JP2005528917A - Aldehyde dehydrogenase - Google Patents

Abstract

The present invention relates to a novel aldehyde dehydrogenase having the following physicochemical properties: a molecular weight of 190,000 ± 15,000 Da (including two α subunits and a subunit structure of one β subunit) or 250,000 ± 20,000 Da Molecular weight (including subunit structure of two α subunits and two β subunits) (where α subunit has a molecular weight of 75,000 ± 3,000 Da and β subunit has a molecular weight of 55,000 ± 2,000 Da) Dehydrogenase activity against L-sorbosone, D-glucosone, D-glucose, and D-xylose; utilizes pyrroloquinoline quinone and heme c as cofactors; optimum pH for vitamin C production from about 6.5 to about 8.0, Optimum pH when producing 2-keto-L-gulonic acid from L-sorbosone: about 9.0; and Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , Mg ²⁺ , monoiodoacetate And to sodium azide More disturbed.

Description

  The present invention converts L-sorbosone to L-ascorbic acid (hereinafter referred to as vitamin C) at neutral pH and L-sorbosone to 2-keto-L-gulonic acid (hereinafter 2- It relates to a novel enzyme involved in both conversion to KGA), namely an aldehyde dehydrogenase (hereinafter referred to as SNDH III). The present invention also provides a process for producing the enzyme and a process for producing vitamin C and / or 2-KGA directly from an aldose such as L-sorbosone using the enzyme.

  Vitamin C is one of the most important and essential nutrients for humans. The metabolic pathways for producing vitamin C have been extensively studied in various organisms. However, there are no reports on purified enzymes related to direct conversion of L-sorbosone to vitamin C. Thus, the enzymes of the present invention are very useful for new vitamin C production processes that replace current processes such as the Reichstein method (Helvetica Chimica Acta 17: 311 (1934)).

The present invention provides a purified aldehyde dehydrogenase having the following physicochemical properties:
a) 190,000 ± 15,000 Da molecular weight (consisting of 2 α subunits and 1 β subunit subunit structure), or 250,000 ± 20,000 Da molecular weight (2 α subunits and 2 β subunits) (Consisting of the subunit structure of the unit) (where the molecular weight of the α subunit is 75,000 ± 3,000 Da and the molecular weight of the β subunit is 55,000 ± 2,000 Da),
b) Substrate specificity: active against aldehyde compounds,
c) Cofactors: pyrroloquinoline quinone (PQQ) and heme c,
d) Optimal pH: about 6.5 to about 8.0 (when producing vitamin C from L-sorbosone) or about 9.0 (when producing 2-keto-L-gulonic acid from L-sorbosone),
e) Inhibitors: Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , Mg ²⁺ , monoiodoacetate, and sodium azide.

  In one embodiment, the present invention relates to an aldehyde dehydrogenase having a molecular weight of 190,000 ± 15,000 Da, having the aforementioned physicochemical properties.

  In a further embodiment, the present invention relates to an aldehyde dehydrogenase having a molecular weight of 250,000 ± 20,000 Da, having the physicochemical properties described above.

  The source of SNDH III of the present invention is not critical. Thus, the SNDH III of the present invention may be produced, for example, by isolation from Gluconobacter or another microorganism capable of producing an aldehyde dehydrogenase having the properties described above, recombinant or It may be produced by chemical synthesis.

  Another object of the present invention is a process for producing the above-mentioned SNDH III. A microorganism belonging to the genus Gluconobacter capable of producing an aldehyde dehydrogenase having the above-mentioned characteristics is added to a nutrient culture solution under aerobic conditions. A process comprising the steps of: culturing the microbial cell, destroying the microbial cell, and isolating the aldehyde dehydrogenase from the cell-free extract of the disrupted microbial cell.

  In one aspect of the present invention, the process of producing SNDH III is performed by culturing a microorganism belonging to the genus Gluconobacter that can produce an aldehyde dehydrogenase having the characteristics described above, wherein a reaction Is carried out at a pH of about 4.5 to about 9.0 and a temperature of about 20 ° C to about 50 ° C. SNDH III produced in this way is useful for both vitamin C production and 2-KGA production.

  A further object of the present invention is a process for producing a carboxylic acid and / or its lactone from its corresponding aldose, producing an aldehyde and purified SNDH III having the aforementioned characteristics or an aldehyde dehydrogenase having the aforementioned characteristics. A process comprising the step of contacting a cell-free extract prepared from a microorganism belonging to the genus Gluconobacter in the presence of an electron acceptor.

  The term “aldose” means an aldehyde in which every carbon atom except the carbonyl carbon atom has a hydroxyl group.

  As used herein, aldoses include, but are not limited to, L-sorbosone, D-glucosone, D-glucose, and D-xylose.

  A preferred lactone is vitamin C, a preferred carboxylic acid is 2-KGA, and a preferred aldose is L-sorbosone.

