D-Glucaric Acid Producing Bacterium, and Method for Manufacturing D-Glucaric Acid

Abstract

The present invention provides a D-glucaric acid-producing bacterium and a method for producing D-glucaric acid. The present invention is characterized in that D-glucaric acid or a salt thereof is produced from one or more saccharides selected from the group consisting of D-glucose, D-gluconic acid and D-glucuronic acid with catalytic action of a specific alcohol dehydrogenase PQQ-ADH (1) and a specific aldehyde dehydrogenase PQQ-ALDH (2), and that D-glucaric acid or a salt thereof is produced by using a microorganism having the PQQ-ADH (1) and the PQQ-ALDH (2) or a processed product thereof in the presence of the one or more saccharides. The present invention can provide a microorganism having improved productivity of D-glucaric acid to be used for production of D-glucaric acid and a method for efficiently producing D-glucaric acid.

Claims

1-12. (canceled)

13. A method for producing D-glucaric acid and a salt thereof, which comprises obtaining an alcohol dehydrogenase ADH (1) and an aldehyde dehydrogenase ALDH (2) in the form of (a) a cultivated strain of Pseudogluconobacter saccharoketogeneses Rh47-3 that expresses the alcohol dehydrogenase ADH (1) and the aldehyde dehydrogenase ALDH (2), said cultivated strain has reduced or eliminated activity of an aldehyde dehydrogenase ALDH (3), or (b) a processed product of the cultivated strain, mixing the cultivated strain or the processed product with an aqueous solution of one or more saccharides selected from the group consisting of D-glucose, D-gluconic acid, and L-guluronic acid, adjusting the saccharides concentration within the range of 1 to 5% (w/v), adding one or more alkali compounds selected from the group consisting of sodium hydroxide, potassium hydroxide, and calcium carbonate, whereby producing D-glucaric acid from L-guluronic acid without generating D-glucaraldehyde or D-glucuronic acid, and recovering the D-glucaric acid or the salt thereof resulting therefrom.

14. The method according to claim 13, wherein the processed product thereof is a cell disruptate, a cell extract or an acetone powder thereof.

15. The method according to claim 13, which further comprises adjusting the concentration of D-glucose in the reaction mixture to 1 to 2% (w/v).

16. The method according to claim 13, which further comprises adjusting the pH of the reaction mixture to 5 to 8, preferably about 7.0.

17. The method according to claim 13, wherein the activity of the aldehyde dehydrogenase ALDH(2), which is expressed by the strain of Pseudogluconobacter saccharoketogenes Ph47-3 (FERM BP-10820), is higher than that of the alcohol dehydrogenase ADH(1) expressed thereby.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] FIG. 1 is a figure showing reaction properties of PQQ-ADH (1) and PQQ-ALDH (2). An arrow having a larger size indicates a higher reactivity;

[0116] FIG. 2 is a diagram showing the result of mass spectrometry of intermediate A;

[0117] FIG. 3 is a diagram showing the result of mass spectrometry of intermediate B;

[0118] FIG. 4 is a diagram showing the result of .sup.1H-NMR of intermediate B;

[0119] FIG. 5 is a diagram showing the result of .sup.13C-NMR of intermediate B;

[0120] FIG. 6 a diagram showing the result of H,H-COSY NMR of intermediate B;

[0121] FIG. 7 a diagram showing the result of C,H-COSY NMR of intermediate B;

[0122] FIG. 8 shows elution patterns on ion exchange chromatography. A: Rh47-3 strain and B: K591s strain;

[0123] FIG. 9 shows a profile over time of oxidation reaction with a substrate D-glucose;

[0124] FIG. 10 shows a profile over time of oxidation reaction with a substrate sodium D-gluconate;

[0125] FIG. 11 shows the base sequence and the amino acid sequence of SEQ ID NO: 1;

[0126] FIG. 12 is the continuation of SEQ ID NO: 1;

[0127] FIG. 13 shows the base sequence and the amino acid sequence of SEQ ID NO: 2;

[0128] FIG. 14 is the continuation of SEQ ID NO: 2;

[0129] FIG. 15 shows the base sequence and the amino acid sequence of SEQ ID NO: 3;

[0130] FIG. 16 is the continuation of SEQ ID NO: 3; and

[0131] FIG. 17 shows the base sequences of SEQ ID NOs: 4 to 9.

BEST MODE FOR CARRYING OUT THE INVENTION

[0132] The present invention is hereinafter specifically described based on Examples which do not limit the present invention.

EXAMPLE 1

[0133] In the present Example, the production ability of D-glucaric acid was compared between strains of Pseudogluconobacter saccharoketogenes.

[0134] (1) Cultivation of Pseudogluconobacter saccharoketogenes

[0135] The strains used were Pseudogluconobacter saccharoketogenes Rh47-3 strain, Pseudogluconobacter saccharoketogenes TH14-86 strain, Pseudogluconobacter saccharoketogenes 12-5 strain, Pseudogluconobacter saccharoketogenes K591s strain, Pseudogluconobacter saccharoketogenes 12-4 strain, Pseudogluconobacter saccharoketogenes 12-15 strain and Pseudogluconobacter saccharoketogenes 22-3 strain.

[0136] One platinum loop of each strain grown on an agar slant medium was inoculated in a test tube containing 10 mL of a preculture medium containing 1.0% of lactose, 1.0% of yeast extract, 2.0% of corn steep liquor and 0.3% ammonium sulphate (pH 7.0) and was subjected to the shake culture that carried out at 30° C. for 72 hours to prepare a preculture solution. The preculture solution (1 mL) was then inoculated into a Sakaguchi flask containing 100 mL of a main culture medium containing 2.0% of lactose, 0.5% of yeast extract, 1.0% of corn steep liquor, 0.5% of ammonium sulphate, 0.1% of ferrous sulphate and 0.01% of lanthanum chloride (pH 7.0) and was subjected to the shake culture that carried out at 30° C. for 72 hours.

[0137] (2) Measurement of ADH (1) Activity and ALDH (2 and 3) Activity

[0138] The ADH (1) activity and the ALDH (2 and 3) activity of the cultivated strains were measured as follows.

[0139] 1) Measurement of Activity of ADH (1)

[0140] <Reagents>

[0141] Substrate solution: 0.2 M glucose solution

[0142] Buffer: Mcllvaine buffer (pH 5.0)

[0143] Potassium ferricyanide solution: 0.1 M potassium ferricyanide solution

[0144] Reaction termination solution: 5 g of ferric sulphate and 95 mL of phosphoric acid were dissolved in pure water and adjusted to 1 L.

