JP2003159079A - Alcohol/aldehyde dehydrogenase, method for producing the same and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain alcohol/aldehyde dehydrogenase (AADH) in which since AADH can catalyze both oxidation reactions of two stages from an alcohol to a carboxylic acid, for example, 2KLGA (2-keto-L-gulonic acid) can directly be formed from L-sorbose by using the enzyme and a large amount of 2KLGA can simply be mass produced from L-sorbose by culturing the transformant of this invention. <P>SOLUTION: This new protein has AADH activity. This DNA encodes the protein. This recombinant vector contains the DNA. This transformant contains the vector. This new method for producing the AADH comprises culturing the transformant. This method for producing a carboxylic acid, especially the method for producing 2KLGA comprises culturing the protein or the transformant. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein having alcohol dehydrogenase activity and aldehyde dehydrogenase activity (hereinafter referred to as alcohol / aldehyde dehydrogenase or AADH), a DNA encoding the same, and an expression vector containing the DNA. The present invention relates to a method for producing the protein by culturing a transformed host cell, and a method for producing 2-keto-L-gulonic acid using a culture of the enzyme or the transformant.

[0002]

2. Description of the Related Art 2-keto-L-gulonic acid (2KLGA)
Is an important intermediate in the synthesis of L-ascorbic acid. In conventional industrial production, 2KLGA is synthesized by chemically oxidizing L-sorbose according to the Reichstein method. On the other hand, in microorganisms such as Acetobacter and Gluconobacter, L-sorbose dehydrogenase (hereinafter,
It is known that some have the ability to convert L-sorbose into 2KLGA via a two-step enzymatic oxidation reaction by SDH) and L-sorbosone dehydrogenase (hereinafter referred to as SNDH). If fermentative production of 2KLGA by these microorganisms becomes possible, a large amount of 2KLGA can be produced at a lower cost than before.
However, all of the microorganisms that have been attempted to be used conventionally have low production efficiency of 2KLGA and have not yet been applied to industrial production.

On the other hand, Pseudogluconobacter ( Pseu
Microorganisms belonging to dogluconobacter ) are also known to produce 2KLGA from L-sorbose (JP-A-62-228288, JP-A-64-8508).
(See No. 8). In addition, pseudoephedrine saccharoketogenes (Pseudogluconobacter saccharoketoge
nes ), an alcohol dehydrogenase (ADH), which oxidizes a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group, has been isolated (Japanese Patent Laid-Open Publication No. Hei 5)
-68542). However, there is no report that the ADH also has aldehyde dehydrogenase activity.
Alcohol from microorganisms belonging to the genus Gluconobacter /
Although a protein having an aldehyde dehydrogenase activity has been isolated (see JP-A-7-182), it has not been reported whether L-sorbose can be used as a substrate.

[0004]

Therefore, the object of the present invention is to obtain 2K from L-sorbose to L-sorbosone.
Novel protein capable of catalyzing a two-step oxidation reaction of an alcohol to a carboxylic acid including a reaction for producing LGA, that is, a novel protein having AADH activity and DN encoding the same
And to provide a method for producing the novel AADH by culturing a host cell transformed with an expression vector containing the DNA. Still another object of the present invention is to provide a method for producing a carboxylic acid such as 2KLGA using the culture of the novel AADH or the transformant.

[0005]

Means for Solving the Problems As a result of intensive studies aimed at achieving the above object, the present inventors succeeded in purifying a protein having AADH activity from a bacterium belonging to the genus Pseudogluconobacter. . As a result of further characterization of the protein, it was found that the protein is a novel AADH having physicochemical characteristics completely different from conventionally known ADH and AADH. Next, the present inventors cloned the DNA encoding the novel AADH and determined the entire nucleotide sequence. Furthermore, the present inventors produced the AADH by culturing host cells transformed with the expression vector containing the DNA, and contacted the resulting culture with L-sorbose to produce 2KL.
It succeeded in producing GA and came to complete this invention.

That is, the present invention is as follows. [1] A protein having the following characteristics (a) to (c). (a) Action: It can catalyze the reaction of oxidizing a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group and the reaction of oxidizing an aldehyde group of a compound having an aldehyde group to a carboxyl group. (b) Molecular weight: 65,000 ± 2,000 (SDS-PA
GE), 55,000 ± 4,000 (gel filtration) (c) Heme iron and rare earth elements are not contained in the molecule [2] Furthermore, at least one of the following physicochemical properties (a) to (d) The protein of the above-mentioned [1] having one. (a) Optimum pH: 4.5 to 5.5 (b) Isoelectric point: 4.1 ± 0.3 (c) Pyrroloquinoline quinone is included in the molecule as a prosthetic group (d) K _m for sorbose Value: about 40 mM [3] The following protein (a) or (b). (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, A protein that can catalyze the reaction of oxidizing a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group and the reaction of oxidizing an aldehyde group of a compound having an aldehyde group to a carboxyl group [4] Bacteria belonging to the genus Pseudogluconobacter The protein of any of the above-mentioned [1]-[3], which can be purified from [5] A DNA encoding the protein according to any one of the above [1] to [4], particularly the DNA comprising the following nucleotide sequence (a) or (b). (a) Base sequence shown in SEQ ID No. 2 in the Sequence Listing (b) DN consisting of base sequence shown in Sequence No. 2 in the Sequence Listing
A nucleotide sequence capable of hybridizing with A under stringent conditions [6] A recombinant vector containing the DNA of [5] above. [7] A host cell transformed with the recombinant vector of [6] above. [8] The above-mentioned [1], which comprises culturing the cell of the above-mentioned [7] in a medium and collecting a protein having an alcohol / aldehyde dehydrogenase activity from the obtained culture.
~ The method for producing a protein according to any one of [4]. [9] By contacting the protein of any of [1] to [4] above or the culture of [8] above or a treated product thereof with a compound containing a hydroxymethyl group or an aldehyde group, the hydroxymethyl group or the A method for producing a compound containing a carboxyl group, which comprises the step of oxidizing an aldehyde group to a carboxyl group, in particular, the compound containing a hydroxymethyl group or an aldehyde group is L-sorbose or L-sorbosone, and the compound containing a carboxyl group is 2-. The method is keto-L-gulonic acid.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The novel protein of the present invention catalyzes a reaction of oxidizing a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group and a reaction of oxidizing an aldehyde group of a compound having an aldehyde group to a carboxyl group. It is an enzyme to obtain. In particular, the enzyme can be preferably used as an enzyme that catalyzes the reaction of converting L-sorbose into 2KLGA via L-sorbosone. The AADH
Has a molecular weight of 65,000 ± by SDS-PAGE.
2,000, and 55,000 ± by gel filtration using a TSK gel G3000 column (Tosoh).
Since it is 4,000, it is presumed that the enzyme is a monomer protein that does not have a subunit structure. Further, the AADH does not contain heme iron or rare earth element in the molecule.

The origin of the AADH of the present invention is not particularly limited as long as it has the above-mentioned characteristics, and its ability to produce the enzyme can be obtained by natural mutation or artificial mutagenesis treatment in addition to organisms that naturally produce the enzyme. It also includes mutants obtained or further enhanced in the ability to produce the enzyme, and recombinants introduced with the AADH gene isolated from these organisms by genetic engineering.

The organisms which naturally produce AADH of the present invention include, for example, microorganisms, preferably bacteria, more preferably bacteria belonging to the genus Pseudogluconobacter, and above all Pseudogluconobacter saccharoketgenes. In addition, examples of mutants in which the enzyme-producing ability is further enhanced by mutagenesis treatment include, for example, JP-A-62-2282.
P. 88. Saccaro Ketogenes K591S
Strains (FERM BP-1130, IFO14464),
P. Saccharoket genez 12-5 strain (FERMBP-1
129, IFO 14465), P.I. Saccharoketgenes TH14-86 strain (FERM BP-1128, IFO
14466), P.I. Saccharoketogenes 12-15 strain (FERM BP-1132, IFO14482),
P. Saccharoket genesis 12-4 strain (FERM BP-
1131, IFO 14483), P. Saccharoket genesis strain 22-3 (FERM BP-1133, IFO14
484).

In a more preferred embodiment, A of the present invention
ADH further has at least one of the following physicochemical properties. (a) Optimum pH: 4.5 to 5.5 (b) Isoelectric point: 4.1 ± 0.3 (c) Contains pyrroloquinoline quinone in the molecule as a prosthetic group (d) K _m for sorbose Value: about 40 mM

In a further preferred embodiment, the AADH of the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. And a protein having AADH activity.

The AADH of the present invention includes (1) a method of isolating and purifying a culture of cells or tissues that produce the enzyme as a raw material, (2) a method of chemically synthesizing, or (3) a gene recombination technique. It can be obtained by appropriately using the publicly known method of. The following method is exemplified as a preferred embodiment of the method (1).

First, the AADH-producing microorganism of the present invention, such as Pseudogluconobacter saccharoketgenes, is cultivated in an appropriate liquid medium, and the fraction having AADH activity is separated and recovered from the resulting culture ( For example,
P. When Saccharoketgenes is used as an isolation source, A
Since ADH activity is found in the intracellular (cytoplasmic) fraction,
After collecting the cells by centrifuging the culture, the cells are disrupted by ultrasonic treatment, lysozyme and osmotic shock treatment,
Obtain the supernatant by centrifugation). If AADH activity is found in the medium, the culture supernatant may be used as it is or after concentration. If AADH activity is present in the cell membrane, the cell disruption supernatant is further subjected to ultracentrifugation. The precipitate thus obtained may be solubilized by adding an appropriate surfactant or the like, and then subjected to the next purification operation. AAD of purpose
H can be purified from the active fraction obtained as described above by appropriately combining separation techniques generally used for isolating and purifying enzyme proteins.
Specifically, for example, a method utilizing a difference in solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing P
Method utilizing difference in molecular weight such as AGE, SDS-PAGE, method utilizing charge such as ion exchange chromatography, hydroxylapatite chromatography, method utilizing specific affinity such as affinity chromatography, reverse phase high performance liquid Examples thereof include a method utilizing a difference in hydrophobicity such as chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing.

The production of AADH of the present invention by chemical synthesis can be carried out, for example, by synthesizing all or part of the sequence using a peptide synthesizer based on the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. it can.

Further, the production of AADH of the present invention using a gene recombination technique is carried out by
It can be carried out by culturing a host cell transformed with an expression vector containing a DNA encoding ADH in a medium and collecting the enzyme from the resulting culture.

The DNA of the present invention is not particularly limited as long as it is the DNA encoding AADH of the present invention, but preferably encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing or its equivalent. It is a DNA, more preferably the DNA having the nucleotide sequence shown in SEQ ID NO: 2 of the Sequence Listing or the nucleotide sequence capable of hybridizing with the DNA having the nucleotide sequence under stringent conditions. Here, the “equivalent” means a polypeptide having an amino acid sequence in which one or several amino acids have been deleted, substituted or added within a range that does not change the above-mentioned characteristics of AADH of the present invention. The "stringent condition" means a condition under which a DNA having a homology of about 60% or more in the nucleotide sequence can hybridize.

The DNA of the present invention may be obtained by any method. For example, cDNA prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, RNA or DN obtained by amplification by a PCR method using RNA or DNA as a template
A, and DNA constructed by appropriately combining these methods are included.