  In one embodiment, the process of producing a carboxylic acid and / or its lactone from its corresponding aldose was prepared from an aldehyde and purified SNDH III having the characteristics described above or a microorganism belonging to the genus Gluconobacter as described above. Including contacting the cell-free extract, wherein the molecular weight of SNDH III is 190,000 ± 15,000 Da.

  In one embodiment, the process of producing a carboxylic acid and / or its lactone from its corresponding aldose was prepared from an aldehyde and purified SNDH III having the characteristics described above or a microorganism belonging to the genus Gluconobacter as described above. Including contacting the cell-free extract, wherein the molecular weight of SNDH III is 250,000 ± 20,000 Da.

  In one aspect, the present invention is a process for producing a carboxylic acid and / or its lactone from its corresponding aldose, producing an aldehyde and purified SNDH III having the above characteristics or an aldehyde dehydrogenase having the above characteristics It relates to a process comprising the step of contacting a cell-free extract prepared from a microorganism belonging to the genus Gluconobacter. Here, the reaction is carried out at a pH of about 4.5 to about 9.0 and a temperature of about 20 ° C to about 50 ° C. For the production of vitamin C, the reaction is preferably performed at a pH of about 6.5 to about 8.0. For the production of 2-KGA, the reaction is preferably performed at a pH of about 9.0.

  The invention also features the use of a purified aldehyde dehydrogenase having the aforementioned properties in a process for producing a carboxylic acid and / or its lactone from its corresponding aldose comprising the following steps: an aldehyde and a purified aldehyde dehydrogenase or Contacting a cell-free extract prepared from a microorganism belonging to the genus Gluconobacter capable of producing the aldehyde dehydrogenase in the presence of an electron acceptor.

  The physicochemical properties of the purified SNDH III sample prepared according to the examples described below are as follows.

1) Enzyme activity SNDH III of the present invention has the following reaction formula:
According to L-sorbosone + electron acceptor → vitamin C and / or 2-KGA + reduced electron acceptor, the oxidation of L-sorbosone to vitamin C and / or 2-KGA is catalyzed in the presence of the electron acceptor.

  SNDH III does not act using oxygen as an electron acceptor. This was confirmed by SNDH III not converting vitamin C and / or 2-KGA from L-sorbosone using oxygen as a potential electron acceptor. Furthermore, oxygen consumption in the reaction mixture was not detected (oxygen consumption was detected using a dissolved oxygen probe). Furthermore, NAD and NADP are not suitable electron acceptors. However, other conventional electron acceptors can be used with the SNDH III of the present invention. Preferred electron acceptors are 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate (PMS), ferricyanide, and cytochrome c. There is no minimum amount of electron acceptor that must be present to convert at least a portion of the aldehyde substrate to its corresponding acid. However, the amount of oxidizable substrate depends on the amount of a particular electron acceptor and its electron accepting properties.

The enzyme assay was performed as follows.
a) Assay for enzyme activity to convert L-sorbosone to vitamin C or 2-KGA products.
The reaction mixture consisted of 1.0 mM PMS, 25 mM potassium phosphate buffer (pH 7.0), 1.0 μM PQQ, 1.0 mM CaCl ₂ , 50 mM L-sorbosone, and a final volume of 100 μl water containing the enzyme solution. This reaction mixture was prepared immediately prior to the assay. The reaction was carried out at 30 ° C. for 60 minutes unless otherwise specified. The amount of vitamin C, which is an indication of enzyme activity, was measured by a high performance liquid chromatography apparatus (HPLC) at a wavelength of 264 nm. The HPLC system consists of a UV detector (TOSOH UV8000; Tosoh Co., Ltd., 3-2-4 Kyobashi, Chuo-ku, Tokyo, Japan), a dual pump (TOSOH CCPE; Tosoh Corporation), an integrator (Shimadzu C- R6A; Shimadzu Co., Ltd., Nishinokyo Kuwabaramachi, Nakagyo-ku, Kyoto, Japan, and Column (YMC-Pack Polyamine II; YMC, Inc., 3233 Burnt Mill Drive Wilimington, NC 28403, USA) It was. The amount of 2-KGA produced, another indication of enzyme activity, was measured by HPLC as described above. One unit of enzyme activity for each production was defined as the amount of enzyme that produced 1 mg vitamin C and 1 mg 2-KGA, respectively, in the reaction mixture.

b) SNDH III photometric assay The reaction mixture consisted of 0.1 mM DCIP, 1.0 mM PMS, 50 mM potassium phosphate buffer (pH 7.0), 1.0 μM PQQ, 2-100 mM substrate (L-sorbosone, D-glucosone, D- Glucose), and a final volume of 100 μl water containing the enzyme solution. This reaction mixture was prepared immediately prior to the assay. The reaction was started with L-sorbosone at 25 ° C., and the enzyme activity was measured at 600 nm as the initial reduction rate of DCIP. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the reduction of 1 μmol of DCIP per minute. The extinction coefficient of DCIP at pH 7.0 was 14.2 mM- ¹ . The reference cuvette included all the ingredients except L-sorbosone.