[0145] <Measurement Procedures>

[0146] The culture solution (1 mL) was centrifuged at 10,000 rpm for 5 minutes to recover the cells. The cells were resuspended in 1 mL of 0.9% saline. The cell suspension was appropriately diluted in 0.9% saline to obtain a crude enzyme solution. In a test tube 250 μL of the buffer, 500 μL of the substrate solution and 50 μL of the crude enzyme solution were placed and preliminarily heated to 30° C. for 5 minutes.

[0147] To the mixture 200 μL of the potassium ferricyanide solution was added to initiate oxidation reaction. After 10 minutes, 500 μL of the reaction termination solution was added to terminate the reaction. The reaction solution was added with 3.5 mL of pure water, left to stand in the dark at room temperature for 20 minutes and measured for the absorbance at 660 nm. For the control, pure water was used instead of the substrate solution. In this measurement, 1 U is defined as the amount of enzyme required to oxidize 1 μM of the substrate per minute.

[0148] 2) Measurement of Activity of ALDH (2 and 3)

[0149] <Reagents>

[0150] Substrate solution: 0.2 M sodium glyoxylate solution (adjusted to pH 8.0 with sodium hydroxide)

[0151] Buffer: McIlvaine buffer (pH 8.0)

[0152] Other reagents and preparation methods thereof and measurement procedures are in accordance with the measurement of ADH (1) activity.

[0153] The results of measurement of enzyme activities of the strains are shown in Table 1. All strains had equivalent ADH (1) activity. Rh47-3 strain showed lower ALDH (2 and 3) activity than other strains.

TABLE-US-00001 TABLE 1 ADH (1) activity and ALDH (2 and 3) activity of strains ADH (1) ALDH (2 and 3) Strain activity (U/mL) activity (U/mL) Rh47-3 3.8 27.3 TH14-86 4.1 278.8 12-5 4.3 170.0 K591s 3.6 286.6 12-4 4.0 118.6 12-15 4.3 115.0 22-3 3.7 143.3

[0154] (3) Production of D-glucaric Acid

[0155] The culture solution (10 mL) was centrifuged at 10,000 rpm for 5 minutes to recover the cells. A substrate solution (10 mL; 60 mM D-glucose, 100 mM sodium D-gluconate, 50 mM sodium D-glucuronate) containing the equivalent amount of calcium carbonate was added to the cells of each strain and was subjected to the oxidation reaction that carried out at 30° C. for 60 hours while shaking. To the reaction solution (20 μL) 980 μL of 0.2 N hydrochloric acid was added to dissolve calcium carbonate followed by analysis for reaction products under the following conditions:

[0156] <Analysis of Reaction Products>

[0157] Instrument: carbohydrate analysis system ICS-3000, available from DIONEX

[0158] Analytical column: CarboPac PA-1 (inner diameter 4 mm×250 mm)

[0159] Detector: a pulsed amperometric detector

[0160] Eluent A: 100 mM sodium hydroxide

[0161] Eluent B: 100 mM sodium hydroxide containing 1 M sodium acetate

[0162] Analysis period: 12 minutes

[0163] Gradient condition: the concentration of eluent B was linearly increased from 0% to 100% over 12 minutes from the start of the analysis.

[0164] Column temperature: 35° C.

[0165] Flow rate: 1.0 mL/min

[0166] Standard substances used were D-glucose (Wako Pure Chemical Industries, Ltd.), D-gluconic acid sodium salt (Wako Pure Chemical Industries, Ltd.), D-glucuronic acid sodium salt monohydrate (Wako Pure Chemical Industries, Ltd.), 2-keto-D-gluconic acid hemicalcium salt (Wako Pure Chemical Industries, Ltd.) and D-glucaric acid monopotassium salt (SIGMA). Intermediate A and a sodium salt of intermediate B were prepared according to Examples 2 and 3.

[0167] The molar yield of each product after reaction with D-glucose is shown in Table 2 for Rh47-3 strain as well as other strains. The molar yield of each product after reaction with sodium D-gluconate and the molar yield of each product after reaction with sodium D-glucuronate are shown in Tables 3 and 4, respectively. With any of the substrates, Rh47-3 strain produced the highest amount of D-glucaric acid. Each strain other than Rh47-3 strain produced mainly 2-keto-D-gluconic acid after reaction with D-glucose or sodium D-gluconate and mainly two unknown substances after reaction with sodium D-glucuronate.

TABLE-US-00002 TABLE 2 Molar yield (%) of products after reaction with D-glucose D-glucuronic D-glucaric 2-keto-D- Other Strain acid acid gluconic acid substances Rh47-3 25.6 50.3 1.3 22.8 TH14-86 6.7 12.9 60.8 19.6 12-5 18.2 10.4 49.5 21.9 K591s 9.4 19.1 53.7 17.8 12-4 16.3 8.4 57.0 18.3 12-15 12.1 11.0 61.2 15.7 22-3 11.5 13.1 55.5 19.9

TABLE-US-00003 TABLE 3 Molar yield (%) of products after reaction with sodium D-gluconate Intermediate D-glucaric 2-keto-D- Other Strain B acid gluconic acid substances Rh47-3 1.6 81.2 2.8 14.4 TH14-86 0 9.7 61.3 29.0 12-5 0 22.3 53.7 24.0 K591s 0 27.2 47.3 25.5 12-4 0 6.4 65.9 27.7 12-15 0 22.1 51.6 26.3 22-3 0 16.2 55.3 28.5

TABLE-US-00004 TABLE 4 Molar yield (%) of products after reaction with sodium D-glucuronate Strain D-glucuronic acid D-glucaric acid Other substances Rh47-3 58.6 36.1 5.3 TH14-86 32.1 12.4 55.5 12-5 41.2 16.5 42.3 K591s 20.8 21.3 57.9 12-4 42.5 18.8 38.7 12-15 39.3 11.7 49.0 22-3 37.7 10.9 51.4

EXAMPLE 2

[0168] In the present Example, intermediate A was prepared during production of D-glucaric acid by Pseudogluconobacter saccharoketogenes and the structure of intermediate A was examined. (1) Preparation of Intermediate A

[0169] To 500 mL of 5% (w/v) D-glucose solution, washed cells (from 500 mL of main culture) of Pseudogluconobacter saccharoketogenes Rh47-3 strain cultivated according to the method described above were added and was subjected to the oxidation reaction that initiated at 30° C., 150 rpm, aeration of 0.2 L/min.