The DNA of the present invention can be isolated, for example, by the following method. First, the enzyme is completely or partially purified from the cell or tissue producing AADH according to the method described above, and the N-terminal partial amino acid sequence thereof is determined by the Edman method. Further, the amino acid sequence of an oligopeptide obtained by partially decomposing the enzyme with a sequence-specific protease is similarly determined by the Edman method. An oligonucleotide having a nucleotide sequence corresponding to the determined partial amino acid sequence is synthesized and used as a primer or a probe to perform PCR or colony (or plaque) from RNA or DNA prepared from cells or tissues producing AADH. ) Cloning the DNA encoding the enzyme protein by the hybridization method.

Alternatively, fully or partially purified AA
CDNA or genomic DN prepared from cells or tissues that produce AADH by preparing an antibody against the enzyme using all or part of DH as an antigen according to a conventional method
DNA encoding the enzyme can also be cloned from the A library by antibody screening.

Alternatively, the DNA of the present invention can be prepared by synthesizing all or part of the sequence using a fully automatic DNA synthesizer, for example, based on the base sequence shown in SEQ ID NO: 2 in the Sequence Listing. .

The present invention also relates to a recombinant vector containing any of the above DNAs. The recombinant vector of the present invention is not particularly limited as long as it can be replication-retained or autonomously propagated in various host cells such as prokaryotic cells and / or eukaryotic cells, and includes plasmid vectors and phage vectors. The recombinant vector can be conveniently prepared by inserting any of the above-mentioned DNAs into a cloning vector or expression vector available in the art using an appropriate restriction enzyme site.

In particular, the recombinant vector of the present invention comprises AAD
It is an expression vector functionally containing H-encoding DNA. As used herein, the term "functionally comprises" means that the DNA is arranged such that the DNA can be transcribed and a polypeptide encoded thereby can be produced in a host cell compatible with the vector. Preferred is a vector having an expression cassette in which a promoter region, a start codon, a DNA encoding AADH, a stop codon and a terminator region are sequentially arranged. Examples of the expression vector used include a promoter region that functions in various host cells of prokaryotic cells and / or eukaryotic cells and that can express a gene located downstream thereof, and a signal that terminates transcription of the gene, that is, There is no particular limitation as long as it contains a terminator region, and the promoter region and the terminator region are linked via a sequence containing at least one unique restriction enzyme recognition site, but for the selection of transformants It is preferable to further contain a selectable marker gene. Further, if desired, the expression vector may include a start codon and a stop codon downstream of the promoter region and upstream of the terminator region, respectively.

When bacteria are used as the host cell, the expression vector generally needs to contain a replicable unit capable of autonomously replicating in the host cell in addition to the above promoter region and terminator region. -3 in the promoter region
5 areas, -10 areas, operator and Shine-Dalgar
The no (SD) sequence is included. For example, when the host is E. coli, the promoter regions are tufB, λPL, t
rp, lacUV5, lap, lac, ompA, ph
oA, recA and rrnB promoters, and when the host is Bacillus subtilis, SLO1 promoter, SPO2 promoter, pen
P promoter etc. are mentioned. As the terminator region, a commonly used natural or synthetic terminator can be used. As the selectable marker gene, a resistance gene against various drugs such as tetracycline, ampicillin, kanamycin, chloramphenicol, hygromycin, a gene complementing auxotrophy, or the lacZ gene can be used. ATG is usually used as the start codon, but GTG can also be used in some cases. Commonly used TGA, TAA and TAG are used as stop codons.

Genomic DNA derived from cells or tissues in which the DNA encoding AADH of the present invention produces the enzyme
When obtained from a form containing the original promoter and terminator region, the expression vector of the present invention is a suitable cloning vector known in the art that can be replication-retained or autonomously propagated in the host cell to be transformed. It can be prepared by inserting the DNA into the site. As the cloning vector to be used, when the host is bacteria, E. coli-derived pBR-based vector, pUC-based vector, etc., or Bacillus subtilis-derived pUB
110, pTP5, pC194 and the like are exemplified.

Oxidation reaction from alcohol to carboxylic acid,
In particular, considering the application to the oxidation reaction of L-sorbose to 2KLGA, cells originally having a high ability to oxidize alcohol to carboxylic acid, for example, SDH and SNDH-producing Gluconobacter bacteria (for example, G. oxyde). It is more preferable to use DANCE T-100 etc. as a host cell. Specific examples of the expression vector preferably used in such an embodiment include G. A plasmid having the promoter region of the Oxidance T-100-derived SNDH gene or the above-mentioned Escherichia coli promoter and derived from the genus Gluconobacter (for example, pF3 derived from Gluconobacter IAM12138,
G. Oxidance T-100-derived pF4 and the like). Furthermore, the vector is a plasmid derived from a common microorganism such as Escherichia coli (for example, pBR322 or pBR322 derived from Escherichia coli) so that the expression vector containing the AADH gene can be easily constructed and mass-produced.
It is preferably in the form of a shuttle vector having a replicable unit (UC18, pHSG298 or the like or a yeast-derived 2μ plasmid). Examples of suitable shuttle vectors include chimeric plasmids pFG14A and pFG14B of pHSG298 and pF3, and chimeric plasmids pFG15A and pFG of pHSG298 and pF4.
15B and the like.

The transformant of the present invention is the AADH of the present invention.
It can be prepared by transforming a host cell with a recombinant vector containing a DNA encoding The host cell is not particularly limited as long as it is compatible with the recombinant vector used and can be transformed, and naturally occurring cells usually used in the art (for example, bacteria, yeast, mold,
Various cells such as animal cells such as COS and CHO, plant cells such as tobacco culture cells), or artificially produced mutant or recombinant cells can be used. Preferably bacteria, especially E. coli (eg DH5α, HB101
Etc.), Bacillus subtilis, Pseudogluconobacter bacteria, Gluconobacter bacteria, Pseudomonas bacteria, Acetobacter bacteria and the like.

The recombinant vector can be introduced into the host cell by a conventionally known method. For example, when the host is bacteria such as Escherichia coli and Bacillus subtilis, the method of Cohen et al. [P
Roc.Natl. Acad. Sci. USA, 69: 2110 (1972)], protoplast method [Mol. Gen. Genet., 168: 111 (1979)]
And the competent method [J. Mol. Biol., 56: 209 (197
1)] and the like. In particular, when a Pseudogluconobacter bacterium, a Gluconobacter bacterium, a Pseudomonas bacterium, an Acetobacter bacterium, or the like is used as a host, the transformation efficiency is low in a usual transformation method, and thus the electroporation method [Nucleic Acids Res., 16:
6127 (1988)] is advantageous.

In particular, the transformant of the present invention is the A of the present invention.
A host cell transformed with an expression vector functionally containing DNA encoding ADH. AADH of the present invention
Can be produced by culturing the cells in an appropriate medium and collecting AADH from the resulting culture.

The nutrient medium used contains saccharides such as glucose and fructose as a carbon source, glycerol, preferably L-sorbose and D-sorbitol. It may also contain an inorganic or organic nitrogen source (for example, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, beef extract, etc.). Further, if desired, other nutrients [for example, inorganic salts (for example, sodium diphosphate or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (for example, vitamin B ₁ ), Antibiotics (eg ampicillin, kanamycin)]
May be added to the medium. For example, when Gluconobacter bacteria are used as a host, D-sorbitol,
Preferable examples include a medium containing yeast extract, CaCO ₃ , glycerol and the like.

Cultivation of the transformant is usually pH 5.5-
It is carried out at 8.5, preferably pH 7 to 7.5, 18 to 40 ° C, preferably 20 to 30 ° C for 5 to 50 hours.

Purification of AADH from the culture is
After separation of the active fractions, various separation techniques commonly used in the art can be appropriately combined.

The present invention also oxidizes a compound containing a hydroxymethyl group or an aldehyde group using the AADH of the present invention, or the culture of AADH-producing cells artificially produced as described above or a treated product thereof. And a method for producing a compound containing a carboxyl group, particularly a method for producing 2KLGA by oxidizing L-sorbose or L-sorbosone. In the method, a compound containing hydroxymethyl group or aldehyde group, particularly L-sorbose or L, is added to AADH, a culture of the enzyme-producing cells or an AADH active fraction obtained by treating the culture.
-Including contacting the sorbosons. When AADH activity is found in the culture supernatant of the culture, the method of contacting the culture with the substrate also includes a method of culturing the enzyme-producing cells in a medium containing an appropriate amount of the substrate. The medium used in this case may be a medium suitable for ordinary culture except that it contains a reaction substrate. Further, the culture conditions such as temperature and pH may be appropriately selected so as to balance the optimum conditions for cell culture and the optimum conditions for AADH activity.

When AADH activity is found in the cytoplasm, the culture after completion of the culture is centrifuged or filtered to recover the cells, which are then collected in an appropriate buffer such as an acetate buffer (about pH 4-6). The supernatant obtained by suspending the cells in), disrupting the cells by ultrasonication or the like, and then centrifuging the cells may be used.

The compound containing a carboxyl group thus produced, particularly 2KLGA, is a purification method generally used from the reaction solution (the culture supernatant of AADH-producing cells when the cells are cultured in a medium containing a substrate). (For example,
Dialysis, gel filtration, column chromatography on an appropriate adsorbent, high performance liquid chromatography, etc.).

[0035]

EXAMPLES Examples will be shown below for specifically explaining the present invention, but the present invention is not limited to these examples. The plasmids, enzymes such as restriction enzymes, T4 DNA ligase, and other commercial products used in the following examples were used according to the instructions in the attached instructions unless otherwise specified. Operations such as cloning of DNA, transformation of host cells, culturing of transformants, and recovery of 2KLGA from the resulting culture are well known to those skilled in the art, or can be known from literatures. It is a thing.

Example 1 Alcohol from Pseudogluconobacter saccharoketgenes IFO 14464 /
Purification of aldehyde dehydrogenase (1) P. Culture of Saccharoketgenes IFO14464 P. Saccharoketgenes IFO14464 strain was obtained from the Fermentation Research Institute (2-17-85, Jusohonmachi, Yodogawa-ku, Osaka). 500 ml of a single colony of the strain
Medium in a Erlenmeyer flask of volume (10% sorbose, 2
% Corn steep liquor, 0.5% dry yeast, 0.
5% ammonium sulfate, 0.05% Na ₂ S ₂ O ₃ ,
_{0.2% FeSO 4 · 7H 2 O} , 4.0% CaCO 3;
The cells were inoculated into 100 ml (8 strains) of pH 7.0. Culture is 3
It carried out at 0 degreeC, 250 rpm, and 5.0 cm-throw for 48 hours. Combine the resulting culture broths (800 m
l) It was centrifuged at 4 ° C. and 6,000 rpm for 10 minutes. The obtained cells were stored at -20 ° C or lower until just before use. (2) Preparation of bacterial cell soluble fraction The cells obtained in (1) were treated with 50 mM phosphate buffer (p
H7.5) was suspended in 200 ml and then dispensed into 8 bottles.
One of them was centrifuged at 500 rpm for 10 minutes and Ca
After removing CO ₃ , the cells were disrupted by sonication.
The cell lysate was centrifuged at 4 ° C. and 10,000 rpm for 10 minutes to collect the supernatant. The resulting supernatant is further stored at 4 ° C for 3
Ultracentrifugation was performed at 2,000 rpm for 1 hour, and the supernatant was collected to obtain a cell-soluble fraction. (3) Ion-exchange chromatography 2 ml of the cell-soluble fraction was equilibrated with 25 mM Tris-hydrochloric acid (pH 8.0) containing 0.1 M sodium chloride to prepare a TSK gel DEAE-5PW column (7.5 m).
m inner diameter × 75 mm, Tosoh) was used for ion exchange chromatography. Immediately, the NaCl concentration is 50
Elution was performed with a linear gradient of 0.35 M per minute (flow rate 1.0 ml / min), and 1.5 ml each was collected. The AADH activity was determined by the method of Sugizawa et al. [Agric. Biol. Che.
m., 55: 363-370 (1991)], 0.1 mM 2,6-dichlorophenolindophenol (0.2 mM L-sorbose as a substrate in 27.5 mM phosphate buffer (pH 7.0) DCIP) was used as the electron acceptor. 1 unit of enzyme is 1 μmo per minute
It was defined as the amount of enzyme that catalyzes the reduction of 1 DCIP. The reduction of DCIP is measured by an absorptiometer (Model UV-16
0, Shimadzu Corporation) was used to measure the degree of decrease in absorbance at 600 nm. Collect the AADH active fractions and collect them with Molcat L (LGC: molecular weight cut off 10,000; Millipore)
Concentrated twice. (4) TSK-gel obtained by equilibrating a part (100 μl) of the concentrated active fraction obtained by gel filtration chromatography (3) with a 25 mM phosphate buffer (pH 7.5) containing 0.1 M sodium chloride. G30
It was subjected to gel filtration chromatography using a 00 SWXL column (6.0 mm inner diameter × 40 cm, Tosoh).
Elution was performed using the same buffer (flow rate 0.5 ml / min), and each fraction (0.5 ml) was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE:
Acrylamide 12.5%), and enzymatic assay. AADH activity corresponded to a protein of approximately 64 kDa in SDS-PAGE analysis.