  The protein concentration was measured using a protein assay CBB solution (Nacalai tesque, Inc., Kyoto, Japan).

2) The substrate specificity of the substrate-specific enzyme is the same enzyme assay as described in 1b) except that 100 mM potassium phosphate (pH 7.5) or 100 mM Tris-HCl (pH 9.0) was used as the buffer. Determined using the method. The relative activity of SNDH III for D-glucosone (2 mM), D-glucose (100 mM), and D-xylose (100 mM) is as follows: SNDH III for L-sorbosone (2 mM) at both pH 7.5 and pH 9.0. It was higher than the relative activity. However, the relative activity for L-sorbose (100 mM), D-sorbitol (100 mM), and L-gulono-γ-lactone (100 mM) is comparable to that for L-sorbosone at both pH 7.5 and pH 9.0. It was less than 1%. These results are shown in Table 1A.

(Table 1A)
Substrate specificity of purified SNDH III

(Table 1B)
Estimated oxidation products of the substrates shown in Table 1A

3) Optimum pH
The correlation between the reaction rate of SNDH III and the pH value of the reaction mixture was determined by the same assay as described in 1a) except that various pH and 100 mM buffer solutions were used.

  The SNDH III of the present invention showed a relatively high vitamin C production activity at a pH of about 6.5 to about 8.0, and a high 2-KGA production activity at a pH of about 9.0.

4) Influence of temperature The influence of temperature on the enzyme reaction was tested by the same assay as described in 1a) except that various temperatures were used. In both vitamin C and 2-KGA production, the enzyme reaction was stable up to at least 50 ° C.

5) Effect of metal ions and inhibitors The effect of metal ions and inhibitors on the L-sorbosone dehydrogenase activity of the enzyme was investigated by measuring the activity using the same assay as described in 1b) above. . Each compound solution was stirred into a basic reaction mixture and the reaction was initiated by the addition of SNDH III of the present invention. The results are shown in Table 2.

(Table 2)
Effects of inhibitors and metals on the activity of purified SNDH III.

Each compound was added to the reaction mixture at a concentration of 1.0 mM (with the exception that the concentrations of EDTA, NaN ₃ , and monoiodoacetate were 5.0 mM).

As shown in Table 3, Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , and Mg ²⁺ inhibited enzyme activity. The enzyme activity was strongly inhibited by the addition of 5 mM monoiodoacetate. Enzymatic activity was weakly inhibited by the addition of 5 mM sodium azide.

6) Molecular weight
The molecular weight of SNDH III was measured using a size exclusion gel column (TSK-Gel G3000 SWXL; Tosoh Corporation, 1-7-7 Akasaka, Minato-ku, Tokyo, Japan). In this chromatography, the enzyme showed two peaks corresponding to an apparent molecular weight of about 190,000 ± 15,000 Da and about 250,000 ± 20,000 Da. When purified SNDH III treated with 2% SDS was subjected to 10% SDS-polyacrylamide gel electrophoresis and then analyzed by CBB staining, SNDH III was found to have two different subunits (each with a molecular weight of 75,000 ± 3,000 (α subunit Unit) and a molecular weight (β subunit) of 55,000 ± 2,000 Da. This is because SNDH III has two subunit structures (two subunits with a molecular weight of 75,000 ± 3,000 Da, one subunit with a molecular weight of 55,000 ± 2,000 Da, one subunit structure, or an α with a molecular weight of 75,000 ± 3,000 Da. 2 subunits, and a β subunit having a molecular weight of 55,000 ± 2,000 Da contains two subunit structures).

  Both the trimeric and tetrameric forms of SNDH III are active.

7) Prosthetic group
Purified SNDH III (0.1 mg) dissolved in 50 μl of 100 mM NaH ₂ PO ₄ —HCl (pH about 1.0) was added to an equal volume of methanol and mixed well. The sample was centrifuged to remove the precipitate. The resulting supernatant was used for prosthetic group analysis. The absorption spectrum of the extract was almost the same as that of PQQ standard (Mitsubishi Gas Chemical, Japan). The absorbance peaks were found to be at 251 nm and 348 nm. Furthermore, by HPLC analysis using a reverse phase column (YMC-Pack Pro C18 AS-312; YMC Co., Ltd) at a wavelength of 313 nm, the SNDH III extract with methanol showed the same retention time as the PQQ sample.