[0170] The analysis of the sample was carried out over time while adjusting pH to 7.0 with 1 M sodium hydroxide solution. When D-glucose of the sample was completely oxidized, the sample was centrifuged at 10,000 rpm for 10 minutes to collect a reaction solution (intermediate A: 17.8%, intermediate B: 2.8%, D-glucuronic acid: 75.2%, D-glucaric acid: 1.9% and other substances: 2.3%).

[0171] A portion of the collected reaction solution (solid matter: 5.0 g) was sequentially applied to a column containing 1 L of strong acid ion exchange resin PK-216 (Mitsubishi Chemical Corporation) and a column containing 3 L of weak base ion exchange resin IRA-96SB (Organo Corporation) in order to elute only intermediate A. The eluate was added with 1% (w/w) of active carbon to decolorize followed by freeze-drying to obtain 0.8 g powder of intermediate A with a purity of 98.0%.

[0172] (2) Examination of Atructure of Intermediate A

[0173] A methanol solution of intermediate A was used for mass spectrometry under the following analytical conditions.

[0174] Instrument: FINNIGAN LCQ-DECA mass spectrometer available from Thermo Quest Corporation

[0175] Ion source: ESI

[0176] Spray voltage: 7 kV

[0177] Capillary voltage: -11 V

[0178] Capillary temperature: 150° C.

[0179] Sample concentration: 50 μg/mL

[0180] Sample introduction rate: 10 μL/min

[0181] The result of mass spectrometry of intermediate A is shown in FIG. 2. As a result, negative ion peaks, [M−H].sup.− 176.9 and [M.sub.2−H].sup.− 354.8, were detected. In the presence of formic acid, two negative ion peaks, [M+HCOO].sup.− 222.8 and [M.sub.2+HCOOO].sup.− 400.7, were detected. Based on the intensity of peaks, it was predicted that the complex with formic acid was produced more easily than negative ions produced by deprotonation and it was confirmed that the complex was not a dicarboxylic acid but a dialdehyde having a molecular mass of 178.

[0182] An aqueous solution of intermediate A sample was used for measurement of reducing ability by the Somogyi-Nelson method and also subjected to a measurement of total sugar amount by the phenol-sulphuric acid method. The results showed that the ratio of (amount of reducing sugar)/(amount of total sugar) was 8.02, confirming the strong reducing ability. From these results, it was predicted that intermediate A had a structure of D-glucaraldehyde which is a derivative of D-glucose having aldehyde groups at both C1 and C6 positions.

EXAMPLE 3

[0183] In the present Example, intermediate B was prepared during production of D-glucaric acid by Pseudogluconobacter saccharoketogenes and the structure of intermediate B was examined.

[0184] (1) Preparation of Sodium Salt of Intermediate B

[0185] To 500 mL of 3% (w/v) sodium D-gluconate solution, centrifuged cells (from 500 mL of main culture) of Pseudogluconobacter saccharoketogenes Rh47-3 strain cultivated according to the method described above were added and was subjected to the oxidation reaction that initiated at 30° C., 150 rpm, aeration of 0.2 L/min. The analysis of the sample was carried out over time while adjusting pH to 7.0 with 1 M sodium hydroxide solution. When D-gluconic acid was completely oxidized, the sample was centrifuged at 10,000 rpm for 10 minutes to collect a reaction solution (intermediate B: 71.4%, D-glucaric acid: 26.0% and other substances: 2.6%).

[0186] A portion of the collected reaction solution (solid matter: 5.0 g) was concentrated to a solid matter of 70% followed by cooling to 20° C. to crystallize the sodium salt of intermediate B. The crystal was washed with 30% (v/v) ethanol and then dried under reduced pressure at 30° C. for 3 hours to obtain 1.3 g of the sodium salt of intermediate B with a purity of 99.5%.

[0187] (2) Examination of Structure of Intermediate B

[0188] A methanol solution of intermediate B was used for mass spectrometry under the following analytical conditions.

[0189] Instrument: FINNIGAN LCQ-DECA mass spectrometer available from Thermo Quest Corporation

[0190] Ion source: ESI

[0191] Spray voltage: 5 kV

[0192] Capillary voltage: -11 V

[0193] Capillary temperature: 250° C.

[0194] Sample concentration: 50 μg/mL

[0195] Sample introduction rate: 10 μL/min

[0196] The result of mass spectrometry of intermediate B is shown in FIG. 3.

[0197] As a result, negative ion peaks, [M.sub.2−H].sup.− 193.0 and [M.sub.2−H].sup.− 386.8 and Na[M.sub.2−H.sub.2].sup.− 408.9 were detected. It was predicted that the molecular mass of intermediate B was 194.

[0198] FIGS. 4, 5, 6 and 7 respectively show the results of .sup.1H-NMR (300 MHz, D.sub.2O), .sup.13C-NMR (75 MHz, D.sub.2O), H,H-COSY (300 MHz, D.sub.2O) and C,H-COSY (75 MHz, D.sub.2O) of intermediate B.

[0199] According to the results of .sup.1H-NMR and .sup.13C-NMR, the spectra were assigned as follows: .sup.1H-NMR (300 MHz, D.sub.2O) δ4.86 (d, 1H, .sup.3J.sub.1,2=8.6 Hz, H1), 4.33 (s, 1H, H5), 4.08 (s, 2H, H3 and H4), 3.63 (d, 1H, .sup.3J.sub.1,2=8.6 Hz, H1). .sup.13C-NMR (75 MHz, D.sub.2O) δ178.6 (C6), 96.2 (C1), 77.1 (C5), 74.0 (C3 or C4), 73.8 (C4 or C3), 71.5 (C2).

[0200] From these results, intermediate B had a structure derived from D-glucose having a carboxyl group and an aldehyde group on each end that was different from the structure of D-glucuronic acid, and thus it was predicted that intermediate B was L-guluronic acid (uronic acid of L-gulose).

EXAMPLE 4

[0201] In this Example, ADH (1), ALDH (2) and ALDH (3) were purified.

[0202] (1) Cultivation of Pseudogluconobacter saccharoketogenes

[0203] One platinum loop of each of Pseudogluconobacter saccharoketogenes Rh47-3 strain and Pseudogluconobacter saccharoketogenes K591s strain grown on an agar slant medium was inoculated in a test tube containing 10 mL of a seed culture medium containing 1.0% of lactose, 1.0% of yeast extract, 2.0% of corn steep liquor and 0.3% ammonium sulphate (pH 7.0) and was subjected to the shake culture that carried out at 30° C. for 72 hours.