Example 2 Identification of Enzymatic Reaction Product Using an enzyme solution purified by gel filtration chromatography, the enzymatic reaction product using sorbose and sorbosone as substrates was subjected to high performance liquid chromatography (HPLC).
Was analyzed by. The reaction conditions are a substrate concentration of 10 mg / ml,
100 mM phosphate buffer (pH 7.5) and 1 mM phenazine mesosulfate were used and left at 30 ° C. for 24 hours. HPLC analysis conditions are columns: Capcell pa
k NH2 (4.6 mm inner diameter x 250 mm, Shiseido),
Mobile phase: 20 mM sodium phosphate, 30% acetonitrile (pH 3.0), flow rate: 1.2 ml / min, injection amount: 5 μl, detection wavelength: 210 nm. as a result,
10 mg / ml sorbose produced 0.45 mg / ml 2KLGA and 10 mg / ml sorbose produced 1.0 mg / ml 2KLGA. Therefore, it was found that the enzyme is a kind of AADH.

Example 3 Characteristic analysis of AADH (1) Optimum pH The activity of AADH purified in Example 1 was measured using reaction solutions having various pH values. As a result, it was found that the optimum pH range of this enzyme was pH 4.5 to 5.5 (Table 1).

[0039]

[Table 1]

(2) Substrate specificity Basic reaction solution composition 0.2 M Tris-hydrochloric acid (pH 7.5) 0.3 ml 1 mM DCIP 0.12 ml 6 mM PMS 0.12 ml Enzyme solution 0.024 ml H ₂ O 0. After adding 0.48 ml of various substrates having a hydroxymethyl group to the basic reaction solution of 156 ml, the decrease in the absorbance of DCIP at 600 nm was measured by a spectrophotometer. As a control, the same amount of distilled water was used instead of the substrate. The results are shown in Table 2. The enzyme activity for each substrate was expressed as a relative activity with the enzyme activity as a substrate when 0.2 M ethanol was used as 100.

[0041]

[Table 2]

[0042] (3) After addition of various concentrations of sorbose 0.48ml basic reaction solution shown in K _m value (2) with respect to sorbose, by measuring the decrease in absorbance at 600nm of DCIP at spectrophotometer K _{The m} value was derived. As a result, the K _m value of this enzyme for sorbose was 40 mM.

(4) Isoelectric point Full-automatic electrophoresis system (Past) manufactured by Pharmacia
System) was used to determine the isoelectric point (pI) of AADH. The pI of this enzyme is PhastGe manufactured by Pharmacia.
1 IEF 3-9 was used to determine pI calibration kit 3-10 as a pI marker. as a result,
The isoelectric point of this enzyme was 4.1.

(5) Molecular weight The molecular weight of AADH was determined by gel filtration column chromatography. The measurement was carried out under the following conditions using a high performance liquid chromatograph manufactured by Millipore. Column: TSK gel G3000 SWXL (manufactured by Tosoh Corporation) x 2 Mobile phase: 25 mM phosphate buffer (pH 7.5) + 0.
1M NaCl flow rate: 0.5 ml / min Detector: UV-230 nm As a molecular weight marker, ovalbumin (43,00)
0), bovine serum albumin (67,000) and aldolase (158,000). As a result, the molecular weight of this enzyme was determined to be 55,000 ± 4,000. Next, this enzyme was treated with SDS in the presence of 2-mercaptoethanol, and its molecular structure was analyzed by SDS-PAGE. As molecular weight markers, α-lactalbumin (14,400), trypsin inhibitor (20,
100), carbonic anhydrase B (30,00)
0), ovalbumin (43,000), bovine serum albumin (67,000) and phosphorylase b (9).
4,000) was used. As a result, the molecular weight of this enzyme is 6
It was determined to be 5,000 ± 2,000. Therefore, this enzyme was presumed to be a monomeric protein having no subunit structure.

(6) Amino acid composition AADH solution was placed on a PVDF membrane (Immobilon P; manufactured by Millipore), air-dried, washed with distilled water, and then the membrane was dried and cut into small pieces of about 2 mm × 5 mm. The membrane piece was placed in a container containing 6N hydrochloric acid + 7% thioglycolic acid, sealed under reduced pressure, and hydrolyzed with hydrochloric acid at 110 ° C. for 22 hours. Transfer only the hydrolyzed solution to another test tube, and remove the membrane strip with 0.1N.
It was washed with hydrochloric acid + 30% methanol. The hydrolyzed solution and the washing solution were combined and dried under reduced pressure, and then 100 μl of distilled water was added and dissolved to obtain a sample solution. 50 μl of the sample solution was injected into an amino acid analyzer (L-850 type manufactured by Hitachi) and analyzed. The results are shown in Table 3.

[0046]

[Table 3]

(7) To 100 μl of the prosthetic group AADH solution, 10 μl of 1N hydrochloric acid and 100 μl of methanol were added and mixed. 15,000 of the mixture
After centrifuging at 0 rpm for 10 minutes to remove the precipitate, the supernatant was analyzed under the following conditions using a high performance liquid chromatograph manufactured by Millipore. Column: Puresil 5 μC18 120Å (4.6
Mobile phase: 27% (v / v) methanol + 1% (v / v) 85% phosphoric acid flow rate: 1.0 ml / min Detector: UV-254 nm As a result, pyrroloquinoline quinone (PQQ) The shape and retention time of the peak were completely the same as those of the standard sample (made by Ube Industries, Ltd.). In addition, PQQ is 0.4 m / mol of this enzyme protein obtained from amino acid analysis by the ninhydrin method.
It was calculated to be ol-bonded.

(8) Inductively coupled plasma mass spectrometry (ICP-MS) for the metal element AADH
Method), iron and rare earth elements were not detected. From this, it was presumed that this enzyme is not a heme protein. In addition, when this enzyme was analyzed by inductively coupled plasma emission spectrometry (ICP-AES method), calcium was detected, and it was calculated that 1.9 mol of calcium was bound to 1 mol of this enzyme protein determined by amino acid analysis by the ninhydrin method. It was

Example 4 Amino Acid Sequence Analysis of AADH (1) A part (200 μl) of the concentrated active fraction obtained in (2) of Example 2 was added with 0.14 g of urea and 50 mM Tris-hydrochloric acid (pH 9.0). ) 300 μl, 1 mg / ml Achromobacter protease I (Wako Pure Chemical Industries, Ltd.) 2 μl were added, and the mixture was digested at 37 ° C. for 3 hours. The processing liquid is COSMOS
Reversed phase chromatography on an IL 5C4-AR-300 column eluting with a linear gradient of 0% to 60% acetonitrile in 0.05% trifluoroacetic acid (flow rate 1.0 ml / min) of P1 to P6. Six types of peptide fragments were separated. These peptides were run directly on an automated protein sequencer model 477 (Applied Biosystems) to determine the amino acid sequence of each fragment. The results are shown in Table 4.

[0050]

[Table 4]

(2) From the result of (1) above, it was suggested that a disulfide bond was present in the AADH protein molecule. Next, in the presence of 2-mercaptoethanol, fragmentation with Achromobacter protease I (Wako Pure Chemical Industries) was performed. I went. That is, in the concentrated AADH active fraction (150 μl), 0.105 g of urea and 50 mM were added.
Tris-hydrochloric acid (pH 9.0) 225 μl, 1 mg / ml
Achromobacter protease I (Wako Pure Chemical Industries)
1.5 μl was added, and fragmentation was performed at 37 ° C. for 4 hours. The treatment liquid was subjected to reverse phase chromatography (CO
SMOSIL 5C4-AR-300), 0.0
5% acetonitrile in trifluoroacetic acid 0% to 60%
Elute with a linear gradient of (flow rate 1.0 ml / min)
Six kinds of peptides P11 to P16 were separated. These peptides were run directly on an automated protein sequencer model 476A (Applied Biosystems) to determine the amino acid sequence. The results are shown in Table 5.

[0052]

[Table 5]

Example 5 Isolation of AADH Gene Fragment by PCR Method (1) Synthesis of PCR Primer A pair of mixed primers, 3FS (GAR GTB TGG TCS ACS C
G; Sequence listing SEQ ID NO: 15) and 3ARS (VACS GC VAC
RTA CTG; Sequence Listing, SEQ ID NO: 16) was designed, and each of these oligonucleotides was synthesized by the phosphoamidite method using a DNA synthesizer model 392 (Applied Biosystems). The synthesized oligonucleotide was desorbed from a CPG polymer carrier (CPG: controlled pore glass) with 28% ammonia water to give 6
All protecting groups were removed by heating at 0 ° C. for 9 hours. The solvent was distilled off from the reaction mixture under reduced pressure, and the residue was mixed with 200 μl of TE buffer [10 mM Tris-HCl (pH 7.4), 1 mM ED].
TA]. The resulting solution was washed once with ether and precipitated with ethanol. The oligonucleotide obtained by precipitation was used for PCR as it was without further purification.

(2) Preparation of chromosomal DNA Pseudogluconobacter saccharoketgenes IFO
14464 single colonies were incubated at 37 ° C. for 4 days in 80 ml of medium (pH 6.0) consisting of 2.5% mannitol, 0.3% polypeptone and 0.5% yeast extract.
After culturing for 8 hours, the cells were collected by centrifugation at 6,000 rpm for 10 minutes and suspended in 1 ml of sterilized water. The suspension was diluted with 1 ml of STE buffer [20% sucrose, 50 mM Tris-hydrochloric acid (pH 8), 1 mM EDTA], mixed with 2 mg of lysozyme, and incubated at 37 ° C for 30 minutes. Sarkosyl solution [1% lauroyl sarcosylate, 100 mM EDTA (pH 8.5)] 2.
5 ml and proteinase K (final concentration 100 μg / m
After the addition of l), the mixture was incubated at 50 ° C. for 2 hours. Cesium chloride 5.5g and 5mg
0.3 ml of ethidium bromide / ml was dissolved in the mixture, and ultracentrifugation was performed at 20 ° C. and 50,000 rpm for 16 hours. The portion containing the chromosomal DNA was isolated, and TE buffer [10 mM Tris-HCl (pH 7.4), 0.1 mM was added.
EDTA] After dissolving with 30 ml, 2 L of 1 mM ED
It was dialyzed twice with TA. The dialysate was washed 4 times with 15 ml of isobutanol, 2 times with 10 ml of phenol, and 3 times with 5 ml of chloroform, and then dried in a centrifugal concentrator.
The dried solid was dissolved in 0.5 ml of TE buffer and then purified by ethanol precipitation. Add it to TE buffer 0.
Dissolve in 5 ml, and dissolve the desired chromosomal DNA solution (234 μg
/ Ml) was obtained.