  Attempts were made to detect heme c in purified SNDH III by measuring a reduced-minus-oxidized difference spectrum with an ultraviolet-visible spectrophotometer (Shimadzu UV-2200; Shimadzu Corporation). SNDH III was suspended in 50 mM potassium phosphate buffer (pH 7.0) at a concentration of 50 μg / ml and oxidized with SNDH III reduced by dithionite and ammonium persulfate to measure the difference spectrum. The form of SNDH III was prepared. This spectrum gave a maximum difference at 552 nm and 523 nm.

  These results strongly suggest that SNDH III has PQQ and heme c as prosthetic groups.

8) Effect of substrate concentration
In order to determine the Km value for L-sorbosone, the oxidation reaction rate using various concentrations of L-sorbosone from 1 mM to 8 mM was measured. The Michaelis constant was calculated to be 6.5 mM at pH 7.5 and 16.8 mM at pH 9.0 from the Lineweaver-Burk plot based on the reaction rate when DCIP was used as the electron acceptor for the reaction.

9) Purification procedure
The purification of SNDH III is performed by any combination of known purification methods such as ion exchange column chromatography, hydrophobic column chromatography, salting out, and dialysis.

  SNDH III provided by the present invention comprises culturing appropriate microorganisms in nutrient broth under aerobic conditions, destroying microbial cells, isolating aldehyde dehydrogenase from cell-free extracts of destroyed microbial cells and It can be prepared by purifying.

  The microorganism used in the process of the present invention is a microorganism belonging to the genus Gluconobacter that can produce the aldehyde dehydrogenase defined above.

  A preferred strain is Gluconobacter oxydans. The most preferably used strain in the present invention is Gluconobacter oxydans DSM 4025. Based on the provisions of the Budapest Treaty, this strain was deposited as DSM No. 4025 on March 17, 1987 in the German microbial collection in Göttingen, Germany (Deutsche Sammlung von Mikroorganismen). The depositors were: Oriental Scientific Instruments Import and Export Corporation for Institute of Microbiology, Academia Sinica, 52 San-Li-He Rd., Beijing, People's Republic of China. The actual depositor was the Institute. The full address of the institute is The Institute of Microbiology, Academy of Sciences of China, Haidian, Zhongguancun, Beijing 100080, People's Republic of China.

  In addition, the subculture of this strain was instituted by the National Institute of Advanced Industrial Science and Technology as deposit number Gluconobacter-oxydans DSM No. 4025 (FERM BP-3812) on March 30, 1992, based on the provisions of the Budapest Treaty. (AIST), also deposited in Japan. Depositors are Nippon Roche Co., Ltd., 2-6-1 Shiba, Minato-ku, Tokyo, Japan. Most preferably, this subculture is also used in the present invention.

  The object of the present invention is therefore derived from Gluconobacter oxydans having the identifying characteristics of the strain Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), its subcultures or variants. It is to provide a defined aldehyde dehydrogenase.

  Mutants of Gluconobacter-oxydans DSM 4025 (FERM BP-3812) or microorganisms belonging to the genus Gluconobacter and having the identifying characteristics of Gluconobacter-oxydans DSM 4025 (FERM BP-3812) For example, by treating cells with ultraviolet or X-ray irradiation or chemical mutagens (eg, nitrogen mustard or N-methyl-n′-nitro-N-nitrosoguanidine).

  Any type of microorganism can be used, such as resting cells, acetone-treated cells, freeze-dried cells, immobilized cells, etc. to act directly on the substrate. Although any means known per se as a method associated with microbial incubation methods can be employed, the use of aeration and agitated submerged fermentors is particularly preferred. A preferred cell concentration range for carrying out the reaction is about 0.01 g cell wet weight / ml to 0.7 g cell wet weight / ml, preferably 0.03 g cell wet weight / ml to 0.5 g cell wet weight / ml. .

  The microorganism “Gluconobacter-oxydans” also includes such species of synonyms or bathons with the same physicochemical properties as defined by the International Code of Nomenclature of Prokaryotes.

The characteristics of Gluconobacter oxydans DSM No. 4025 (FERM BP-3812) are as follows:
a) Producing 2-KGA from sorbose,
b) oxidizing ethanol to acetic acid,
c) oxidizing D-glucose to D-gluconic acid and 2-keto-D-gluconic acid,
d) formation of ketone bodies of polyalcohols,
e) Pellicle and ring growth in pH 4 and 5 mannitol broth (24 hour culture) and fungal growth in pH 4.5 glucose broth,
f) does not substantially oxidize glycerol to dihydroxyacetone,
g) Produces 2-keto-D-glucaric acid from sorbitol and glucaric acid but does not produce 2-keto-D-glucaric acid from glucose, fructose, gluconic acid, mannitol, or 2-keto-D-gluconic acid ,
h) polymorphism, appearance, no flagella,
i) producing brown pigment from fructose,
j) proliferates well when co-cultured in the presence of Bacillus megaterium or cell extract thereof,
k) It is sensitive to streptomycin.

