Agric. Biol. Chern., 52 (2), 419-426, 1988 419 Characterization of D-Sorbitol Dehydrogenase 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-glucose could be achieved by initial isomerization of D-glucoseto D-fructose via xylose isomerase, then reduction to sorbitol via sorbitol dehydrogenase coupled with NADHregeneration from the methanol oxidative enzymes 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 enzyme 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 bacteria, 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 Alcohol oxidase (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.