(3) AADH gene fragment amplification by PCR P. Saccaro Ketogenes IFO 144
PCR was carried out using the mixed primers (3FS and 3RS) synthesized in (1), using 64 chromosomal DNAs as a template. Reaction mixture [in PCR buffer, template DNA100
ng, each primer 1 nmol, 200 μM each dNT
50 units of 2.5 units of Ps and Taq DNA polymerase (Perkin-Elmer Cetus) were used for denaturation at 98 ° C. for 1 minute at 55 ° C. using a hybrid thermal reactor model HB-TR1 (Hydride, UK). Was subjected to PCR for 30 minutes, and the extension reaction was performed at 75 ° C. for 2 minutes. After the amplification was completed, the reaction solution was subjected to 4% agarose gel electrophoresis,
A fragment (84 bp) encoding part of ADH was isolated. The obtained DNA and pGEM-T (Promega) were ligated with T4 DNA ligase (Takara Shuzo). Maniatis et al. [Molecular Cloning, 2nd e
d., Vol. 1, Cold Spring Harbor Laboratory Press, U
SA (1989)] with the ligation mixture according to the method described in E. E. coli DH10B was transformed.

(4) DNA sequence analysis The desired plasmid pUC18 43-18 was isolated from one of the transformants obtained in (3), and the nucleotide sequence of the inserted DNA fragment was analyzed using the 370A DNA sequencer (Applied Biosystems). ) Was used according to the attached protocol by the dideoxy termination method. Universal and reverse primers (New) that are M13 sequencing primers
Sequencing was carried out using England Biolabs, USA). As a result, it was revealed that the inserted DNA had the following base sequence. 5'-GAA GTC TGG TCC ACC CGT CTG CCG GGT GCG GTT TCC
GGC TAC ACC ACC AGC TAC TC C ATC GAT GGC CGC CAG TAC GTC GC
G GTT-3 '(SEQ ID NO: 17 in the Sequence Listing)

(5) Amplification of longer AADH gene fragment Complementary strand sequence 3BRS (GA
GTA GCT GGT GGT GTAGCC; Sequence Listing SEQ ID NO: 18),
It was synthesized in the same manner as (1) using a DNA synthesizer model 392 (Applied Biosystems).
Further, seven kinds of mixed primers were synthesized based on the amino acid sequences of P1, P4 and P5 shown in Table 4 and P11, P14 and P16 shown in Table 5 (Table 6).

[0058]

[Table 6]

PCR was carried out in all combinations using each of the primers shown in Table 6 as a forward primer and 3BRS as a reverse primer. Reaction mixture [PC
Template DNA (P. saccharoketgenes IF in R buffer)
O14464-derived chromosomal DNA) 100 ng, each primer 125 pmol, 200 μM each dNTPs and Taq DNA polymerase 2.5 unit (Perkin Elmer Cetus)] 50 μl at 98 ° C.
PCR was performed for 30 minutes, including annealing for 2 minutes at 55 ° C for 2 minutes, and extension reaction for 2 minutes at 75 ° C. After the reaction was completed, the reaction solution was subjected to 2.0% agarose gel electrophoresis to separate PCR products. Among all combinations, it was found that using 11FS as the forward primer gave a specific amplified fragment of approximately 500 bp. This DNA fragment was purified from gel and used for T4 DN
It was connected to pGEM-T (Promega) using A ligase (Takara Shuzo). E. coli with the binding mixture according to the method of Maniatis et al. (Supra). E. coli DH10B was transformed.
From one of the transformants obtained, the desired plasmid p # 4
Was isolated and characterized by restriction enzyme mapping, and then the nucleotide sequence of the inserted DNA was determined in the same manner as in (4). As a result, it was revealed that the inserted DNA was composed of the base sequences represented by base numbers 1168 to 1688 in the base sequence represented by SEQ ID NO: 2 in Sequence Listing.

Example 6 Isolation of DNA containing the entire coding region of AADH of Pseudogluconobacter saccharoketgenes IFO14464 (1) (1) Preparation of digoxigenin (DIG) -labeled probe Obtained in Example 5. Plasmid p # 4 was digested with NcoI and PstI (Nippon Gene) and purified by 2% agarose gel electrophoresis to isolate the insert DNA. The obtained DNA was subjected to DI using a DIG labeling kit (Boehringer Mannheim) according to the attached protocol.
G-labeled. (2) Preparation of chromosomal DNA library P. Saccaro Ketogenes I
The FO14464-derived chromosomal DNA was partially digested with Sau3AI and subjected to 4% by 0.8% agarose gel electrophoresis.
The 20 kb fragment was isolated and purified by standard methods. The obtained fragment was used as a lambda phage vector E.
It was cloned into the BamHI site of MBL-3 (Stratagene). E. coli XL-1 Blue MRA (P
When 2) was used as the indicator strain, a titer of 190 pfu / μl was obtained. (3) Escherichia coli XL-1 Blue MR as plaque hybridization plate-immobilized bacteria
Preparation of lambda phage plaques with A (P2) and immobilization of the plaques on nitrocellulose filters was performed according to the protocol described by Maniatis et al. (Supra). A filter containing lambda DNA was washed with hybridization buffer [50% formamide, 0.2
% Bovine serum albumin, 0.2% Polyvinylpyrrolidone, 0.2% Ficoll, 20 mM phosphate buffer (p
H6.5), 5 × SSC, 0.1% SDS, 50 μg
/ Ml salmon sperm DNA], and incubated at 42 ° C. for 18 hours to prepare a DIG-ized probe (521 bp, 250
ng / ml) in the same buffer at 42 ° C. for 24 hours. Next, 1 x S containing 0.1% SDS
Excess probe was removed by incubation in SC at 42 ° C. The filter was detected using a detection kit (Boehringer Mannheim), and finally two positive clones were obtained. (4) Southern blotting Phage DNA extracted from one of the positive clones (No. 20) was digested with PstI (Nippon Gene) and subjected to 0.8% agarose gel electrophoresis. The DNA fragments separated on the gel were transferred onto a nitrocellulose filter by electroblotting. A DNA fragment of approximately 3 kbp was found to hybridize with the DIG labeled probe.

(5) DNA sequence analysis of AADH gene The DNA of lambda phage (No. 20) was digested with PstI to obtain a PstI / PstI (3.0 kb) fragment.
This DNA was subcloned into the PstI site of pUC18 to obtain p20PstI. DNA of the template DNA
The sequence was determined by the dideoxy termination method using a 370A DNA sequencer (Applied Biosystems) according to the attached protocol. M
13 Sequencing Primers Universal and Reverse Primers (New Engl
and Biolabs, USA) were used for initial sequencing. Further analysis was performed using the synthetic oligonucleotides listed in Table 7.

[0062]

[Table 7]

As a result, the lambda phage arm part DNA was inserted from the middle of the base sequence corresponding to P15 in Table 5, and the lambda phage (No. 20) insert DNA was deleted at the AADH amino terminal side. I found out.

Example 7 Isolation of DNA containing the entire coding region of AADH of Pseudogluconobacter saccharoketgenes IFO14464 (2) (1) Repreparation of DIG-labeled probe The probe designed in Example 6 To prepare probes at different positions, p20PstI was digested with KpnI and PstI (Nippon Gene) and purified by 2% agarose gel electrophoresis to give the insert DNA (541b
p) was isolated. The obtained DNA was labeled with DIG using a DIG labeling kit (Boehringer Mannheim) according to the attached protocol.

(2) Plaque hybridization Using the DIG-labeled probe re-prepared in (1) above, plaque hybridization was carried out in the same manner as in (6) of Example 6 to finally obtain two plaques. A positive clone was obtained.

(3) DNA sequence analysis of AADH gene One of the positive clones, lambda phage (No. 3)
6) DNA is digested with a restriction enzyme to obtain SalI / SalI
(3.0 kb), SalI / SalI (6.8 kb),
An EcoRI / SacI (3.0 kb) fragment was obtained. These DNAs were subcloned into pUC18 to obtain p36Sal15, p36Sal41, p36ES, respectively.
Got Template DNA (p36Sal15, p36Sal
41, p36ES) and 370 AD
It was carried out according to the attached protocol by the dideoxy termination method using NA sequencer (Applied Biosystems). Universal and reverse primers (New England Biolabs, M13 sequencing primers).
USA) was used for initial sequencing.
Further analysis was also performed using the synthetic oligonucleotides described in Tables 7 and 8.

[0067]

[Table 8]

As a result, lambda phage (No. 36)
An ORF consisting of 1824 bp (SEQ ID NO: 2 in the Sequence Listing) was found in the insert DNA. The ORF contained all the DNA sequences corresponding to the internal amino acid sequences listed in Tables 4 and 5. Furthermore, when the amino acid composition of the polypeptide encoded by the ORF was calculated based on the amino acid sequence deduced from the DNA sequence, it was in good agreement with the numerical value obtained from the amino acid analysis of AADH (see Table 3). From the above results, it was concluded that the ORF encodes AADH. In addition, pyrroloquinoline quinone (PQ is included in the amino acid sequence deduced from the DNA sequence of the ORF.
Q) It was confirmed by homology search that the binding site consensus sequence was highly conserved (amino acid numbers 65 to 92 and 270 to 29 in SEQ ID NO: 1 in the sequence listing).
1), it was strongly suggested that the AADH contains PQQ as a prosthetic group.

Example 8 Expression of AADH gene in Escherichia coli (1) Construction of expression vector a) Plasmid p36ES containing AADH gene was Sa
digested with cI and EcoRI / SacI adapter (GGAA
TTCCAGCT; Sequence Listing SEQ ID NO: 56) was inserted and p36EE
Got This was digested with EcoRI, and the 2.9 kb fragment containing the AADH gene was inserted into the EcoRI restriction site of shuttle vector pFG15A. pHSG298 or
Plasmid pADH3 inserted in the same direction (clock direction) as i and plasmid pADH inserted in the opposite direction (unclock direction) to ori of pHSG298
4 was obtained (FIG. 1). b) PCR was performed using p36ES as a template and the following primers (denaturation: 94 ° C., 30 seconds; annealing: 55 ° C., 2 minutes; extension: 72 ° C., 2 minutes; 25 cycles), and upstream of the start codon of the AADH gene. To N
A coI restriction site was introduced. Sense primer (SEQ ID NO: 57 in Sequence Listing): 5'-TAGCTGAATTCCATGGTCAGTAGGAATATTTCTC ¹ ATGAGATTTGAG
TATTTGCGGCAGA-3 'antisense primer (SEQ ID NO: 58 in Sequence Listing): 5'- ⁷⁸⁵ TCGTAGGGCGCGCCGCCCCA ⁷⁶⁶ -3' (superscript numbers correspond to corresponding AADH gene (SEQ ID NO: 2)
The 0.8 kb fragment specifically amplified was digested with EcoRI and AscI, and p36EE was transformed into E.
4.6 kb obtained by digestion with coRI and AscI
Ligation with the fragment gave p36EE-N. A 2.7 kb fragment containing the AADH coding region obtained by digesting this plasmid with EcoRI and NcoI was added to tu
Expression vector pSDH-tuf containing fB promoter
EcoRI (partial digestion) / Nc of B1-Eco-d9U
Ligation with the 4.2 kb fragment obtained by digestion with oI gave the expression vector pADH-tufB in which AADH was placed under the control of the tufB promoter (FIG. 2).