  Microorganisms can be cultured under aerobic conditions in a culture solution to which appropriate nutrients are added. Culturing can be performed at a pH of about 4.0 to about 9.0, preferably at a pH of about 6.0 to about 8.0. The culture period varies depending on the pH, temperature, and nutrient medium to be used, and is preferably about 1 to 5 days. A preferred temperature range for carrying out the cultivation is from about 13 ° C to about 36 ° C, preferably from about 18 ° C to about 33 ° C. Temperatures up to about 50 ° C. may also be suitable for culturing microorganisms.

  Usually the medium is an assimilable carbon source (e.g. glycerol, D-mannitol, D-sorbitol, erythritol, ribitol, xylitol, arabitol, inositol, dulcitol, D-ribose, D-fructose, D-glucose, and sucrose) (Preferably D-sorbitol, D-mannitol, and glycerol); and digestible nitrogen sources such as organic substances (e.g., peptone, yeast extract, baker's yeast, urea, amino acids, and corn steep liquor) It is required to contain nutrients. Various inorganic materials (eg, nitrates and ammonium salts) can also be used as nitrogen sources. In addition, the medium usually contains inorganic salts (eg, magnesium sulfate, potassium phosphate, calcium carbonate).

One embodiment for isolating and purifying SNDH III from microorganisms after culture is briefly described below.
(1) Harvest cells from liquid culture broth by centrifugation or filtration.
(2) The collected cells are washed with water, physiological saline, or a buffer solution having an appropriate pH.
(3) Suspend the washed cells in a buffer solution and destroy the cells with a homogenizer, a sonicator, a French press, or a treatment with lysozyme in order to obtain a solution of broken cells.
(4) Isolating and purifying SNDH III from a cell-free extract of disrupted cells (preferably from a soluble fraction of microorganisms).

  Cell-free extracts can be obtained from disrupted cells by any conventional technique, including but not limited to centrifugation.

  SNDH III provided by the present invention is useful as a catalyst for producing vitamin C and / or 2-KGA from L-sorbosone. This reaction is carried out at a pH value of about 4.5 to about 9.0 for both vitamin C production and 2-KGA production in an electron acceptor (e.g., DCIP, PMS) in a solvent such as phosphate buffer, Tris-buffer. Etc.). For vitamin C production, best results are usually obtained when the pH is set to about 6.5 to about 8.0 and the temperature is set to about 20 ° C to about 40 ° C. For 2-KGA production, best results are usually obtained when the pH is set to about 9.0 and the temperature is set to about 20 ° C to about 50 ° C.

  The concentration of L-sorbosone in the reaction mixture may vary depending on other reaction conditions, but is generally about 0.5-50 g / l, most preferably about 1 to about 30 g / l.

  In this reaction, SNDH III can also be used in an immobilized state on a suitable carrier. Any means for immobilizing enzymes generally known in the art can be used. For example, the enzyme may be directly attached to a resin film, granule, etc. having one or more functional groups, via a cross-linking compound (e.g., glutaraldehyde) having one or more functional groups. It may be bonded to a resin.

  In addition to the above, the cultured cells also produce carboxylic acids and / or their lactones from their corresponding aldoses (particularly to produce 2-KGA and / or vitamin C from L-sorbosone). Useful. Other carboxylic acids and / or their lactones are produced from their corresponding aldose under the same conditions (including substrate concentration) as the conversion of L-sorbosone to 2-KGA and / or vitamin C as described above. .

  The following examples further illustrate the invention.

Example 1: Preparation of SNDH III Unless otherwise specified, all operations were performed at 8 ° C and the buffer was 0.05 M potassium phosphate (pH 7.0).

(1) Culture of Gluconobacter-oxydans DSM No. 4025 (FERM BP-3812) Gluconobacter-oxydans DSM No. 4025 (FERM BP-3812) was mixed with 5.0% D-mannitol, 0.25% MgSO _4. Grow for 4 days at 27 ° C. on agar plates containing 7H ₂ O, 1.75% corn steep liquor, 5.0% baker's yeast, 0.5% urea, 0.5% CaCO ₃ , and 2.0% agar. 1 Platinum ear volume of cells in a 500 ml Erlenmeyer flask 2% L-sorbose, 0.2% yeast extract, 0.05% glycerol, 0.25% MgSO ₄ · 7H ₂ O, 1.75% corn steep liquor, 0.5% urea And 50 ml of a seed culture medium containing 1.5% CaCO ₃ , and placed on a rotary shaker at 180 ° C. for 1 day. The seed culture thus prepared was prepared in 15 liters of medium (8.0% L-sorbose, 0.05% glycerol, 0.25% MgSO ₄ · 7H ₂ O, 3.0% corn steep liquor, 0.4% in a 30 l jar fermenter. Used to inoculate yeast extract and 0.15% antifoam). The fermentation parameters were 800 rpm for the stirring speed and 0.5 vvm (air volume / medium volume / min) for aeration at a temperature of 30 ° C. The pH was maintained at 7.0 with sodium hydroxide during the fermentation. After 48 hours of culture, 30 liters of culture broth containing Gluconobacter oxydans DSM No. 4025 (FERM BP-3812) cells using two sets of fermenters were collected by continuous centrifugation. The pellet containing the cells was collected and suspended in an appropriate amount of physiological saline. After centrifuging the suspension at 2,500 rpm (1,000 x g), a supernatant containing slightly reddish cells to remove insoluble material from the corn steep liquor and yeast extract, which are medium components Was recovered. The supernatant was then centrifuged at 8,000 rpm (10,000 × g) to obtain a cell pellet. As a result, Gluconobacter-oxydans DSM No. 4025 (FERM BP-3812) cells having a wet weight of 123 g were obtained from 30 liters of broth.