[0204] Then 1 mL of each seed culture solution was inoculated into three Sakaguchi flasks respectively containing 100 mL of a preculture medium containing 1.0% of lactose, 1.0% of yeast extract, 2.0% of corn steep liquor and 0.3% of ammonium sulphate (pH 7.0) and was subjected to the shake culture that carried out at 30° C. for 72 hours.

[0205] Finally the whole amount of preculture solution was inoculated to a fermenter containing 30 L of a main culture medium containing 2.0% of lactose, 0.5% of yeast extract, 1.0% of corn steep liquor, 0.5% of ammonium sulphate, 0.1% of ferrous sulphate and 0.01% lanthanum chloride (pH 7.0) and was subjected to the main culture that carried out under the conditions of 30° C., aeration of 10 L/min, stirring of 250 rpm for 72 hours. The culture solution was centrifuged at 10,000 rpm for 10 minutes. The recovered cells were washed twice with 0.9% saline. Accordingly from the 30-L culture solution, about 90 g of wet cells of each strain were obtained.

[0206] (2) Preparation of Cell Extract Fraction

[0207] The recovered wet cells (30 g) were suspended in 100 mL of 10 mM phosphate buffer (pH 6.5) and disrupted on a French press. The solution obtained by disruption was centrifuged at 18,000 rpm for 15 minutes and the supernatant thereof was recovered. The obtained supernatant was further centrifuged at 30,000 rpm for 60 minutes and the supernatant thereof was recovered as a cell extract fraction.

[0208] (3) Ion Exchange Chromatography

[0209] Each cell extract fraction of Rh47-3 strain and K591s strain was applied to a column (inner diameter 5.6 cm×5 cm) containing TOYOPEARL DEAE-650M (Tosoh Corporation) which had been previously equilibrated with 10 mM phosphate buffer (pH 6.5) (hereinafter abbreviated as “buffer”) containing 100 mM glycerol.

[0210] After washing the column with 150 mL of the buffer, enzymes were eluted with a linear gradient so as to attain the concentration of sodium chloride of 0.35 M in the buffer over 500 mL. Fractions (5 mL each) were collected and measured for ADH (1) activity and ALDH (2 and 3) activity according to the methods for activity measurement shown in Example 1.

[0211] The elution pattern is shown in FIG. 8 (A: Rh47-3 strain, B: K591s strain). The peak having ADH (1) activity was eluted at almost the same position for both Rh47-3 and K591s strains. The fractions having ADH (1) activity were collected for each strain and were subjected to concentration and desalting with an ultrafiltration membrane having a molecular weight cut off of 10,000.

[0212] Meanwhile with regard to the peaks having ALDH (2 for Rh47-3 strain and 3 for K591s strain) activity, one peak was observed before the ADH (1) active fractions in Rh47-3 strain and two peaks were observed before and after the ADH (1) active fractions in K591s strain. Among these active peaks, fractions having high activities, namely fractions of peak A (Rh47-3 strain) and peak B (K591s strain) were collected and were subjected to concentration and desalting with an ultrafiltration membrane having a molecular weight cut off of 10,000. For both Rh47-3 and K591s strains, ALDH (3) activity was observed in non-adsorbed fractions. However because the enzyme activity was low, purification was not carried out.

[0213] (4) Hydrophobic Chromatography

[0214] To each fraction (about 3 mL) obtained by ion exchange chromatography, an equivalent amount of the buffer containing 3 M ammonium sulphate was added followed by centrifugation at 10,000 rpm for 20 minutes to remove insoluble substances. The obtained supernatant was applied to a column (inner diameter 3.0 cm×10 cm) containing TOYOPEARL Butyl-650 (Tosoh Corporation) which had been previously equilibrated with the buffer containing 1.5 M ammonium sulphate.

[0215] After washing the column with 100 mL of the buffer containing 1.5 M ammonium sulphate, adsorbed enzymes were eluted with a linear gradient so as to attain the concentration of ammonium sulphate of 0 M over 300 mL. Fractions having ADH (1) activity and ALDH (2 for Rh47-3 strain and 3 for K591s strain) activity were collected and were subjected to concentration and desalting with an ultrafiltration membrane having a molecular weight cut off of 10,000. For K591s strain, ALDH (3) activity was observed in non-adsorbed fractions. However because the enzyme activity was low, further purification was not carried out.

[0216] (5) Gel Filtration Chromatography

[0217] The fractions obtained by hydrophobic chromatography were subjected to a TSK-gel G3000SW column (inner diameter 6.0 mm×40 cm, Tosoh Corporation) which had been previously equilibrated with 10 mM phosphate buffer (pH 6.5) containing 0.1 M sodium chloride. Elution was carried out at a flow rate of 0.6 mL/min and detection was carried out with a UV detector (280 nm).

[0218] Accordingly PQQ-ADH (1) having ADH (1) activity was purified from Rh47-3 and K591s strains, PQQ-ALDH (2) having ALDH (2) activity was purified from Rh47-3 strain and PQQ/Heme-ALDH (4) having ALDH (3) activity was purified from K591s strain. The total activity, total amount of protein and specific activity of the purified enzymes is shown in Table 5.

TABLE-US-00005 TABLE 5 Total activity, total amount of protein and specific activity of purified enzymes Total Total amount of Specific activity protein activity Enzyme (U) (mg) (U/mg) Rh47-3 PQQ-ADH (1) 2,054 53.2 38.6 strain PQQ-ALDH (2) 22,320 2.5 8,928 K591s PQQ-ADH (1) 1,408 39.1 36.0 strain PQQ/Heme-ALDH (4) 132,775 4.8 27,661

EXAMPLE 5

[0219] In the present Example, PQQ-ADH (1) was characterized.

[0220] ADH (1) enzymes purified respectively from Pseudogluconobacter saccharoketogenes Rh47-3 strain and Pseudogluconobacter saccharoketogenes K591s strain exhibited the identical properties and were identified as PQQ-ADH (1).

[0221] (1) Molecular Weight

[0222] Based on the result of SDS-PAGE analysis of the purified enzyme, it was determined that the enzyme had a molecular weight of 64,000±5,000. The molecular weight determined by gel filtration chromatography as described in Example 4 (5) was 120,000±10,000. Therefore it was predicted that the enzyme is a dimer of identical subunits.