(2) Transformation of Escherichia coli Competent cells for electroporation (ELECTROM
AX DH10B; manufactured by GIBCOBRL) 20 μl of the three plasmids pADH3 and pAD constructed in (1) above
H4 and pADH-tufB, respectively, and mixed with E.
E. coli DH10B / pADH3 and E. coli DH10B / pADH4 and E. coli DH10B / pADH4
E. coli DH10B / pADH-tufB was obtained respectively.

(3) Measurement of AADH activity a) E. coli DH10B / pADH3 and E. coli DH10B / pADH4
Were cultured in 50 ml of L medium containing 50 μg / ml kanamycin at 20 ° C. for 48 hours and then collected. Fifty
Suspend in 10 ml of mM Tris-HCl buffer (pH 7.5), sonicate for 5 minutes, and then centrifuge (4 ° C, 10,
(000 rpm, 10 minutes) and the supernatant was collected to obtain a cell-free extract. E. coli DH10B was cultured in L medium and treated in the same manner as a control. 1M C was added to the cell-free extract
aCl ₂ and 100 mM PQQ were each added to a final concentration of 10
mM and 1 mM were added, and the mixture was incubated at 4 ° C. for 2 hours, and then the ultrafiltration membrane (centriprep-
50; manufactured by Amicon) and concentrated about 6-fold and subjected to SDH / SNDH activity measurement using L-sorbose as a substrate. Standard reaction solution [35 m in a cuvette with an optical path length of 1 cm
M phosphate buffer (pH 7.0), 0.13 mM DCI
P, 0.82 mM PMS] 0.915 ml and sample solution 0.2 ml were added, and 1M L-sorbose aqueous solution 0.225 ml was added to start the reaction, and the decrease of the absorbance at 600 nm due to the reduction of DCIP was taken with time. Measured. The amount of enzyme that reduced 1 μmol of DCIP in 1 minute was defined as 1 unit (U). DCIP 600
The molecular extinction coefficient at ¹ nm is 14.5 mM ^-1 (pH 7.
0). As a result, only E. coli DH10B / pADH3 had S
DH / SNDH activity was observed (Table 9). Moreover, in SDS-PAGE of the cell-free extract, E. coli DH10B /
A band was detected only in pADH3 at the same molecular weight position as AADH.

[0072]

[Table 9]

B) 50 μg of E. coli DH10B / pADH-tufB
/ Ml Kanamycin-containing L medium 100 ml at 25 ℃,
After culturing for 48 hours, the cells were collected. After suspending in 20 ml of 50 mM Tris-HCl buffer (pH 7.5) and sonicating for 5 minutes, centrifugation (4 ° C., 10,000 rpm, 10)
The cell-free extract was obtained. E. col
iDH10B was cultured in L medium and treated in the same manner as a control. After adding CaCl ₂ and PQQ at various concentrations to the cell-free extract and incubating at 4 ° C. for 1 hour,
Ultrafiltration membrane (Centriprep-50; manufactured by Amicon)
Was concentrated about 6-fold using and was subjected to SDH / SNDH activity measurement using L-sorbose as a substrate. The activity was measured in the same manner as in the above a). As a result, PQQ independently activated AADH in a concentration-dependent manner. CaCl ₂ is PQ
AADH concentration-dependently in the presence of Q and synergistically with PQQ
Were activated (Table 10). Cell-free extract SDS-PA
As a result of GE, it was revealed that the expression level of the AADH protein was higher when the promoter was replaced with the tufB promoter.

[0074]

[Table 10]

[0075]

INDUSTRIAL APPLICABILITY Since the AADH of the present invention can catalyze both of the two-step oxidation reaction of alcohol to carboxylic acid, the use of the enzyme can directly generate carboxylic acid from alcohol. Therefore, for example, L-
It can be used to generate 2KLGA directly from L-sorbose in the synthesis of ascorbic acid. Further, by using the transformant of the present invention, it becomes possible to fermentatively produce 2KLGA from L-sorbose, and 2KLGA can be easily produced in a large amount.

[0076]

[Sequence list free text] SEQ ID NO: 3 Amino acid number 2: Gln or Leu. Amino acid number 4: Gly or Leu. Amino acid number 5: Pro or Val. Amino acid number 6: Arg or Pro. Amino acid number 8: Asn or Lys. Amino acid number 13: unidentified amino acid. Amino acid number 18: unidentified amino acid. Amino acid number 20: unidentified amino acid. Amino acid number 23: unidentified amino acid. Amino acid number 24: unidentified amino acid. Amino acid number 27: unidentified amino acid. Amino acid number 30: unidentified amino acid. SEQ ID NO: 4 Amino acid number 1: Asn or Ala. Amino acid number 2: Thr or Pro. Amino acid number 3: Ala or Val. Amino acid number 4: Asn or Glu. Amino acid number 5: Gln or Trp. Amino acid number 6: Asn or Pro. Amino acid number 7: Ser or Ile. Amino acid number 8: Ile or Leu. Amino acid number 9: Ala or Arg. Amino acid number 10: Ser or Gly. Amino acid number 11: Asn or Ile. Amino acid number 12: Asp or Tyr. Amino acid number 13: Asp or Gln. Amino acid number 14: Thr or Gly. Amino acid number 15: Gly or Trp. Amino acid number 17: Tyr or Val. Amino acid number 18: Ser or Thr. Amino acid number 19: Pro or Val. Amino acid number 20: Asn or Leu. Amino acid number 21: Asp or Glu. Amino acid number 23: Met or Ile. Amino acid number 24: Asn or Ile. SEQ ID NO: 6 Amino acid number 1: Thr or Ser. Amino acid number 2: Tyr or Asn. Amino acid number 3: Asn or Leu. Amino acid number 5: Val or unidentified amino acid. Amino acid number 6: Pro or Ile. Amino acid number 7: Thr or Pro. Amino acid number 8: Phe or Leu. Amino acid number 9: Ser or Leu. Amino acid number 10: Asn or Gly. Amino acid number 11: Gly or Ala. Amino acid number 12: Arg or unidentified amino acid. Amino acid number 13: Asp or Tyr. Amino acid number 14: Asp or Trp. Amino acid number 15: Pro or Val. Amino acid number 16: Ser or Met. Amino acid number 18: Gly or Arg. Amino acid number 19: Thr or Tyr. Amino acid number 20: Thr or Leu. Amino acid number 21: Glu or Pro. Amino acid number 22: Ala or Lys. Amino acid number 23: Thr or Ala. Amino acid number 24: Ser or Pro. SEQ ID NO: 7 Amino acid number 1: Unidentified amino acid. Amino acid number 6: unidentified amino acid. SEQ ID NO: 8 Amino acid number 1: Asp or Leu. Amino acid number 2: Asn or Ala. Amino acid number 3: Val or Trp. Amino acid number 4: Gly or Glu. Amino acid number 5: Asp or Thr. Amino acid number 6: Asn or Leu. Amino acid number 7: Gln or Arg. Amino acid number 8: Gly or Leu. Amino acid number 9: Gln or Val. Amino acid number 10: Gly or Trp. Amino acid number 11: Ser or Val. Amino acid number 12: Glu or Arg. Amino acid number 13: Thr or Glu. Amino acid number 14: Gly or Met. Amino acid number 15: Glu or Val. Amino acid number 16: Ala or Pro. Amino acid number 17: Asn or Gly. Amino acid number 20: Gly or Glu. Amino acid number 21: Gly or Pro. Amino acid number 22: Ala or Ile. Amino acid number 23: Ala or Val. Amino acid number 24: Val or Ile. Amino acid number 25: Asp or Ala. Amino acid number 26: Gly or Tyr. Amino acid number 27: Asn or Val. Amino acid number 28: Gly or Val. Amino acid number 29: Val or Ile. Amino acid number 30: Ala or Ile. SEQ ID NO: 9 Amino acid number 7: Ser or Arg. SEQ ID NO: 10 Amino acid number 4: Ala or Val. Amino acid number 5: Ala or Trp. Amino acid number 6: Gln, Trp or Glu. Amino acid number 7: Phe or Gln. Amino acid number 8: Phe or Asp. Amino acid number 9: Ala or Asp. Amino acid number 10: Ala or Lys. SEQ ID NO: 11 Amino acid number 1: Thr or Ala. Amino acid number 3: Val or Asn. Amino acid number 4: Tyr or Glu. Amino acid number 6: Pro, Asn or Ala. Amino acid number 8: Phe or Ile. Amino acid number 9: Leu or Ala. Amino acid number 11: Gly or Ile. Amino acid number 16: Leu or Ser. Amino acid number 17: Ala or Val. Amino acid number 20: Leu or Asn. SEQ ID NO: 12 Amino acid number 1: Unidentified amino acid. Amino acid number 9: unidentified amino acid. Amino acid number 11: unidentified amino acid. Amino acid number 23: unidentified amino acid. Amino acid number 24: unidentified amino acid. Amino acid number 27: unidentified amino acid. Amino acid number 28: unidentified amino acid. SEQ ID NO: 13 Amino acid number 1: Ala, Glu or Asp. Amino acid number 2: Val or Gly. Amino acid number 5: Ala or Thr. Amino acid number 6: Ile or Arg. Amino acid number 7: Glu or Leu. Amino acid number 8: Asn or Pro. Amino acid number 9: Phe or Gly. Amino acid number 10: Ala or Gln. Amino acid number 11: Val or Pro. Amino acid number 13: Gly or Thr. Amino acid number 14: Tyr or Ala. Amino acid number 15: Asp or Thr. Amino acid number 18: Ala or Tyr. Amino acid number 21: Asn or Asp. Amino acid number 23: Ala or Arg. Amino acid number 24: Asn or Gln. Amino acid number 29: unidentified amino acid. SEQ ID NO: 14 Amino acid number 1: Ser, Ala, Val, Asp or Trp. Amino acid number 12: unidentified amino acid. Amino acid number 23: unidentified amino acid. SEQ ID NO: 15 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 16 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 17 AADH gene fragment amplified by PCR. SEQ ID NO: 18 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 19 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 20 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 21 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 22 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 23 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 24 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 25 An oligonucleotide designed to function as a PCR primer for amplifying AADH gene fragment. SEQ ID NO: 26 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 27 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 28 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 29 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 30 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 31 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 32 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 33 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 34 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 35 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 36 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 37 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 38 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 39 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 40 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 41 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 42 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 43 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 44 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 45 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 46 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 47 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 48 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 49 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 50 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 51 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 52 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 53 An oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 54 Oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 55 oligonucleotide designed to function as a primer for determining AADH gene sequence. SEQ ID NO: 56 EcoRI / SacI adapter. SEQ ID NO: 57 Oligonucleotide designed to function as a PCR primer for introducing NcoI site upstream of AADH coding sequence. SEQ ID NO: 58 An oligonucleotide designed to function as a PCR primer for introducing an NcoI site upstream of AADH coding sequence.