(2) Preparation of cytosol fraction A part of the cell paste (64.2 g) was suspended in 280 ml of buffer solution and subjected to French press. The supernatant after centrifugation to remove intact cells was used as a cell-free extract. The cell free extract was centrifuged at 100,000 × g for 60 minutes. The resulting supernatant (227 ml) was used as the soluble fraction of Gluconobacter oxydans DSM No. 4025 (FERM BP-3812). After dialyzing this fraction against buffer, 105 ml of the dialysis fraction was used for the next purification step (specific activity to produce vitamin C from L-sorbosone in the dialysis fraction was 0.107 units / mg protein) ).

(3) Diethylaminoethyl (DEAE) -cellulose column chromatography Dialysate (150 ml) was equilibrated with a buffered DEAE-cellulose column (Whatman DE-52, 3 × 50 cm; Whatman BioSystems Ltd .), Springfield MIII, James Whatman Way, Maidstone, Kent, UK) and washed with buffer to elute minor proteins. Next, the protein bound to the resin was eluted stepwise with a buffer containing 0.28M NaCl, a buffer containing 0.32M NaCl, and a buffer containing 0.36M NaCl. Major enzyme activity was eluted with 0.36M NaCl. This active fraction (143 ml) was collected.

(4) Carboxymethyl-cellulose column chromatography To concentrate a portion (127 ml) of the active fraction obtained from the previous step, an ultrafiltrator (Centriprep-10, Amicon ); Filtered through Amicon Inc., Cherry Hill Drive, Beverly, MA 01915, USA). After dialyzing the concentrated sample (28 ml) against buffer, 28 ml of the dialysis fraction (31 ml) was carboxymethyl-cellulose column equilibrated with buffer (Whatman CM-52, 3 × 23 cm; Whatman Biosystems). The protein that passed through the column without binding to the resin was collected.

(5) Q-Sepharose column chromatography (first stage)
The pooled active fraction (43 ml) was filtered through an ultrafilter (centriprep-10) to concentrate. A portion (9.5 ml) of the concentrated active fraction (10 ml) obtained from the previous step was loaded onto a Q-Sepharose column (Pharmacia, 1.5 × 50 cm) equilibrated with buffer. After the column was washed with a buffer containing 0.3M NaCl, a 0.3-0.6M linear gradient of NaCl was added to the buffer. The active fraction was eluted at a NaCl concentration of 0.55-0.57M.

(6) Q-Sepharose column chromatography (second stage)
The pooled active fraction (22 ml) obtained from the previous step was filtered through an ultrafilter (centriprep-10) for concentration. The concentrated sample (3.0 ml) was dialyzed against buffer. The dialyzed sample (3.5 ml) was placed on a Q-Sepharose column (Pharmacia, 1.5 × 50 cm) equilibrated with buffer. After the column was washed with a buffer containing 0.35M NaCl, a 0.35-0.7M linear gradient of NaCl was added to the buffer. The active fraction was eluted at a NaCl concentration of 0.51 to 0.53M.

(7) Gel filtration column chromatography The pooled active fractions (20 ml) obtained from the previous step were filtered through an ultrafilter (Centreprep-10) to concentrate and desalinate. A portion (1.5 ml) of a concentrated and desalted (less than 0.1 M NaCl) sample (2.0 ml) was equilibrated with a buffer containing 0.1 M NaCl, Sephacryl S-300 High Resolution (High Resolution) column (Pharmacia, 1.5 × 120 cm). The active fraction (12 ml) was collected and dialyzed against buffer.

