WO1999020763A1 - D-sorbitol deshydrogenase, genes et utilisation de celle-ci - Google Patents

D-sorbitol deshydrogenase, genes et utilisation de celle-ci Download PDF

Info

Publication number
WO1999020763A1
WO1999020763A1 PCT/JP1998/004612 JP9804612W WO9920763A1 WO 1999020763 A1 WO1999020763 A1 WO 1999020763A1 JP 9804612 W JP9804612 W JP 9804612W WO 9920763 A1 WO9920763 A1 WO 9920763A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
amino acid
acid sequence
polypeptide
seq
Prior art date
Application number
PCT/JP1998/004612
Other languages
English (en)
Japanese (ja)
Inventor
Yoshimasa Saito
Yoshinori Ishii
Yuji Noguchi
Koji Yoshikawa
Shinsuke Soeda
Original Assignee
Fujisawa Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fujisawa Pharmaceutical Co., Ltd. filed Critical Fujisawa Pharmaceutical Co., Ltd.
Publication of WO1999020763A1 publication Critical patent/WO1999020763A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/99021—D-Sorbitol dehydrogenase (acceptor) (1.1.99.21)

Definitions

  • the present invention relates to a protein having D-sorbitol dehydrogenase (hereinafter referred to as SLDH) activity, each subunit thereof, a gene encoding them, L-sorbose and 2-keto by genetic manipulation using the genes.
  • the present invention relates to a method for producing L-dalonic acid (hereinafter referred to as 2KLGA) and an expression system involved in the production thereof.
  • 2KLGA L-dalonic acid
  • L-sorbose is a key intermediate in the synthesis of L-ascorbic acid (vitamin C) by the Reichstein method (see Figure 1).
  • D-sorbitol is chemically oxidized, about half of the product is converted to D-sorbose, whereas when microorganisms having S LDH activity are contacted with D-sorbitol, L-sorbitol is produced in about 95% yield. Fermentation has been used in the process of converting D-sorbitol to L-sorbose since only the body can be obtained.
  • 2KLGA is industrially synthesized by chemically oxidizing L-sorbose.
  • SDH L-sorbose dehydrogenase
  • SNDH L-sorbosone dehydrogenase
  • an object of the present invention is to provide an SLDH gene for establishing the above-described method for producing 2KLGA by fermentation, and furthermore, to reduce host microorganisms transformed with the gene, particularly SDH and SNDH activities. Is to provide a microorganism having the same.
  • an object of the present invention is to provide a method for producing L-sorbose or 2KLGA from D-sorbitol using the microorganism.
  • Another object of the present invention is to provide a method for producing recombinant SLDH by culturing a host microorganism transformed with the SLDH gene and a method for producing L-sorbose by an enzymatic method using the SLDH. It is to be. Disclosure of the invention
  • the present inventors have conducted intensive studies in order to achieve the above object, and as a result, a DNA containing the three subunit coding regions of the enzyme and their promoter regions from acetic acid bacteria having SLDH activity. was successfully cloned. Furthermore, the present inventors transformed host cells with an expression vector having the DNA, cultured the obtained recombinant host cells to obtain SLDH, and used the culture to convert L-sorbitol into L-LDH. Successful conversion to sorbose efficiently led to the completion of the present invention.
  • N-terminal amino acid sequence is Ser Ser Ser Asn Ser Leu Ser Ala Asp Val Val lie Val Gly Ser Gly Val Ala Gly Ala (SEQ ID NO: 8) or one or several amino acids in the amino acid sequence Deleted, substituted or added amino acids Is an array
  • amino acid sequence at the N-terminal is Glu Glu Ala Lys Ser Pro Leu Ala Ser Arg Asp Glu Tyr Glu Arg Phe Phe Glu Val Ser (SEQ ID NO: 9) or one or several amino acids in the amino acid sequence Is a deleted, substituted or added amino acid sequence
  • N-terminal amino acid sequence is Glu Glu Ala Lys Ser Pro Leu Ala Ser Arg Asp Glu Tyr Glu Arg Phe Phe Glu Val Ser (SEQ ID NO: 9) or one or several amino acids in the amino acid sequence Is a deleted, substituted or added amino acid sequence
  • amino acid sequence at the N-terminal is Ser Ser Ser Asn Ser Leu Ser Ala Asp Val Val lie Val Gly Ser Gly Val Ala Gly Ala (SEQ ID NO: 8) or one or several amino acids in the amino acid sequence Amino acid sequence with deleted, substituted or added acid
  • the amino acid sequence at the N-terminal side is Met Arg Glu Gly Asn Lys Ala Glylie Arg Arg Leu Phe Leu Pro Ala Ala lie Ala Ser (SEQ ID NO: 10) or one or several in the amino acid sequence Is an amino acid sequence in which amino acids are deleted, substituted or added, and has a consensus sequence of three heme binding sites (Cys Xaa Xaa Cys
  • An oligomer protein comprising the polypeptide of [1] or [2], and capable of catalyzing a reaction for converting D-sorbitol to L-sorbose.
  • (b) consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing, and the following (c) or (d) Reaction to convert D-sorbitol to L-sorbose with some polypeptides Polypeptides that can form soluble oligomeric proteins
  • An oligomer protein comprising the polypeptide of the above [7] and [8] and capable of catalyzing a reaction for converting D-sorbitol to L_sorbose.