[0223] (2) N-terminal Amino Acid Sequence

[0224] After transferring the purified enzyme on a PVDF membrane, an N-terminal amino acid sequence of the enzyme was analyzed on an automated protein primary structure analyzer PPSQ-21A (Shimadzu Corporation). It was found that the amino acid sequence was Ala-Glu-Thr-Thr-Ser-Glu-Arg-Leu-Leu-Asn.

[0225] (3) Prosthetic Group

[0226] To a solution (50 μL) of the purified enzyme (360.0 μg), 50 μL of 1N hydrochloric acid and 250 μL of methanol were added. The mixture was thoroughly mixed and then centrifuged at 12,000 rpm for 5 minutes. The supernatant (20 μ) thereof was analyzed by HPLC under the following conditions. Separately, 10 μL of 2 mM dithiothreitol (DTT) was added to 50 μL of the supernatant. The mixture was then analyzed by HPLC in the similar manner to examine whether or not the enzyme contained pyrroloquinoline quinone (PQQ).

[0227] Column: Cadenza CD-C18 (inner diameter 4.6 mm×7.5 cm, Imtakt Corporation)

[0228] Mobile phase: 30% (v/v) methanol containing 1% (v/v) of 85% phosphoric acid

[0229] Flow rate: 1.0 mL/min

[0230] Temperature: 35° C.

[0231] Detector: UV (254 nm)

[0232] As a result, the enzyme extract showed the same retention time as a standard PQQ (pyrroloquinoline quinone disodium salt, Kanto Chemical Co., Inc.). The enzyme extract after reducing treatment with DTT also had the same retention time as the standard PQQ after reducing treatment with DTT. From these results, it was suggested that PQQ-ADH (1) contains PQQ as a component.

[0233] (4) Optimal pH

[0234] Oxidation activity was measured in 0.15 M GTA buffer having pH ranging from 3.0 to 10.0. As a result, PQQ-ADH (1) had an optimal pH of 5.0 to 5.5.

[0235] (5) Substrate Specificity

[0236] Each substrate (D-glucose, sodium D-gluconate, sodium D-glucuronate, intermediate A, sodium salt of intermediate B) was dissolved in Mcllvaine buffer (pH 5.0) and the activity of ADH was measured as described in Example 1 to study the substrate specificity of PQQ-ADH (1) . The results are shown in Table 6. The enzyme activity for each substrate is expressed as a relative activity to the activity measured with D-glucose as a substrate that was set to be 100.

TABLE-US-00006 TABLE 6 Substrate specificity of PQQ-ADH (1) Substrate Relative activity (%) 0.2M D-glucose 100 0.2M sodium D-gluconate 88.6 0.2M sodium D-glucuronate <1 0.02M intermediate A 59.1 0.2M sodium salt of intermediate B 2.7

[0237] (6) Analysis of Reaction Products

[0238] To each substrate solution (100 μL; 50 mM D-glucose, 50 mM sodium D-gluconate, 50 mM sodium D-glucuronate, 50 mM intermediate A or 50 mM sodium salt of intermediate B) containing 50 mM calcium carbonate, 90 μL of 100 mM potassium ferricyanide solution and 10 μL (46 U) of a solution of purified PQQ-ADH (1) were added and were subjected to the oxidation reaction that initiated at 30° C. The reaction products were quantified on the carbohydrate analysis system indicated in Example 1. The molar yield of the reaction products at 16 hours after the initiation of the reaction is shown in Table 7.

TABLE-US-00007 TABLE 7 Molar yield (%) of reaction products D-gluconic Intermediate Intermediate D-glucuronic D-glucaric Substrate D-glucose acid A B acid acid D-glucose 1.3 0 68.8 0 29.9 0 Sodium — 22.6 — 77.4 — 0 D-gluconate Sodium — — — — 100.0  0 D-glucuronate Intermediate A — — 32.7 0.8 66.5 0 Sodium salt of — — — 97.9 — 2.1 intermediate B

EXAMPLE 6

[0239] In the present Example, PQQ-ALDH (2) was characterized.

[0240] PQQ-ALDH (2) purified from Pseudogluconobacter saccharoketogenes Rh47-3 strain exhibited the following properties.

[0241] (1) Molecular Weight

[0242] From the result of SDS-PAGE analysis of a solution of the purified enzyme, it was determined that the enzyme had a molecular weight of 61,000±5,000. The molecular weight determined by gel filtration chromatography as described in Example 4 (5) was 180,000 ±10,000. Therefore it was predicted that the enzyme is a trimer of identical subunits.

[0243] (2) N-terminal Amino Acid Sequence

[0244] After transferring the purified enzyme on a PVDF membrane, an N-terminal amino acid sequence of the enzyme was analyzed on an automated protein primary structure analyzer. However, the amino acid sequence could not be determined because the N-terminal was blocked.

[0245] (3) Internal Amino Acid Sequence

[0246] To 100 μL of a solution of the purified enzyme (220.8 μg), 10 μL of a V8 protease solution (0.1 mg of V8 protease dissolved in 1 mL of 0.1 M Tris-hydrochloric acid buffer (pH 8.0)) was added and was subjected to the reaction that allowed to proceed at 30° C. for 16 hours. Peptide fragments were separated by SDS-PAGE and transferred onto a PVDF membrane.

[0247] The internal amino acid sequence of the enzyme was analyzed on an automated protein primary structure analyzer and it was found that a peptide fragment having a molecular weight of 17 kDa had the amino acid sequence: Phe-Xaa-Ser-Asn-Thr-Asp-Val-Asn-Pro-Leu.

[0248] (4) Prosthetic Group

[0249] According to the result of the analysis carried out as the method described in Example 5 (3), it was suggested that PQQ-ALDH (2) contains PQQ as a component.

[0250] (5) Optimal pH

[0251] Oxidation activity was measured in GTA buffer having pH ranging from 4.0 to 10.0. As a result, PQQ-ALDH (2) had an optimal pH of 7.5 to 8.0.

[0252] (6) Substrate Specificity

[0253] Each substrate (D-glucose, sodium D-gluconate, sodium D-glucuronate, intermediate A, sodium salt of intermediate B, sodium glyoxylate) was dissolved in Mcllvaine buffer (pH 8.0) and the activity of ALDH was measured as described in Example 1 to study the substrate specificity of PQQ-ALDH (2). The results are shown in Table 8. The enzyme activity for each substrate is expressed as a relative activity to the activity measured with the sodium salt of intermediate B as a substrate that was set to be 100.