(8) Hydrophobic column chromatography In order to concentrate the dialyzed active fraction obtained from the previous step, it was filtered with an ultrafilter (centriprep-10). A portion (1.5 ml) of the concentrated sample (1.75 ml) was added to an equal volume (1.5 ml) of buffer containing 3 M (final concentration: 1.5 M) ammonium sulfate. After centrifuging the sample (15,000 × g), the supernatant was loaded onto a RESOURCE ISO column (Pharmacia, 1.0 ml) equilibrated with a buffer containing 1.5 M ammonium sulfate. After the column was washed with a buffer containing 1.5M ammonium sulfate, the protein was eluted with a buffer containing a 1.5-0.75M linear gradient of ammonium sulfate. The active fraction corresponding to SNDH III was eluted at ammonium sulfate concentrations of 1.15 to 1.13M. Using a dialysis cup (Dialysis-cup MWCO 8000, Daiichi pure chemicals, 3-13-5 Nihonbashi, Chuo-ku, Tokyo, Japan) And dialyzed. The fractions were then collected and stored at -20 ° C.

  A summary of the enzyme purification steps is shown in Table 3.

酵素1単位^*は、前記1a)に記載の反応混合物において1時間にビタミンC 1mgを産生する酵素の量と定義した。 (Table 3)
Purification of SNDH III from Gluconobacter oxydans DSM No. 4025 (FERM BP-3812)

One unit of enzyme ^* was defined as the amount of enzyme that produced 1 mg of vitamin C per hour in the reaction mixture described in 1a) above.

(9) Purification of isolated enzyme Purified enzyme (0.12 mg / ml) with a specific activity of 34.5 units / mg protein for producing vitamin C and a specific activity of 7.82 units / mg protein for producing 2-KGA Used for the following analysis.

  The molecular weight of native SNDH III was 280 nm using a size exclusion gel column (TSK gel G3000 SWXL column, 7.8 × 300 mm) (equilibrated with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.3 M NaCl). And evaluated by high performance liquid chromatography at a flow rate of 1.5 ml / min. Cyanocobalamin (1.35 kDa), myoglobin (17 kDa), ovalbumin (44 kDa), γ-globulin (158 kDa), and thyroglobulin (670 kDa) were used as molecular weight standards. Purified SNDH III showed two peaks (molecular weight 190,000 ± 15,000 Da and molecular weight 250,000 ± 20,000 Da, respectively).

  According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), SNDH III consisted of two subunits. The molecular weight of one subunit is 75,000 ± 3,000 Da, and the molecular weight of the other subunit is 55,000 ± 2,000 Da.

(10) Identification of reaction product A reaction mixture containing purified SNDH III (1.21 μg), L-sorbosone (50 mM), PMS (1 mM), CaCl ₂ (1 mM), and PQQ (1 μM) in 100 μl of buffer at 30 ° C. Incubated for 1 hour. The reaction products were analyzed by thin layer chromatography (silica gel 60F ²⁵⁴ , MERCK, 64271 Darmstadt, Germany) and HPLC. Two products vitamin C and 2-KGA were obtained from the enzymatic reaction. For vitamin C, the samples were assayed using an amino-column (YMC-Pack polyamine-II), YMC, Inc.) on an HPLC instrument. For 2-KGA, samples were assayed using a C-18 column (YMC-Pack Pro C18, YMC, Inc.) on an HPLC instrument.

Example 2: Effect of pH on the production of L-sorbosone from vitamin D or 2-KGA by SNDH III The effect of pH on the enzymatic reaction was tested. A reaction mixture containing purified SNDH III (607 ng), L-sorbosone (50 mM), PMS (1 mM), CaCl ₂ (1 mM), and PQQ (1 μM) in 100 μl of buffer (100 mM) was incubated at 30 ° C. for 1 hour. The reaction product was analyzed by HPLC. The results are shown in Table 4.

(Table 4)
Effect of pH on production of L-sorbosone to vitamin C or 2-KGA by SNDH III

Example 3: Effect of temperature on production of L-sorbosone from vitamin D or 2-KGA by SNDH III The effect of temperature on enzyme activity was tested. Various reaction mixtures containing purified SNDH III (607 ng), L-sorbosone (50 mM), PMS (1 mM), CaCl ₂ (1 mM), and PQQ (1 μM) in 100 μl of 25 mM potassium phosphate buffer (pH 7.0) Incubated for 1 hour at various temperatures (20-60 ° C). The reaction product was analyzed by HPLC. The results are shown in Table 5.