[0077]

[Sequence Listing] SEQUENCE LISTING <110> Fujisawa Pharmaceutical Co., Ltd. <120> Alcohol / Aldehyde Dehydrogenase, Production Method Thereof and Use Thereof <130> A4950 <160> 58 <170> PatentIn version 3.0 <210> 1 <211 > 608 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <400> 1 Met Arg Phe Glu Tyr Leu Arg Gln Asn Val Val Gly Leu Ala Leu Ser 1 5 10 15 Thr Ala Leu Ile Ala Ser Leu Ser Gly Pro Ala Phe Ala Gln His Asp 20 25 30 Ala Asn Ala Ala Ala Glu Pro Ser Lys Ala Gly Gln Ser Ala Ile Glu 35 40 45 Asn Phe Gln Pro Val Thr Ala Asp Asp Leu Ala Gly Lys Asn Pro Ala 50 55 60 Asn Trp Pro Ile Leu Arg Gly Asn Tyr Gln Gly Trp Gly Tyr Ser Pro 65 70 75 80 Leu Asp Gln Ile Asn Lys Asp Asn Val Gly Asp Leu Gln Leu Val Trp 85 90 95 Ser Arg Thr Met Glu Pro Gly Ser Asn Glu Gly Ala Ala Ile Ala Tyr 100 105 110 Asn Gly Val Ile Phe Leu Gly Asn Thr Asn Asp Val Ile Gln Ala Ile 115 120 125 Asp Gly Lys Thr Gly Ser Leu Ile Trp Glu Tyr Arg Arg Lys Leu Pro 130 135 140 Ser Ala Ser Lys Phe Ile Asn Ser Leu Gly Ala Ala Lys Arg Ser Ile 145 150 155 160 Ala Leu Phe Gly Asp Lys Val Tyr Phe Val Ser Trp Asp Asn Phe Val 165 170 175 Val Ala Leu Asp Ala Lys Thr Gly Lys Leu Ala Trp Glu Thr Asn Arg 180 185 190 Gly Gln Gly Val Glu Glu Gly Val Ala Asn Ser Ser Gly Pro Ile Val 195 200 205 Val Asp Gly Val Val Ile Ala Gly Ser Thr Cys Gln Phe Ser Gly Phe 210 215 220 Gly Cys Tyr Val Thr Gly Thr Asp Ala Glu Ser Gly Glu Glu Leu Trp 225 230 235 240 Arg Asn Thr Phe Ile Pro Arg Pro Gly Glu Glu Gly Asp Asp Thr Trp 245 250 255 Gly Gly Ala Pro Tyr Glu Asn Arg Trp Met Thr Gly Ala Trp Gly Gln 260 265 270 Ile Thr Tyr Asp Pro Glu Leu Asp Leu Val Tyr Tyr Gly Ser Thr Gly 275 280 285 Ala Gly Pro Ala Ser Glu Val Gln Arg Gly Thr Glu Gly Gly Thr Leu 290 295 300 Ala Gly Thr Asn Thr Arg Phe Ala Val Lys Pro Lys Thr Gly Glu Val 305 310 315 320 Val Trp Lys His Gln Thr Leu Pro Arg Asp Asn Trp Asp Ser Glu Cys 325 330 335 Thr Phe Glu Met Met Val Val Ser Thr Ser Val Asn Pro Asp Ala Lys 340 345 350 Ala Asp Gly Met Met Ser Val Gly Ala Asn Val Pro Arg Gly Glu Thr 355 360 365 Arg Lys Val Leu Thr Gly Val Pro Cys Lys Thr Gly Val Ala Trp Gln 370 375 380 Phe Asp Ala Lys Thr Gly Asp Tyr Phe Trp Ser Lys Ala Thr Val Glu 385 390 395 400 Gln Asn Ser Ile Ala Ser Ile Asp Asp Thr Gly Leu Val Thr Val Asn 405 410 415 Glu Asp Met Ile Leu Lys Glu Pro Gly Lys Thr Tyr Asn Tyr Cys Pro 420 425 430 Thr Phe Leu Gly Gly Arg Asp Trp Pro Ser Ala Gly Tyr Leu Pro Lys 435 440 445 Ser Asn Leu Tyr Val Ile Pro Leu Ser Asn Ala Cys Tyr Asp Val Met 450 455 460 Ala Arg Thr Thr Glu Ala Thr Pro Ala Asp Val Tyr Asn Thr Asp Ala 465 470 475 480 Thr Leu Val Leu Ala Pro Gly Lys Thr Asn Met Gly Arg Val Asp Ala 485 490 495 Ile Asp Leu Ala Thr Gly Glu Thr Lys Trp Ser Tyr Glu Thr Arg Ala 500 505 510 Ala Leu Tyr Asp Pro Val Leu Thr Thr Gly Gly Asp Leu Val Phe Val 515 520 525 Gly Gly Ile Asp Arg Asp Phe Arg Ala Leu Asp Ala Glu Ser Gly Lys 530 535 540 Glu Val Trp Ser Thr Arg Leu Pro Gly Ala Val Ser Gly Tyr Thr Thr 545 550 555 560 Ser Tyr Ser Ile Asp Gly Arg Gln Tyr Val Ala Val Val Ser Gly Gly 565 570 575 Ser Leu Gly Gly Pro Thr Phe Gly Pro Thr Thr Pro Asp Val Asp Ser 580 585 590 Ala Ser Gly Ala Asn Gly Ile Tyr Val Phe Ala Leu Pro Glu Lys Lys 595 600 605 <210> 2 <211> 1827 <212> DNA <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> CDS <222> (1) .. (1824) <400> 2 atg aga ttt gag tat ttg cgg cag aac gtc gtg ggc ctc gcc ctg tcg 48 Met Arg Phe Glu Tyr Leu Arg Gln Asn Val Val Gly Leu Ala Leu Ser 1 5 10 15 acg gcc ttg atc gca tcg ctg agc ggg ccc gcg ttc gca cag cac gac 96 Thr Ala Leu Ile Ala Ser Leu Ser Gly Pro Ala Phe Ala Gln His Asp 20 25 30 gcc aac gcc gct gcc gag ccg agc aag gcc ggc cag agc gct atc gag 144 Ala Asn Ala Ala Ala Glu Pro Ser Lys Ala Gly Gln Ser Ala Ile Glu 35 40 45 aac ttc cag ccg gtc acg gct gac gat ctc gcc ggc aag aac ccg gcc 192 Asn Phe Gln Pro Val Thr Ala Asp Asp Leu Ala Gly Lys Asn Pro Ala 50 55 60 aac tgg ccg atc ctg cgc ggt aac tac cag ggc tgg ggt tac tnc asg Trp Pro Ile Leu Arg Gly Asn Tyr Gln Gly Trp Gly Tyr S er Pro 65 70 75 80 ctc gac cag atc aac aag gac aac gtt ggc gac ctc cag ctc gtc tgg 288 Leu Asp Gln Ile Asn Lys Asp Asn Val Gly Asp Leu Gln Leu Val Trp 85 90 95 tcg cgc acg atg gag ccg ggc t aac gaa ggt gct gcc atc gcc tat 336 Ser Arg Thr Met Glu Pro Gly Ser Asn Glu Gly Ala Ala Ile Ala Tyr 100 105 110 aac ggc gtc atc ttc ctg ggc aac acc aac gac gtg atc cag gcg atc 384 Asn Gly Val Ile Leu Gly Asn Thr Asn Asp Val Ile Gln Ala Ile 115 120 125 gac ggc aag acg ggt tcc ctc atc tgg gaa tat cgt cgc aag ctg ccg 432 Asp Gly Lys Thr Gly Ser Leu Ile Trp Glu Tyr Arg Arg Lys Leu Pro 130 135 140 tcg gcc tcc aag ttc atc aac tcg ctc ggc gcc gcc aag cgt tcg atc 480 Ser Ala Ser Lys Phe Ile Asn Ser Leu Gly Ala Ala Lys Arg Ser Ile 145 150 155 160 gca ctc ttc ggc gac aag gtc tat ttc aac ttc gtt 528 Ala Leu Phe Gly Asp Lys Val Tyr Phe Val Ser Trp Asp Asn Phe Val 165 170 175 gtc gcc ctc gat gcc aag acc ggc aag ctg gcc tgg gaa acc aat cgc 576 Val Ala Leu Asp Ala Lyu Thr Gly Lys Ala Trp Glu Thr Asn Arg 180 185 190 ggc cag ggc gtt gaa gaa ggc gtt gcg aac tct tcc ggc ccg atc gtg 624 Gly Gln Gly Val Glu Glu Gly Val Ala Asn Ser Ser Gly Pro Ile Val 195 200 205 gtc gat ggc gtc gtg atg ggc tcc acc tgc cag ttc tcg ggc ttc 672 Val Asp Gly Val Val Ile Ala Gly Ser Thr Cys Gln Phe Ser Gly Phe 210 215 220 ggt tgc tat gtg acc ggt acc gac gct gag tcc ggt gaa gaa ctg tgg 720 Gly Cys Thr Gly Thr Asp Ala Glu Ser Gly Glu Glu Leu Trp 225 230 235 240 cgc aac acc ttc atc ccc cgt ccg ggc gaa gaa ggt gac gac acc tgg 768 Arg Asn Thr Phe Ile Pro Arg Pro Gly Glu Glu Gly Asp Asp Thr Trp 245 250 255 ggc ggc gcg ccc tac gaa aac cgt tgg atg acc ggt gcc tgg ggc cag 816 Gly Gly Ala Pro Tyr Glu Asn Arg Trp Met Thr Gly Ala Trp Gly Gln 260 265 270 atc acc tat gtt ccg gaa ctc gac tac gtt ggc tcg acc ggc 864 Ile Thr Tyr Asp Pro Glu Leu Asp Leu Val Tyr Tyr Gly Ser Thr Gly 275 280 285 gcc ggc ccg gct tcg gaa gtc cag cgc ggc acc gaa ggc ggc act ctc 912 Ala Gly Pro Ala Ser Glu Pro Ala Glu Pro Ala Ser Glu Gly Thr Glu Gly Gly Thr Leu 290 295 300 gca ggc acc aat acc cgc ttt gcg gtg aag ccc aag acc ggt gaa gtc 960 Ala Gly Thr Asn Thr Arg Phe Ala Val Lys Pro Lys Thr Gly Glu Val 305 310 315 320 gtc tgg aag cac cag acc ctg ccc cgc gac aac tgg gac tcg gaa tgc 1008 Val Trp Lys His Gln Thr Leu Pro Arg Asp Asn Trp Asp Ser Glu Cys 325 330 335 acg ttc gaa atg atg gtc gtc tcg acg tcc gtc aac ccgacg Thr Phe Glu Met Met Val Val Ser Thr Ser Val Asn Pro Asp Ala Lys 340 345 350 gcc gat ggc atg atg tcc gtc ggt gcc aac gtg ccg cgt ggc gaa acc 1104 Ala Asp Gly Met Met Ser Val Gly Ala Asn Val Pro Arg Gly Glu Thr 355 360 365 cgc aag gtg ctg acc ggc gtg ccg tgc aag acc ggc gtc gcc tgg cag 1152 Arg Lys Val Leu Thr Gly Val Pro Cys Lys Thr Gly Val Ala Trp Gln 370 375 380 ttc gat gcc aag acc ggc gac tgg tcc aag gca acc gtc gag 1200 Phe Asp Ala Lys Thr Gly Asp Tyr Phe Trp Ser Lys Ala Thr Val Glu 385 390 395 400 cag aac tcg atc gct tcg atc gat gac acg ggc ctc gtt acg gtc aat 1248 Gln Asn Ser Il e Ala Ser Ile Asp Asp Thr Gly Leu Val Thr Val Asn 405 410 415 gag gac atg atc ctc aag gag ccg ggc aag acc tac aat tac tgc ccg 1296 Glu Asp Met Ile Leu Lys Glu Pro Gly Lys Thr Tyr Asn Tyr Cys Pro 420 425 430 acc ttc ctc ggc ggt cgt gac tgg ccg tcg gcc ggc tac ctg ccg aag 1344 Thr Phe Leu Gly Gly Arg Asp Trp Pro Ser Ala Gly Tyr Leu Pro Lys 435 440 445 tcg aac ctc tac agt gct ac cc tac gac gtg atg 1392 Ser Asn Leu Tyr Val Ile Pro Leu Ser Asn Ala Cys Tyr Asp Val Met 450 455 460 gcc cgt acg acc gaa gcc act ccg gct gac gtc tac aac acc gac gcc 1440 Ala Arg Thr Thr Glu Ala Thr Pro Ala Asp Val Tyr Asn Thr Asp Ala 465 470 475 480 acg ctc gtg ctg gct ccg ggc aag acc aat atg ggt cgc gtg gat gcc 1488 Thr Leu Val Leu Ala Pro Gly Lys Thr Asn Met Gly Arg Val Asp Ala 485 490 495cc atc gcc acc ggc gag acc aag tgg tcc tac gaa acc cgt gcg 1536 Ile Asp Leu Ala Thr Gly Glu Thr Lys Trp Ser Tyr Glu Thr Arg Ala 500 505 510 gct ctc tac gac ccg gtc ctg acc acc ggc ggc gac ctcgt 1584 Ala Leu Tyr Asp Pro Val Leu Thr Thr Gly Gly Asp Leu Val Phe Val 515 520 525 ggc ggt atc gat cgt gac ttc cgc gct ctg gac gcc gag tcc ggc aag 1632 Gly Gly Ile Asp Arg Asp Phe Arg Ala Leu Asp Ala Glu Ser Gly Lys 530 535 540 gaa gtc tgg tcc acc cgt ctg ccg ggt gcg gtt tcc ggc tac acc acc 1680 Glu Val Trp Ser Thr Arg Leu Pro Gly Ala Val Ser Gly Tyr Thr Thr 545 550 555 560 agc tac tcc atc gat ggc cag tac gtc gcg gtt gtc tcg ggc ggc 1728 Ser Tyr Ser Ile Asp Gly Arg Gln Tyr Val Ala Val Val Ser Gly Gly 565 570 575 tcg ctc ggt ggc ccg acc ttc ggc ccg acc acc ccg gc Gcgcg Pro Thr Phe Gly Pro Thr Thr Pro Asp Val Asp Ser 580 585 590 gct tcg ggc gcg aac ggc atc tac gtc ttc gct ctt ccc gag aag aag 1824 Ala Ser Gly Ala Asn Gly Ile Tyr Val Phe Ala Leu Pro Glu Lys Lys 595 600 605 taa 1827 <210> 3 <211> 30 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (2) .. (2) <223> Gln or Leu. <220><221> UNSURE <222> (4) .. (4) <223> Gly or Le u. <220><221> UNSURE <222> (5) .. (5) <223> Pro or Val. <220><221> UNSURE <222> (6) .. (6) <223> Arg or Pro. <220><221> UNSURE <222> (8) .. (8) <223> Asn or Lys. <220><221> UNSURE <222> (13) .. (13) <223> Unidentified amino acid. <220><221> UNSURE <222> (18) .. (18) <223> Unidentified amino acid. <220><221> UNSURE <222> (20) .. (20) <223> Unidentified amino acid. <220><221> UNSURE <222> (23) .. (23) <223> Unidentified amino acid. <220><221> UNSURE <222> (24) .. (24) <223> Unidentified amino acid. <220><221> UNSURE <222> (27) .. (27) <223> Unidentified amino acid. <220><221> UNSURE <222> (30) .. (30) <223> Unidentified amino acid. <400> 3 Val Xaa Thr Xaa Xaa Xaa Asp Xaa Trp Asp Ser Glu Xaa Thr Phe Glu 1 5 10 15 Met Xaa Val Xaa Ser Thr Xaa Xaa Asn Pro Xaa Ala Lys Xaa 20 25 30 <210> 4 <211> 25 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Asn or Ala. <220><221> UNSURE <222> (2). . (2) <223> Thr or Pro. <220><221> UNSURE <222> (3) .. (3) <223> A la or Val. <220><221> UNSURE <222> (4) .. (4) <223> Asn or Glu. <220><221> UNSURE <222> (5) .. (5) <223> Gln or Trp. <220><221> UNSURE <222> (6) .. (6) <223> Asn or Pro. <220><221> UNSURE <222> (7) .. (7) <223> Ser or Ile. <220><221> UNSURE <222> (8) .. (8) <223> Ile or Leu. <220><221> UNSURE <222> (9) .. (9) <223> Ala or Arg. <220><221> UNSURE <222> (10) .. (10) <223> Ser or Gly. <220><221> UNSURE <222> (11) .. (11) <223> Asn or Ile. <220><221> UNSURE <222> (12) .. (12) <223> Asp or Tyr. <220><221> UNSURE <222> (13) .. (13) <223> Asp or Gln. <220><221> UNSURE <222> (14) .. (14) <223> Thr or Gly. <220><221> UNSURE <222> (15) .. (15) <223> Gly or Trp. <220><221> UNSURE <222> (17) .. (17) <223> Tyr or Val. <220><221> UNSURE <222> (18) .. (18) <223> Ser or Thr. <220><221> UNSURE <222> (19) .. (19) <223> Pro o Val. <220><221> UNSURE <222> (20) .. (20) <223> Asn or Leu. <220><221> UNSURE <222> (21) .. (21) <223> Asp or Glu. <220><221> UNSURE <222> (23) .. (23) <223> Met or Ile. <220><221> UNSURE <222> (24). . (24) <223> Asn or Ile. <400> 4 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Lys 20 25 <210> 5 <211> 30 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <400> 5 Glu Val Trp Ser Thr Arg Leu Pro Gly Ala Val Ser Gly Tyr Thr Thr 1 5 10 15 Ser Tyr Ser Ile Asp Gly Arg Gln Tyr Val Ala Val Val Ser 20 25 30 <210> 6 <211> 30 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Thr or Ser. <220 ><221> UNSURE <222> (2) .. (2) <223> Tyr or