TABLE-US-00008 TABLE 8 Substrate specificity of PQQ-ALDH (2) Substrate Relative activity (%) 0.2M D-glucose 11.8 0.2M sodium D-gluconate <1 0.2M sodium D-glucuronate 18.6 0.02M intermediate A 476 0.2M sodium salt of intermediate B 100 0.2M sodium glyoxylate 3,050

[0254] (7) Analysis of Reaction Products

[0255] To each substrate solution (100 μL; 50 mM D-glucose, 50 mM sodium D-gluconate, 50 mM sodium D-glucuronate, 50 mM intermediate A or 50 mM sodium salt of intermediate B) containing 50 mM calcium carbonate, 90 μL of 100 mM potassium ferricyanide solution and 10 μL (188 U) of a solution of purified PQQ-ALDH (2) were added and were subjected to the oxidation reaction that initiated at 30° C. The reaction products were quantified on the carbohydrate analysis system indicated in Example 1. The molar yield of the reaction products at 16 hours after the initiation of the reaction is shown in Table 9.

TABLE-US-00009 TABLE 9 Molar yield (%) of reaction products D-gluconic Intermediate Intermediate D-glucuronic D-glucaric Substrate D-glucose acid A B acid acid D-glucose 97.8 2.2 0   0 0  0 Sodium — 100.0 — 0 — 0 D-gluconate Sodium — — — — 93.5 6.5 D-glucuronate Intermediate A — — 0.8 23.4 59.8 16.0 Sodium salt of — — — 67.7 — 32.3 intermediate B

EXAMPLE 7

[0256] In the present Example, PQQ/Heme-ALDH (4) was characterized.

[0257] PQQ/Heme-ALDH (4) purified from Pseudogluconobacter saccharoketogenes K591s strain exhibited the following properties.

[0258] (1) Molecular Weight

[0259] From the result of SDS-PAGE analysis of a solution of the purified enzyme, it was determined that the enzyme had a molecular weight of 59,000±5,000. The molecular weight determined by gel filtration chromatography under the conditions described in Example 4 (5) was 130,000±10, 000. Therefore it was predicted that the enzyme is a dimer of identical subunits.

[0260] (2) N-terminal Amino Acid Sequence

[0261] After transferring the purified enzyme on a PVDF membrane, an N-terminal amino acid sequence of the enzyme was analyzed on an automated protein primary structure analyzer. However, the amino acid sequence could not be determined because the N-terminal was blocked.

[0262] (3) Internal Amino Acid Sequence

[0263] To 100 μL of a solution of the purified enzyme (200.5 μg), 10 μL of a V8 protease solution (0.1 mg of V8 protease dissolved in 1 mL of 0.1 M Tris-hydrochloric acid buffer (pH 8.0)) was added and was subjected to the reaction that allowed to proceed at 27° C. for 16 hours. Peptide fragments were separated by SDS-PAGE and transferred onto a PVDF membrane. The internal amino acid sequence of the enzyme was analyzed on an automated protein primary structure analyzer and it was found that a peptide fragment having a molecular weight of 37 kDa had the amino acid sequence: Ala-Ser-Trp-Asn-Gly-Val-Pro-Pro-Glu-Asn.

[0264] (4) Prosthetic Group

[0265] According to the result of the analysis carried out as the method described in Example 5 (3), it was suggested that PQQ/Heme-ALDH (4) contains PQQ as a component. Separately a solution of the purified enzyme (100.4 μg/mL) was added with 1/20 amount of 1M Tris-hydrochloric acid buffer (pH 9.0) followed by addition of sodium dithionite up to a final concentration of 5 mM. The absorption spectrum of the solution was measured. The result showed the absorption maximum at 522 and 550 nm, and thus it was suggested that PQQ/Heme-ALDH (4) contains, in addition to PQQ, Heme c as a prosthetic group.

[0266] (5) Optimal pH

[0267] Oxidation activity was measured in GTA buffer having pH ranging from 4.0 to 10.0. As a result, PQQ-ALDH (4) had an optimal pH of 7.5 to 8.0.

[0268] (6) Substrate Specificity

[0269] Each substrate (D-glucose, sodium D-gluconate, sodium D-glucuronate, intermediate A, sodium salt of intermediate B, sodium glyoxylate) was dissolved in Mcllvaine buffer (pH 8.0) and the activity of ALDH was measured as described in Example 1 to study the substrate specificity of PQQ/Heme-ALDH (4). The results are shown in Table 10. The enzyme activity for each substrate is expressed as a relative activity to the activity measured with sodium D-gluconate as a substrate that was set to be 100.

TABLE-US-00010 TABLE 10 Substrate specificity of PQQ/Heme-ALDH (4) Substrate Relative activity (%) 0.2M D-glucose 35.1 0.2M sodium D-gluconate 100 0.2M sodium D-glucuronate 2.9 0.02M intermediate A 11.6 0.2M sodium salt of intermediate B 21.1 0.2M sodium glyoxylate 20,260

[0270] (7) Analysis of Reaction Products

[0271] To each substrate solution (100 μL; 50 mM D-glucose, 50 mM sodium D-gluconate, 50 mM sodium D-glucuronate, 50 mM intermediate A or 50 mM sodium salt of intermediate B) containing 50 mM calcium carbonate, 90 μL of 100 mM potassium ferricyanide solution and 10 μL (1,680 U) of a solution of purified PQQ/Heme-ALDH (4) were added and was subjected to the oxidation reaction that initiated at 30° C. The reaction products were quantified on the carbohydrate analysis system indicated in Example 1. The molar yield of the reaction products at 16 hours after the initiation of the reaction is shown in Table 11. A slight amount of D-glucaric acid was produced only when sodium D-glucuronate was used as a substrate.

TABLE-US-00011 TABLE 11 Molar yield (%) of reaction products 2-keto- D-gluconic Intermediate Intermediate D-gluconic D-glucuronic D-glucaric Other Substrate D-glucose acid A B acid acid acid substances D-glucose 22.4 0.8 0  0 72.1 0 0 4.7 Sodium — 0 — 0 84.8 — 0 15.2 D-gluconate Sodium — — — — —  94.1 5.3 0.6 D-glucuronate Intermediate A — — 69.6 12.0 — 0 0 18.4 Sodium salt of — — — 58.1 — 0 0 41.9 intermediate B

EXAMPLE 8

[0272] In the present Example, the purified enzymes were used for production of D-glucaric acid.