(Table 5)
Effect of temperature on production of L-sorbosone from vitamin D or 2-KGA by SNDH III

Claims (13)

Purified aldehyde dehydrogenase with the following physicochemical properties:
a) 190,000 ± 15,000 Da molecular weight (consisting of 2 α subunits and 1 β subunit subunit structure), or 250,000 ± 20,000 Da molecular weight (2 α subunits and 2 β subunits) (Consisting of the subunit structure of the unit) (where the molecular weight of the α subunit is 75,000 ± 3,000 Da and the molecular weight of the β subunit is 55,000 ± 2,000 Da),
b) Substrate specificity: active against aldehyde compounds,
c) Cofactors: pyrroloquinoline quinone (PQQ) and heme c,
d) Optimal pH: about 6.5 to about 8.0 (when producing vitamin C from L-sorbosone) or about 9.0 (when producing 2-keto-L-gulonic acid from L-sorbosone),
e) Inhibitors: Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , Mg ²⁺ , monoiodoacetate, and sodium azide.   2. The aldehyde dehydrogenase according to claim 1, which is derived from a microorganism belonging to the genus Gluconobacter, which can produce the aldehyde dehydrogenase according to claim 1.   The aldehyde dehydrogenase according to claim 2, wherein the microorganism is Gluconobacter oxydans having identification characteristics of strain Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), its subcultures or mutants. .   4. The aldehyde dehydrogenase according to claim 3, wherein the microorganism is Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), a subculture or a mutant thereof. The following physicochemical properties:
a) 190,000 ± 15,000 Da molecular weight (consisting of 2 α subunits and 1 β subunit subunit structure), or 250,000 ± 20,000 Da molecular weight (2 α subunits and 2 β subunits) (Consisting of the subunit structure of the unit) (where the molecular weight of the α subunit is 75,000 ± 3,000 Da and the molecular weight of the β subunit is 55,000 ± 2,000 Da),
b) Substrate specificity: active against aldehyde compounds,
c) Cofactors: pyrroloquinoline quinone (PQQ) and heme c,
d) Optimal pH: about 6.5 to about 8.0 (when producing vitamin C from L-sorbosone) or about 9.0 (when producing 2-keto-L-gulonic acid from L-sorbosone),
e) Inhibitors: Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , Mg ²⁺ , monoiodoacetate, and sodium azide,
A process for producing aldehyde dehydrogenase having the following steps:
A step of culturing a microorganism belonging to the genus Gluconobacter capable of producing an aldehyde dehydrogenase having the above-mentioned characteristics in a nutrient medium under aerobic conditions, a step of destroying microbial cells, and the absence of destroyed microbial cells. Isolating and purifying aldehyde dehydrogenase from the cell extract.   6. The process of claim 5, wherein the reaction is conducted at a pH of about 4.5 to about 9.0 and a temperature of about 20 ° C to about 50 ° C. A process for producing a carboxylic acid and / or its lactone from its corresponding aldose and comprising the following steps:
Aldehydes and the following physicochemical properties:
a) 190,000 ± 15,000 Da molecular weight (consisting of 2 α subunits and 1 β subunit subunit structure), or 250,000 ± 20,000 Da molecular weight (2 α subunits and 2 β subunits) (Consisting of the subunit structure of the unit) (where the molecular weight of the α subunit is 75,000 ± 3,000 Da and the molecular weight of the β subunit is 55,000 ± 2,000 Da),
b) Substrate specificity: active against aldehyde compounds,
c) Cofactors: pyrroloquinoline quinone (PQQ) and heme c,
d) Optimal pH: about 6.5 to about 8.0 (when producing vitamin C from L-sorbosone) or about 9.0 (when producing 2-keto-L-gulonic acid from L-sorbosone),
e) Inhibitors: Co ²⁺ , Cu ²⁺ , Fe ³⁺ , Ni ²⁺ , Zn ²⁺ , Mg ²⁺ , monoiodoacetate, and sodium azide,
Contacting a cell-free extract prepared from a microorganism belonging to the genus Gluconobacter capable of producing a purified aldehyde dehydrogenase having the above-mentioned property or an aldehyde dehydrogenase having the aforementioned characteristics in the presence of an electron acceptor.   The microorganism is strain Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), a subculture or variant thereof, and Gluconobacter oxydans having identification characteristics A process according to any one of the above.   9. The process according to claim 8, wherein the microorganism is Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), a subculture or a variant thereof.   8. The process of claim 7, wherein the lactone is vitamin C, the carboxylic acid is 2-keto-L-gulonic acid, and the aldose is L-sorbosone.   The reaction according to any one of claims 7 to 10, wherein the reaction is carried out at a pH of about 4.5 to about 9.0 and a temperature of about 20 ° C to about 50 ° C when producing vitamin C and 2-keto-L-gulonic acid, respectively. The process described in the section.   When the reaction produces vitamin C, at a pH of about 6.5 to about 8.0, and when producing 2-keto-L-gulonic acid, at a pH of about 9.0, for both production, about 20 ° C. to about 50 ° C. The process according to any one of claims 7 to 11, which is carried out at a temperature of   Use of a purified aldehyde dehydrogenase according to claim 1 in a process for producing a carboxylic acid and / or its lactone from its corresponding aldose comprising the steps of: producing an aldehyde and the purified aldehyde dehydrogenase or the aldehyde dehydrogenase Contacting a cell-free extract prepared from a microorganism belonging to the genus Gluconobacter in the presence of an electron acceptor.