Enzymatic Production of D-Glycerate from L-Tartrate

Agric. Biol. Chem., 53 (8), 2101 ~2105, 1989 2101 Enzymatic Production of D-Glycerate from L-Tartrate Setsuo Furuyoshi,* Nariyoshi Kawabata,* Hidehiko Tanaka1" and Kenji SoDA+t Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan * Department of Chemistry, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received February 13, 1989 D-Glyceric acid is a useful chiral synthon in synthetic organic chemistry. To produce D-glycerate cheaply, microorganisms that convert L-tartrate into D-glycerate with good yields and selectivity were isolated from soil samples. Onemicroorganism obtained was identified as a strain of Pseudomonassp. group Ve-2 and found to produce a new enzyme, L-tartrate decarboxylase. This enzymecatalyzes the direct conversion of L-tartrate into D-glycerate, with almost 100 %selectivity. In the presence of a cell- extract of Pseudomonas sp. group Ve-2, the amount of D-glycerate produced from L-tartrate reached 53 g/l under the best conditions examined, with a molar yield of almost 100% and an optical purity of more than 92%e.e. D-Glyceric acid is a useful chiral synthon in semialdehyde in P. ovalis,10) and from hy- synthetic organic chemistry.1'2) The best droxypyruvate in Pseudomonas sp.n) in the knownmethod for the preparation of optically presence of equivalent amounts of NADH. pure D-glyceric acid is the oxidation of a- However, D-glycerate production with micro- methyl-D-glucopyranoside, followed by hy- bial enzymes has not been developed indus- drolysis.3) However, it is rather complicated. trially. D-Glyceric acid is alternatively obtained by L-Tartrate is a cheap by-product of the wine optical resolution of its racemic form with industry and, therefore, is expected to be a penicillia or aspergilli.4) good starting material for D-glycerate pro- D-Glycerate is also synthesized in many duction. To produce D-glycerate cheaply, we microorganisms. In Pseudomonas putida5'6) have screened microorganisms that convert l- and Rhodopseudomonas sphaeroides^ D-glyc- tartrate into D-glycerate with good yields and erate is formed from L-tartrate by a coupled selectivity. Onemicroorganism obtained was enzymatic system in the presence of catalyt- found to produce a new enzyme,L-tartrate ic amounts of NAD+and Mn2+. The first decarboxylase, which catalyzes the direct con- enzyme in this system, L-tartrate dehydro- version of L-tartrate into D-glycerate, with genase (EC 1.1.1.93), catalyzes the NAD+- almost 100%selectivity.12) It seems reasonable linked oxidation of L-tartrate to oxalogly- to produce D-glycerate from L-tartrate using colate.8) The formation of D-glycerate from this enzyme. In this paper, we describe the oxaloglycolate and the oxidation of NADH isolation of a microorganism that produces l- are mediated by the second enzyme, oxalogly- tartrate decarboxylase, the formation of the colate reductive decarboxylase (EC 1. 1. 1.92).9) enzymeand the simple enzymatic production D-Glycerate is also produced from tartronic of D-glycerate from L-tartrate, with emphasis + Present address: Faculty of Agriculture, Okayama University, Okayama 700, Japan. +t To whomall correspondence should be addressed. 2102 S. Furuyoshi et al. on the reaction conditions. partment thermostatically maintained at 30°C. The standard assay mixture for the measurement of L-tartrate dehydrogenase was the same as the standard reaction Materials and Methods mixture for glycerate production, in a total volume of 1.0 ml. The reaction was started by the addition ofenzyme Materials. NAD+and NADHwere purchased from to the reaction mixture previously incubated at 30°C. The Kohjin Co., and glyoxylate reductase (EC 1.1.1.26, from quantity of the enzyme used was such that there would be spinach leaves) and diaphorase (EC 1.8.1.4, NADH: dye a linear change in the absorbance at 340nmfor at least oxidoreductase, from Clostridium kluyveri) from Sigma 2min. One unit of the enzyme was defined as the amount Chemical Co. Other chemicals used were the best available of enzyme that catalyzed the appearance of 1 ^mol of commercial products. NADHper min. Specific activity was expressed as units per mg of protein. Screening of glycerate-producing microorganisms. L- Protein was determined by the method ofLowry et a/.13) Tartrate-assimilating microorganisms were obtained by with crystalline bovineserumalbuminas a standard. using a medium containing 1.0% potassium L-tartrate, 0.3% (NH4)2SO4, 0.3% K2HPO4, 0.01% MgSO4-7H2O and 0.02% yeast extract. The pH was adjusted to 7.5 with Results and Discussion 3n HC1. This mediumwas used as the basal medium. A small amount ofa soil sample was inoculated in 5 ml of the Screening of glycerate-producing microorgan- basal medium. After aerobic cultivation at 25°C until isms growth was detected visually, the microorganisms were To produce D-glycerate cheaply, initially, isolated on agar plates of the basal medium. The isolated