[0273] To each substrate solution (100 μL; 50 mM D-glucose, 50 mM sodium D-gluconate, 50 mM sodium D-glucuronate) containing 50 mM calcium carbonate, 90 μL, of 100 mM potassium ferricyanide solution and 5 μL of a solution of a purified enzyme (PQQ-ADH (1): 23 U, PQQ-ALDH (2): 94 U or PQQ/Heme-ALDH (4): 840 U) or pure water were added and were subjected to the reaction that carried out at 30° C. for 36 hours. The produced D-glucaric acid or 2-keto-D-gluconic acid were quantified on the carbohydrate analysis system indicated in Example 1.

[0274] The results are shown in Table 12. The combination of PQQ-ADH (1) and PQQ-ALDH (2) produced D-glucaric acid from D-glucose or D-gluconic acid. The combination of PQQ-ADH (1) and PQQ/Heme-ALDH (4) did not produce D-glucaric acid but mainly produced 2-keto-D-gluconic acid from D-glucose or D-gluconic acid.

TABLE-US-00012 TABLE 12 Reactions for producing D-glucaric acid and 2-keto-D-gluconic acid using purified enzymes Yield (mM) D-glucaric 2-keto-D-gluconic Substrate Reaction condition acid acid D-glucose PQQ-ADH (1) + 0 0 pure water PQQ-ALDH (2) + 0 0 pure water PQQ-ADH (1) + 12.8 0 PQQ-ALDH (2) PQQ-ADH (1) + 0 18.3 PQQ/Heme-ALDH (4) Sodium PQQ-ADH (1) + 0 0 D-gluconate pure water PQQ-ALDH (2) + 0 0 pure water PQQ-ADH (1) + 21.7 0 PQQ-ALDH (2) PQQ-ADH (1) + 0 24.1 PQQ/Heme-ALDH (4) Sodium PQQ-ADH (1) + 0 0 D-glucuronate pure water PQQ-ALDH (2) + 2.8 0 pure water PQQ-ADH (1) + 2.5 0 PQQ-ALDH (2) PQQ-ADH (1) + 3.3 0 PQQ/Heme-ALDH (4)

EXAMPLE 9

[0275] In the present Example, analysis of the draft genome of Pseudogluconobacter saccharoketogenes Rh47-3 strain was carried out.

[0276] Genomic DNA was recovered from cultivated cells of Pseudogluconobacter saccharoketogenes Rh47-3 strain using GenElute™ Bacterial Genomic DNA kit (SIGMA) . The genomic DNA was fragmented to respectively have a size of about 500 bp and a biotinylated adaptor was ligated to each fragment. Single-stranded DNA was recovered with streptavidin magnetic beads and the size and concentration of DNA were detected on Bioanalyzer 2100 (Agilent).

[0277] Single-stranded DNA was mixed with beads onto which a complementary sequence of the adaptor was immobilized and subjected to emulsion PCR. After PCR, an appropriate amount of beads were added to a plate and the base sequence was analyzed by pyrosequencing using GS Titanium Sequencing kit XLR70 and Genome Sequencer FLX System (Roche Diagnostics).

[0278] The results showed that the number of total bases analyzed were 106,737,181 bases, the number of contigs of 4 kbase or more was 20 and the number of total bases in contigs of 4 kbase or more was 3,875,227 bases.

[0279] The base sequence analyzed contained 4,345 predicted amino acid coding regions, among which 273 coding regions had homology with dehydrogenases based on homology search. These predicted amino acid sequences included the sequences which matched with the N-terminal amino acid sequence of PQQ-ADH (1) , the internal amino acid sequence of PQQ-ALDH (2) and the internal amino acid sequence of PQQ/Heme-ALDH (4) respectively revealed in Examples 5, 6 and 7.

EXAMPLE 10

[0280] In the present Example, the base sequences of PQQ-ADH (1), PQQ-ALDH (2) and PQQ/Heme-ALDH (4) genes were determined.

[0281] Primers were designed by using genomic DNAs of Pseudogluconobacter saccharoketogenes Rh47-3 and K591s strains as templates and based on information of the base sequence of the draft analysis obtained in Example 9.

[0282] The designed primers (PQQ-ADH (1) forward primer: SEQ ID NO: 4, PQQ-ADH (1) reverse primer: SEQ ID NO: 5, PQQ-ALDH (2) forward primer: SEQ ID NO: 6, PQQ-ALDH (2) reverse primer: SEQ ID NO: 7, PQQ/Heme-ALDH (4) forward primer: SEQ ID NO: 8, PQQ/Heme-ALDH (4) reverse primer: SEQ ID NO: 9) and Pfx50 DNA polymerase (Life Technologies) were used in PCR to determine base sequences of gene regions of PQQ-ADH (1), PQQ-ALDH (2) and PQQ/Heme-ALDH (4) of Rh47-3 and K591s strains.

[0283] The base sequence and a predicted amino acid sequence of PQQ-ADH (1) gene derived from Rh47-3 strain are shown in SEQ ID NO: 1. PQQ-ADH (1) gene contains 1,800 bp (599 amino acid residues) and the amino acid sequence thereof has 42% homology with quinoprotein ethanol dehydrogenase derived from Bradyrhizobium sp. PQQ-ADH (1) gene derived from K591s strain had an identical sequence as PQQ-ADH (1) gene derived from Rh47-3 strain.

[0284] The base sequence and a predicted amino acid sequence of PQQ-ALDH (2) gene derived from Rh47-3 strain are shown in SEQ ID NO: 2. PQQ-ALDH (2) gene contains 1,761 bp (586 amino acid residues) and the amino acid sequence thereof has 35% homology with methanol dehydrogenase large subunit protein derived from Pelagibacterium halotolerans.

[0285] PQQ-ALDH (2) gene derived from K591s strain had the base sequence wherein the 1533rd base, A, in the base sequence of PQQ-ALDH (2) gene derived from Rh47-3 strain was replaced by G, which, however, gave the same translated amino acid, leucine (Leu).

[0286] The base sequence and a predicted amino acid sequence of PQQ/Heme-ALDH (4) derived from Rh47-3 strain is shown in SEQ ID NO: 3. PQQ/Heme-ALDH (4) gene contains 1785 bp (594 amino acid residues) and the amino acid sequence thereof has 41% homology with methanol dehydrogenase large subunit protein derived from Pelagibacterium halotolerans.