Asn. <220><221> UNSURE <222> (3) .. (3) <223> Asn or Leu. <220 ><221> UNSURE <222> (5) .. (5) <223> Val or unidentified amino acid. <220><221> UNSURE <222> (6) .. (6) <223> Pro or Ile. <220><221> UNSURE <222> (7) .. (7) <223> Thr or Pro. <220><221> UNSURE <222> (8) .. (8) <223> Phe or Leu. <220><221> UNSURE <222> (9) .. (9) <223> Ser or Leu. <220><221> UNSURE <222> (10) .. (10) <223> Asn or Gly. <220><221> UNSURE <222> (11) .. (11) <223> Gly or Ala. <220><221> UNSURE <222> (12) .. (12) <223> Arg or unidentified amino acid. <220><221> UNSURE <222> (13) .. (13) <223> Asp or Tyr . <220><221> UNSURE <222> (14) .. (14) <223> Asp or Trp. <220><221> UNSURE <222> (15) .. (15) <223> Pro or Val . <220><221> UNSURE <222> (16) .. (16) <223> Ser or Met. <220><221> UNSURE <222> (18) .. (18) <223> Gly or Arg . <220><221> UNSURE <222> (19) .. (19) <223> Thr or Tyr. <220><221> UNSURE <222> (20) .. (20) <223> Thr or Leu . <220><221> UNSURE <222> (21) .. (21) <223> Glu or Pro. <220><221> UNSURE <222> (22) .. (22) <223> Ala or Lys . <220><221> UNSURE <222> (23) .. (23) <223> Thr or Ala. <220><221> UNSURE <222> (24) .. (24) <223> Ser or Pro . <400> 6 Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Asp Val Tyr Asn Thr 20 25 30 <210> 7 <211> 20 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Unidentified amino acid. <220><221> UNSURE <222> (6) .. (6) <223> U nidentified amino acid. <400> 7 Xaa Ser Tyr Glu Ser Xaa Ala Ala Leu Tyr Asp Pro Val Leu Thr Thr 1 5 10 15 Gly Gly Gly Leu 20 <210> 8 <211> 30 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO144464 <220><221> UNSURE <222> (1) .. (1) <223> Asp or Leu. <220><221> UNSURE <222> (2) .. (2) <223> Asn or Ala . <220><221> UNSURE <222> (3) .. (3) <223> Val or Trp. <220><221> UNSURE <222> (4) .. (4) <223> Gly or Glu . <220><221> UNSURE <222> (5) .. (5) <223> Asp or Thr. <220><221> UNSURE <222> (6) .. (6) <223> Asn or Leu . <220><221> UNSURE <222> (7) .. (7) <223> Gln or Arg. <220><221> UNSURE <222> (8) .. (8) <223> Gly or Leu . <220><221> UNSURE <222> (9) .. (9) <223> Gln or Val. <220><221> UNSURE <222> (10) .. (10) <223> Gly or Trp . <220><221> UNSURE <222> (11) .. (11) <223> Ser or Val. <220><221> UNSURE <222> (12) .. (12) <223> Glu or Arg . <220><221> UNSURE <222> (13) .. (13) <223> Thr or Glu. <220><221> UNSURE <222> (14) .. (14) <223> Gly or Met . <220><221> UNSURE <222> (15) .. (15) <223> Glu or Val. <2 20><221> UNSURE <222> (16) .. (16) <223> Ala or Pro. <220><221> UNSURE <222> (17) .. (17) <223> Asn or Gly. <220><221> UNSURE <222> (20) .. (20) <223> Gly or Glu. <220><221> UNSURE <222> (21) .. (21) <223> Gly or Pro. <220><221> UNSURE <222> (22) .. (22) <223> Ala or Ile. <220><221> UNSURE <222> (23) .. (23) <223> Ala or Val. <220><221> UNSURE <222> (24) .. (24) <223> Val or Ile. <220><221> UNSURE <222> (25) .. (25) <223> Asp or Ala. <220><221> UNSURE <222> (26) .. (26) <223> Gly or Tyr. <220><221> UNSURE <222> (27) .. (27) <223> Asn or Val. <220><221> UNSURE <222> (28) .. (28) <223> Gly or Val. <220><221> UNSURE <222> (29) .. (29) <223> Val or Ile. <220><221> UNSURE <222> (30) .. (30) <223> Ala or Ile. <400> 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ser Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 <210> 9 <211> 8 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (7) .. (7). ) <223> Ser or Arg. <400> 9 Thr Gly Asp Ty r Phe Trp Xaa Lys 1 5 <210> 10 <211> 11 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (4) .. (4) <223> Ala or Val. <220><221> UNSURE <222> (5) .. (5) <223> Ala or Trp. <220><221> UNSURE <222> (6) .. (6) <223> Gln, Trp or Glu. <220><221> UNSURE <222> (7) .. (7) <223> Phe or Gln. <220><221> UNSURE <222> (8) .. (8) <223> Phe or Asp. <220><221> UNSURE <222> (9) .. (9) <223> Ala or Asp. <220><221> UNSURE <222> (10) .. (10) <223> Ala or Lys. <400> 10 Thr Gly Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 <210> 11 <211> 20 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1 ) .. (1) <223> Thr or Ala. <220><221> UNSURE <222> (3) .. (3) <223> Val or Asn. <220><221> UNSURE <222> (4 ). (4) <223> Tyr or Glu. <220><221> UNSURE <222> (6) .. (6) <223> Pro, Asn or Ala. <220><221> UNSURE <222> (8) .. (8) <223> Phe or Ile. <220><221> UNSURE <222> (9) .. (9) <223> Leu or Ala. <220><221> UNSURE <222> (11) .. (11) <223> Gly or Ile. <220><221> UN SURE <222> (16) .. (16) <223> Leu or Ser. <220><221> UNSURE <222> (17) .. (17) <223> Ala or Val. <220><221> UNSURE <222> (20) .. (20) <223> Leu or Asn. <400> 11 Xaa Tyr Xaa Xaa Gln Xaa Thr Xaa Xaa Gly Xaa Arg Asp Trp Pro Xaa 1 5 10 15 Xaa Gly Tyr Xaa 20 <210 > 12 <211> 29 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Unidentified amino acid. <220><221> UNSURE <222 > (9) .. (9) <223> Unidentified amino acid. <220><221> UNSURE <222> (11) .. (11) <223> Unidentified amino acid. <220><221> UNSURE <222>> (23) .. (23) <223> Unidentified amino acid. <220><221> UNSURE <222> (24) .. (24) <223> Unidentified amino acid. <220><221> UNSURE <222 > (27) .. (27) <223> Unidentified amino acid. <220><221> UNSURE <222> (28) .. (28) <223> Unidentified amino acid. <400> 12 Xaa Gln Thr Leu Pro Arg Asp Asn Xaa Asp Xaa Glu Glu Thr Phe Glu 1 5 10 15 Met Met Val Val Val Thr Xaa Xaa Asn Pro Xaa Xaa Lys 20 25 <210> 13 <211> 30 <212> PRT <213> Pseudogluconobacter saccharok etogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Ala, Glu or Asp. <220><221> UNSURE <222> (2) .. (2) <223> Val or Gly. <220><221> UNSURE <222> (5) .. (5) <223> Ala or Thr. <220><221> UNSURE <222> (6) .. (6) <223> Ile or Arg. <220><221> UNSURE <222> (7) .. (7) <223> Glu or Leu. <220><221> UNSURE <222> (8) .. (8) <223> Asn or Pro. <220><221> UNSURE <222> (9) .. (9) <223> Phe or Gly. <220><221> UNSURE <222> (10) .. (10) <223> Ala or Gln. <220><221> UNSURE <222> (11) .. (11) <223> Val or Pro. <220><221> UNSURE <222> (13) .. (13) <223> Gly or Thr. <220><221> UNSURE <222> (14) .. (14) <223> Tyr or Ala. <220><221> UNSURE <222> (15) .. (15) <223> Asp or Thr. <220><221> UNSURE <222> (18) .. (18) <223> Ala or Tyr. <220><221> UNSURE <222> (21) .. (21) <223> Asn or Asp. <220><221> UNSURE <222> (23) .. (23) <223> Ala or Arg. <220><221> UNSURE <222> (24) .. (24) <223> Asn or Gln. <220><221> UNSURE <222> (29) .. (29) <223> Unidentified amino acid. <400> 13 Xaa Xaa Gln Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Xaa Asp 1 5 10 15 Leu Xaa Gly Lys Xaa Pro Xaa Xaa Tyr Val Ile Leu Xaa Gly 20 25 30 <210> 14 <211> 29 <212> PRT <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> UNSURE <222> (1) .. (1) <223> Ser, Ala, Val, Asp or Trp. <220><221> UNSURE <222> (12) .. (12) <223> Unidentified amino acid. <220 ><221> UNSURE <222> (23) .. (23) <223> Unidentified amino acid. <400> 14 Xaa Asn Leu Tyr Val Ile Pro Leu Ser Asn Ala Xaa Tyr Asp Val Met 1 5 10 15 Ala Arg Thr Thr Glu Ala Xaa Pro Ala Asp Val Tyr Asn 20 25 <210> 15 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 15 gargtbtggt csacscg 17 <210> 16 <211> 15 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 16 vacsgcvacr tactg 15 <210> 17 <211> 84 <212> DNA <213> Pseudogluconobacter saccharoketogenes IFO14464 <220><221> misc feature <222> () .. () <223> AADH gene fragment amplified by PCR. <400> 17 gaagtctggt ccacccgtct gccgggtgcg gtttccggct acaccaccag ctactccatc 60 gatggccgcc agtacgtcgc ggtt 84 <210> 18 <211> 20 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 18 gagtagctgg tggtgtagcc 20 <210> 19 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 19 gayaaytggg aytcsg 16 <210> 20 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 20 acstayaayt ayccbcc 17 <210> 21 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 21 aayctbtayg tbatccc 17 <210> 22 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 22 taygayccbg tbctbac 17 <210> 23 <211> 15 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 23 ggygaytayt tctgg 15 <210> 24 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 24 acsttcgara tgatgg 16 <210> 25 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for amplifying AADH gene fragment. <400> 25 taygaygtba tggcscg 17 <210> 26 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 26 caactggccg atcctg 16 <210> 27 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 27 caagacgggt tccctc 16 <210> 28 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 28 ggcgttgaag aaggcg 16 <210> 29 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 29 cgttggatga ccggtg 16 <210> 30 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 30 ggactcggaa tgcacg 16 <210> 31 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 31 cgtcgagcag aactcg 16 <210> 32 <211> 15 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 32 gacgccacgc tcgtg 15 <210> 33 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 33 cagtacgtcg cggttg 16 <210> 34 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 34 caggatcggc cagttg 16 <210> 35 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 35 gagggaaccc gtcttg 16 <210> 36 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 36 cgccttcttc aacgcc 16 <210> 37 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 37 caccggtcat ccaacg 16 <210> 38 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 38 cgtgcattcc gagtcc 16 <210> 39 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 39 cgagttctgc tcgacg 16 <210> 40 <211> 15 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 40 cacgagcgtg gcgtc 15 <210> 41 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 41 caaccgcgac gtactg 16 <210> 42 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 42 ctcatgtatc tgcgctc 17 <210> 43 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 43 gctcaactat gtgggag 17 <210> 44 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 44 catcaaagcc cttgcg 16 <210> 45 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 45 gacgtgttgc tgacgg 16 <210> 46 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 46 ggctcgagga cattcc 16 <210> 47 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 47 gacttcgcga ccgatg 16 <210> 48 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 48 ctcaaccgct agagcgc 17 <210> 49 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 49 gagcgcagat acatgag 17 <210> 50 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 50 ctcccacata gttgagc 17 <210> 51 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 51 cgcaagggct ttgatg 16 <210> 52 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 52 ccgtcagcaa cacgtc 16 <210> 53 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 53 ggaatgtcct cgagcc 16 <210> 54 <211> 16 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 54 catcggtcgc gaagtc 16 <210> 55 <211> 17 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as primer for determining AADH gene sequence. <400> 55 gcgctctagc ggttgag 17 <210> 56 <211> 12 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Eco RI / Sac I adaptor. <400> 56 ggaattccag ct 12 <210> 57 <211> 59 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for introducing Nco I site into upstream of AADH coding sequence. <400> 57 tagctgaatt ccatggtcag taggaatatt tctcatgaga tttgagtatt tgcggcaga 59 <210> 58 <211> 20 <212> DNA <213> Artificial / Unknown <220><221> misc feature <222> () .. () <223> Oligonucleotide designed to act as PCR primer for introducing Nco I site into upstream of AADH coding sequence. <400> 58 tcgtagggcg cgccgcccca 20