bacteria were maintained on the basal medium sup- microorganisms that convert L-tartrate into plemented with 2% agar. glycerate in good yields were screened. Among Cells of a microorganism obtained were inoculated into the L-tartrate-assimilating microorganisms ob- 200 ml of the basal medium in a 500ml-flask. After aerobic tained, cells of 7 strains were found to produce cultivation at 25°C for 12hr, the cells were harvested by enzyme(s) that convert L-tartrate into glyc- centrifugation and then washed twice with 0.85% NaCl. erate. Although L-tartrate dehydrogenase ac- The washed cells, about 1 g (wet weight), were suspended in lml of 0.1m Tris-HCl buffer (pH 7.5) and then tivity was found in cell-extracts of the 7 mi- subjected to sonication at 4°C for 2min with a Seiko croorganisms, no relationship was found be- Instruments model 7040 ultrasonic disintegrator. After tween the L-tartrate dehydrogenase activity centrifugation (40,000xg for 20min at 4°C), the super- and the glycerate-producing activity (Table I). natant was used for glycerate production and the assay of L-tartrate dehydrogenase. Since the best yield of glycerate and the best Standard conditions for D-glycerate production. The stan- Table I. Screening of Glycerate-producing dard reaction mixture for the production of glycerate Microorganisms contained 100 /miol ofTris-HCl buffer (pH 7.5), 1 /rniol of The reaction was carried out in the standard reaction MgCl2, 1 jumol ofNAD+, 50 /miol of potassium L-tartrate mixture at 30°C for an hour. The reaction mixture and an appropriate amount of the cell-extract, in a final contained 0.1 ml of the cell extract obtained, in a total volume of 1.0ml. After incubation at 30°C for 1 hr, 0.2ml volume of 1 ml. of 3n HC1 was added. Tartrate and glycerate were determined by liquid chromatography using a TOSOH Glycerate-producing Total HLC-803D with an ULTRON PS-80H column activity L-tartrate (0.8 x 30cm; Shinwa Kako Co.). Elution was carried out Strain dehydrogenase with 0.126% HC1O4at the flow rate of 0.8ml/min. The Glycerate Tartrate formed consumed activity elution times of tartrate and glycerate were 9.2min and (jumol) (/miol) (milliunits) 1 1. 1 min, respectively. 8 507B 29 8 Analytical methods. The experimental conditions for the 509B 21 assay of L-tartrate decarboxylase were identical to those 5D1A 38 16 for glycerate production. One unit of the enzyme was de- 5D8A 23 21 fined as the amount of enzyme that catalyzed the ap- 5D13B 13 6 pearance of 1 /zmol of D-glycerate per min. 704A 23 21 L-Tartrate dehydrogenase activity was assayed with a 7D10C Trace 10 Shimadzu UV-3000spectrophotometer, with the cell com- Enzymatic Production of D-Glycerate 2103 Table II. Taxonomic Characteristics of Strain 5D1A was isolated from the reaction mixture. The cell-extract of strain 5D1A(containing 938 mg Flagella Polar 1 of protein) was added to 1 1 of20mMTris-HCl Motility Motile by polar flagella buffer (pH 7.5) containing lOOmmol of po- Shape Rod, 0.8-1.2x2.0-3.2/an tassium L-tartrate, 2mmol of NAD+ and Gramstrain Negative Oxidase test Negative 0.2 mmol of MgCl2. On incubation at 30°C for Catalase test Positive 24 hr, L-tartrate was converted into glycerate Growth completely. Then the reaction mixture was anaerobic Negative applied to an Amberlite IRA-400 (OH form) 37/41°C Positive/negative Pigments Yellow-orange (non-diffusible) column (5 x21 cm). The column was washed O-F test Oxidative with 1 1 of H2Oand then the glycerate was Gas from glucose Negative eluted with 1 1 of 1n HC1. The solution was /?-Galactosidase test Negative Indol production Negative concentrated in vacuo and the glycerate hemi- Nitrate reduction Negative calcium salt (dihydrate) was obtained accord- Denitrification Negative ing to the method of Baer et al.16) The purity Phenylalanine deaminase Positive of the glycerate hemicalcium salt obtained Levanfromsucrose Negative was estimated to be almost 100% by high- Lecithinase test Negative Urease test Negative performance liquid chromatography. Hydrolysis of The reaction product and authentic dl- starch Positive glycerate showed the same retention time on gelatin Negative high-performance liquid chromatography. The casein Negative nuclear magnetic resonance and infra-red DNA Negative Tween 80 Negative spectra of the product also showed the same esculin Negative patterns as those of the authentic DL-glycerate Tyrosine degradation Negative hemicalcium salt. The specific optical rotation Growth factor value ([a]o°) of the product was +13.3 (re- requirements None ported [oc]d0 values for D-glycerate hemical- Carbonsource utilization test Utilized: acetate, caprate, citrate, malate, malonate, cium, +12.9~+15.517)). About 96% of the L-arabinose, fructose, glucose, mannose, maltose, reaction product reacted with glyoxylate re- xylose, mannitol, gluconate, 2-ketogluconate, l- ductase and NAD+to yield NADHon 24hr serine.

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