[0287] PQQ/Heme-ALDH (4) gene derived from K591s strain had the base sequence wherein the 224th base, T, in the base sequence of PQQ/Heme-ALDH (4) gene derived from Rh47-3 strain was replaced by C, which gave the amino acid threonine (Thr) instead of isoleucine (Ile) .

EXAMPLE 11

[0288] In the present Example, large scale production of D-glucaric acid from D-glucose was carried out.

[0289] According to the method described in Example 4, Pseudogluconobacter saccharoketogenes Rh47-3 strain was cultivated in the scale of 30 L. The cells collected by centrifugation (6,000 rpm, 20 minutes) were added to 30 L of 10 g/L D-glucose solution and were subjected to the reaction that carried out under the conditions of a temperature of 30° C., a stirring speed of 150 rpm, aeration of 10 L/min. During the reaction, pH was adjusted to 7.0 with 12% (w/v) sodium hydroxide solution.

[0290] Samples were taken over time and quantified for generated D-glucaric acid on the carbohydrate analysis system indicated in Example 1. The results show, as shown in FIG. 9, that 7.0 g/L (molar yield: 60.3%) of D-glucaric acid was produced at maximum at 68 hours after initiation of the reaction.

EXAMPLE 12

[0291] In the present Example, large scale production of D-glucaric acid from sodium D-gluconate was carried out.

[0292] According to the method described in Example 2, Pseudogluconobacter saccharoketogenes Rh47-3 strain was cultivated in the scale of 30 L. The cells collected by centrifugation (6,000 rpm, 20 minutes) were added to 30 L of 30 g/L sodium D-gluconate solution and were subjected to the reaction that carried out under the conditions of a temperature of 30° C., a stirring speed of 150 rpm, aeration of 10 L/min.

[0293] During the reaction, pH was adjusted to 7.0 with 12% (w/v) sodium hydroxide solution. Samples were taken over time and quantified for generated D-glucaric acid on the carbohydrate analysis system indicated in Example 1. The results show, as shown in FIG. 10, that 22.5 g/L (molar yield: 78.6%) of D-glucaric acid was produced at maximum at 82 hours after initiation of the reaction.

EXAMPLE 13

[0294] In the present Example, the purified enzyme was used for production of D-glucaraldehyde (intermediate A).

[0295] To a substrate solution (100 μL; 50 mM D-glucose) containing 50 mM calcium carbonate, 90 μL of 100 mM potassium ferricyanide solution and 10 μL of a solution of purified PQQ-ADH (1) (46 U) were added and were subjected to the oxidation reaction that initiated at 30° C. The reaction products were quantified on the carbohydrate analysis system indicated in Example 1. At 16 hours after initiation of the reaction, D-glucaraldehyde (intermediate A) was produced at a molar yield of 68.8%.

EXAMPLE 14

[0296] In the present Example, the purified enzyme was used for production of L-guluronic acid (intermediate B).

[0297] To a substrate solution (100 μL; 50mM sodium D-gluconate) containing 50 mM calcium carbonate, 90 μL of 100 mM potassium ferricyanide solution and 10 μL of a solution of purified PQQ-ADH (1) (46 U) were added and were subjected to the oxidation reaction that initiated at 30° C. The reaction products were quantified on the carbohydrate analysis system indicated in Example 1. At 16 hours after initiation of the reaction, L-guluronic acid (intermediate B) sodium salt was produced at a molar yield of 77.4%.

EXAMPLE 15

[0298] In the present Example, large scale production of L-guluronic acid (intermediate B) from D-glucose was carried out.

[0299] According to the method described in Example 4, Pseudogluconobacter saccharoketogenes Rh47-3 strain was cultivated in the scale of 30 L. The cells collected by centrifugation (6,000 rpm, 20 minutes) were added to 30 L of 10 g/L D-glucose solution and were subjected to the reaction that carried out under the conditions of a temperature of 30° C., a stirring speed of 150 rpm, aeration of 10 L/min.

[0300] During the reaction, pH was adjusted to 7.0 with 12% (w/v) sodium hydroxide solution. Samples were taken over time and quantified for generated L-guluronic acid on the carbohydrate analysis system indicated in Example 1. The results show that 2.2 g/L (molar yield: 18.3%) of L-guluronic acid (intermediate B) sodium salt was produced at maximum at 12 hours after initiation of the reaction.

EXAMPLE 16

[0301] In the present Example, large scale production of L-guluronic acid (intermediate B) from sodium D-gluconate was carried out.

[0302] According to the method described in Example 4, Pseudogluconobacter saccharoketogenes Rh47-3 strain was cultivated in the scale of 30 L. The cells collected by centrifugation (6,000 rpm, 20 minutes) were added to 30 L of 30 g/L sodium D-gluconate solution and were subjected to the reaction that carried out under the conditions of a temperature of 30° C., a stirring speed of 150 rpm, aeration of 10 L/min.

[0303] During the reaction, pH was adjusted to 7.0 with 12% (w/v) sodium hydroxide solution. Samples were taken over time and quantified for generated L-guluronic acid on the carbohydrate analysis system indicated in Example 1. The results show that 14.9 g/L (molar yield: 41.3%) of L-guluronic acid (intermediate B) sodium salt was produced at maximum at 24 hours after initiation of the reaction.

INDUSTRIAL APPLICABILITY

[0304] As specifically described above, the present invention relates to a D-glucaric acid producing bacterium and a method for producing D-glucaric acid. According to the present invention, a microorganism having improved productivity of D-glucaric acid can be provided. In addition, by using the microorganism having improved productivity of D-glucaric acid, D-glucaric acid can be inexpensively and effectively produced and provided. A method for producing D-glucaraldehyde (intermediate A) and L-guluronic acid (intermediate B) can also be provided which are intermediates during production of D-glucaric acid. Further alcohol dehydrogenase ADH (1) having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or higher homology therewith and aldehyde dehydrogenase ALDH (2) having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or higher homology therewith can be provided. The present invention is useful because it can provide a novel technique with regard to a microorganism having improved productivity of D-glucaric acid to be used for production of D-glucaric acid and a method for efficiently producing D-glucaric acid.

[0305] Reference to Deposited Microorganism

[0306] Name of international depositary institution: International Patent Organism Depositary, National Institute of Technology and Evaluation

[0307] Address of international depositary institution: #120, 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818 Japan

[0308] Date of Acceptance: 26 Apr. 2007

[0309] Accession Number: FERM BP-10820

[0310] Indication of Microorganism: Rh47-3