[Brief description of drawings]

FIG. 1 shows a scheme for the construction of pADH3 and pADH4. Process: SacI digestion and EcoRI / SacI adapter addition process: EcoRI digestion and isolation of 2.9 kb fragment: EcoRI digestion and isolation of 7.3 kb fragment: ligation

FIG. 2 shows a scheme for the construction of pADH-tufB. PCR: A NcoI site was introduced upstream of the AADH start codon with a synthetic primer, and a 0.8 kb fragment encoding the N-terminal side of AADH was amplified. Process: EcoRI, AscI double digestion and 0.8k
b fragment isolation step: EcoRI, AscI double digestion and 4.6k
b fragment isolation step: ligation step: EcoRI, NcoI double digestion and 2.7 k
Isolation step of b fragment: EcoRI partial digestion, NcoI digestion and 4.
2 kb fragment isolation step: ligation

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/04 C12N 15/00 ZNAA C12P 19/02 5/00 A (72) Inventor Yoshinori Ishii Osaka Osaka Fujisawa Yakuhin Kogyo Co., Ltd., 3-4 Dosho-cho, Chuo-ku, Japan (72) Inventor Yuji Noguchi, 3-4-7 Dosho-cho, Doo-cho, Chuo-ku, Osaka-shi, Osaka (72) Inventor Yamada Choji 3-4-7 Doshomachi, Chuo-ku, Osaka-shi, Osaka Prefecture F-term in Fujisawa Pharmaceutical Co., Ltd. (reference) 4B024 AA03 AA05 BA08 BA77 CA01 GA11 HA20 4B050 CC03 DD02 EE10 LL05 4B064 AF17 CA21 CB13 CB30 CC24 CD09 DA10 4B065 AA01Y AB01 AC14 BA02 CA20 CA28 CA41 CA44

Claims (12)

[Claims] 1. A protein having the following characteristics (a) to (c): (a) Action: It can catalyze the reaction of oxidizing a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group and the reaction of oxidizing an aldehyde group of a compound having an aldehyde group to a carboxyl group. (b) Molecular weight: 65,000 ± 2,000 (SDS-PA
GE), 55,000 ± 4,000 (gel filtration) (c) Does not contain heme iron and rare earth elements in the molecule 2. The protein according to claim 1, which further has at least one of the following physicochemical properties (a) to (d). (a) Optimum pH: 4.5 to 5.5 (b) Isoelectric point: 4.1 ± 0.3 (c) Contains pyrroloquinoline quinone in the molecule as a prosthetic group (d) K _m for sorbose Value: about 40 mM 3. The following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, A protein capable of catalyzing the reaction of oxidizing a hydroxymethyl group of a compound having a hydroxymethyl group to an aldehyde group and the reaction of oxidizing an aldehyde group of a compound having an aldehyde group to a carboxyl group 4. The protein according to claim 1, which can be purified from a bacterium belonging to the genus Pseudogluconobacter. 5. A DNA encoding the protein according to any one of claims 1 to 4. 6. The DNA according to claim 5, which comprises the following nucleotide sequence (a) or (b): (a) Base sequence shown in SEQ ID No. 2 in the Sequence Listing (b) DN consisting of base sequence shown in Sequence No. 2 in the Sequence Listing
A base sequence capable of hybridizing with A under stringent conditions 7. A recombinant vector containing the DNA according to claim 5 or 6. 8. A host cell transformed with the recombinant vector according to claim 7. 9. The cell according to claim 8 is cultured in a medium,
The method for producing a protein according to claim 1, which comprises collecting a protein having an alcohol / aldehyde dehydrogenase activity from the obtained culture. 10. A step of oxidizing the hydroxymethyl group or the aldehyde group to a carboxyl group by contacting the protein according to claim 1 with a compound containing a hydroxymethyl group or an aldehyde group. , A method for producing a compound containing a carboxyl group. 11. A step of oxidizing the hydroxymethyl group or the aldehyde group to a carboxyl group by contacting the culture or the treated product thereof according to claim 9 with a compound containing a hydroxymethyl group or an aldehyde group, A method for producing a compound containing a carboxyl group. 12. The compound containing a hydroxymethyl group or an aldehyde group is L-sorbose or L-sorbosone, and the compound containing a carboxyl group is 2-keto-L-.
The method according to claim 10 or 11, which is gulonic acid.