphate buffer, pH 7.0, containing 0.25mMDTTat a flow rate of 3ml/hr. The activities of SDH.The preferred coenzyme of amount of protein and the oxidative activity of SDHwere both activities was NAD(H). However, NADP- assayed. (H) also served but much less efficiently and the activity was less 20% than that ofNAD(H) HC1 buffer from pH 7.0- 12.0 and with Tris- as coenzyme. maleate buffer from pH 5.0-8.6. The maxima of the oxidative and reductive activities were Substrate specificity of SDH observed at pH 9.5 and pH 5.6, respectively The substrate specificity of the purified en- (Fig. 5). zyme was investigated as shown in Table III. The effects of temperature on the oxidative Various sorts of ketoses and aldoses were used activity were examined from 25-45°C. as substrates to examine for the reductive Increasing the temperature up to 37°C in- activity. The enzymewas capable of reducing creased the activity with the maximum at not only D-fructose but also D-xylulose, d- 37°C. ribulose, L-sorbose, and D-xylose. The highest activity was obtained with D-xylulose. The Stability of SDH activity enzyme activity for D-glucose was rather low. The stability for the oxidative activity was Moreover, polyols were investigated for the 424 V. Vongsuvanlert and Y. Tani

Table III. Substrate Specificity of SORBITOL DEHYDROGENASE The reaction was done as described in Materials and Methods except for variation of substrate and con- centrations. The purified enzyme from Sephadex G-200 column chromatography of 13 ^ig and 3.4 jug in reaction mixture was used to measure the reductive and oxidative activities, respectively. Specific

o ubstrateS . , . , Concentration activityà", (him) (unit s/mg protein) Reductive activity for ketose and aldose D-Fructose 100 8. 10 D-Glucose 1 00 0.42 L-Sorbose 100 3.98 D-Ribulose 50 62.6 1 D-Xylose 100 1.97 D-Xylulose 16.65 149.45 Fig. 6. Substrate Concentration on Sorbitol De- L-Rhamnose 100 0.3 1 hydrogenase Activity. D-Ribose 100 0.33 D-Arabinose 100 0. 12 Reaction mixtures with 1.95^g or 6.5/zg of the purified 1 00 0.03 enzyme from Sephadex G-200 column chromatography Fucose 100 0.05 were used to measurethe effect of substrate concentration on the oxidative activity of sorbitol (Fig. 6A) and the Oxidative activity for polyol reductive activity of D-fructose (Fig. 6B), respectively, by D-Sorbitol 8.69 9.08 Lineweaver-Burk plots. The assay reaction for both ac- D-Dulcitol 8.69 0.03 tivities was done by variation of substrate concentra- D-Arabitol 8.69 0.09 Adonitol 8.69 6.94 tions (sorbitol and D-fructose), as indicated. D-Mannitol 8.69 17.93 Xylitol 8.69 1.02 L-Iditol 8.69 0. 30 respectively.

Effects of metal ion and inhibitor on SDH oxidative activity. The enzyme could oxidize activity not only sorbitol but also D-mannitol, adoni- Table IV shows the effects of metal ions and tol, xylitol, and L-iditol. Among these com- inhibitors on the oxidative activity. Fe3+ was pounds, D-mannitol and D-xylulose were the the most effective stimulant of the enzyme most favorable substrates for the oxidative activity. Other metal ions, especially Pb2+ and reductive activities, respectively. inhibited the enzyme activity. Among inhib- itors tested, o-phenanthroline and EDTA Effect of substrate concentration on SDH strongly inhibited the enzyme activity. Sodium activity azide, semicarbazide à"HC1, and DTTincreased The Michaelis constants for the oxidative the enzyme activity. However, high concen- and reductive activities were calculated from trations of semicarbazide-HC1 and DTT in- Lineweaver-Burk plots. As shown in Fig. 6, hibited the enzyme activity. straight lines were obtained in double recipro- cal plots of velocities versus substrate con- DISCUSSION centrations. The Kmvalues for the oxidation of sorbitol and the reduction of D-fructose Wesuggested that the mechanismof sor- were 7.7 and 263mM, while Fmax values were bitol production from D-glucose by a methanol 301 and 384 /imol/min/mg of purified enzyme, yeast, C. boidinii No. 2201, was initiated by the D-Sorbitol Dehydrogenase of Methanol Yeast 425

Table IV. Effects of Metal Ions and Inhibitors fructose. It was similar to the NAD-linked ON SORBITOL DEHYDROGENASE ACTIVITY polyol dehydrogenase from C. utilis which was The reaction was done as described in Materials and induced by D-xylose.14) The purified SDH had Methods except for pretreatment with metal ion or different optimal pHs for the oxidative and inhibitor at 4°C for 30min. The purified enzyme from reductive activities, pH 5.6 and pH 9.5, re- Sephadex G-200 column chromatography (18 /ig) was used to measure the oxidative activity. spectively. In comparison with Torulopsis can- dida NCYC576,14) we suggested that the op- Relative Concentration timal pH for the oxidative activity of SDHof Compound (him) activity (%) C. boidinii No. 2201 was significantly lower than pH 7.0 and nearly the same pH value for Metal salt FeCl3 - 6H2O 10 224 the reductive activity at pH9.0 of both strains. 10 75 However,it was related to manyreports of FeSO4 - 7H2O enzymes from other sources.1'3'8~n) The Na2MoO4 - 2H2O 10 62 CoCl2 -6H2O 1 32 NADP-dependent SDH of Acetobacter sub- Pb(CH3COO)2 - 3H2O 10 7 oxydans catalyzes the oxidation of sorbitol to CaCl2 -2H2O 10 69 ZnSO4- 7H2O 1 29 L-sorbose.24) The SDH of C. boidinii No. 2201 MgSO4 - 7H2O 10 87 which catalyzes the oxidation of sorbitol to d- CuSO4 - 5H2O 10 61 fructose, not only NAD+as coenzyme but Ba(CH3COO)2 1 21 also NADP+was slightly active, though much MnCl2 -4H2O 10 less efficiently, similar to the observations of NiCl2 H2O 1 22 Na2SO4 10 50 Jeffer et al.4) and O'Brient et al.8) Substrate K2CO3 10 70 specificities have been reported to be varied for Li2SO4 H2O 10 63 a number of SDHisolated from a variety of Inhibitor mammalian livers, plant tissues, and microbial EDTA 1 26 sources. However, Kmvalues for D-fructose 10 5 were usually higher than that for sorbitol. The Sodium azide 10 231 Potassium ferricyanide 10 83 enzyme in this study had one 34 times higher Semicarbazide HC1 1 177 for D-fructose than sorbitol. Fmaxvalues for 10 ll both substrates were nearly the same. o-Phenanthroline 1 3 DTT 1 106 Moreover, it could widely oxidize various sorts 10 28 of ketoses and aldoses. For the reductive ac- None 100 tivity, D-fructose was a more favorable sub- strate than D-glucose. isomerization of D-glucose to D-fructose via REFERENCES xylose isomerase and subsequent reduction to sorbitol via SDHby coupling with oxidative 1) N. Leissing and E. Me. Guinness, Biochim. Biophys. * enzymessystem for NADHregeneration. The Acta, 524, 254 (1978). sorbitol production was limited by the first 2) H. Jornvall, H. V. Bahr-Lindstrom and J. Jeffery, step for isomerization of D-glucose to d- Eur. J. Biochem., 140, 17 (1984). 3) N. Leissing and E. T. Me. Guiness, in "Methods in fructose and also by enzyme inactivation Enzymology," Vol. 89, ed. by W. A. Wood, during the cell reaction for sorbitol pro- Academic Press, New York, 1982, pp. 135~ 139. duction. The improvement of sorbitol produc- 4) J. Jeffery, L. Cummins, M. Carlguist and H. Jornvall, tion from D-glucose is nowunder investiga- Eur. J. Biochem, 121, 229 (1981). 5) U. Christensen, E. Tuchsen and B. Andersen, Acta tion. Chem. Scand., 29, 81 (1975). The cell-free extract of C. boidinii No. 2201 6) M. A. Alizade, R. Bressler and K. Brendel, Biochim. from D-xylose-grown cells contained an en- Biophys. Acta, 370, 354 (1974). zyme, NAD-linked SDH, which oxidized D- 7) J. Heitz, J. Biol. Chem.; 248, 5790 (1973). 426 V. Vongsuvanlert and Y. Tani

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