  • DNA of the following (c) or (d) (c) a DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 591 in the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing
  • a promoter gene consisting of the following DNA (a) or (b):
  • DNA comprising a base sequence in which one or several bases have been deleted, substituted or added in the base sequence of (a), and having promoter activity in a microorganism.
  • the transformant has an ability to convert L-sorbose into 2KLGA.
  • Host cells transformed with any of the above expression vectors [20], [21], [23] or [24] are cultured in a medium, and D-sorbitol is obtained from the resulting culture.
  • a method for producing an oligomeric protein comprising collecting an oligomeric protein capable of catalyzing a reaction of converting glycerol to L-sorbose.
  • a host cell transformed with the expression vector of any of [20], [21], [23] or [24] above is cultured in a medium, and the resulting culture or its treatment is obtained.
  • a method for producing L-sorbose comprising the step of bringing D-sorbitol into contact with a substance.
  • a host cell capable of converting L-sorbose transformed into the expression vector of any of [20], [21], [23] or [24] into 2 KLG A in a medium 2.
  • a method for producing KLG A comprising the step of contacting D-sorbitol with the obtained culture or a processed product thereof.
  • a cell having the ability to convert L-sorbose into 2KLGA is transformed with an expression vector containing a group of genes encoding the SLDH of the present invention, and the resulting transformant is cultured.
  • 2KLGA can be produced easily and in large quantities directly from D-sorbitol. Therefore, the production process of L-ascorbic acid can be greatly simplified by this method.
  • FIG. 1 is a diagram showing a reaction scheme of L-ascorbic acid synthesis by the Reichstein method.
  • FIG. 2 shows lambda phage clone # 7 DNA and plasmid pSD37R
  • FIG. 3 shows a restriction map of pBL7Sa17 and pBL7Sa17.
  • FIG. 3 is a diagram showing a method for constructing plasmid p S LDH3.
  • the SLDH of the present invention is an oligomer protein comprising two large and small subunits, more preferably an oligomer protein further comprising a cytochrome c-like polypeptide.
  • Each subunit has the following features.
  • amino acid sequence at the N-terminal is Ser Ser Ser Asn Ser Leu Ser Ala Asp Val Val lie Val Gly Ser Gly Val Ala Gly Ala (SEQ ID NO: 8) or one or several amino acids in the amino acid sequence Amino acid sequence in which the acid has been deleted, substituted or added
  • N-terminal amino acid sequence is Glu Glu Ala Lys Ser Pro Leu Ala Ser Arg Asp Glu Tyr Glu Arg Phe Phe Glu Val Ser (SEQ ID NO: 9) or one or several amino acids in the amino acid sequence Is a deleted, substituted or added amino acid sequence
  • N-terminal amino acid sequence is Met Arg Glu Gly Asn Lys Ala Gly lie Arg Arg Leu Phe Leu Pro Ala Ala lie Ala Ser (SEQ ID NO: 10) or one or several amino acids in the amino acid sequence Amino acid with deletion, substitution or addition of amino acid It is an acid sequence and contains a consensus sequence at three heme binding sites (Cys Xaa Xaa Cys His; SEQ ID NO: 10).
  • the source of the LDH of the present invention is not particularly limited as long as it has the above-mentioned characteristics, and can be obtained by introducing a natural or artificial mutant or a heterologous SLDH gene in addition to a naturally occurring biological source. Those derived from transformants are also included. Preferable examples are acetic acid bacteria, particularly bacteria belonging to the genus Darconobacter, more preferably SLDH derived from the strain Dalconobacter oxydans, more preferably the strain DA03254.
  • the SLDH of the present invention comprises a large subunit consisting of the amino acid sequence shown in SEQ ID NO: 1 or an equivalent thereof, and a small subunit consisting of the amino acid sequence shown in SEQ ID NO: 3 or
  • the LDH of the present invention further comprises a cytochrome c-like polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or an equivalent thereof.
  • the term “equivalent” refers to a polypeptide comprising 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 physicochemical properties of the LDH of the present invention. means.
  • the SLDH of the present invention includes: (1) a method of isolating and purifying a cell or tissue culture producing the enzyme as a raw material, (2) a method of chemically synthesizing, or (3) a gene recombination technique Can be obtained by appropriately using the known method described above.
  • a preferred embodiment of the method (1) the following method is exemplified.
  • a microorganism having the ability to convert D-sorbitol to L-sorbose (for example, Darconobacter oxidans IF03254 strain) is cultured in an appropriate liquid medium, and a fraction having SLDH activity is obtained from the resulting culture. Separate and collect (for example, in the case of Darconobacter oxydans I F03254 strain, since the S LDH activity is found in the cell membrane fraction, the culture is centrifuged to collect the cells, and then subjected to sonication or lysophilization.
  • D-sorbitol to L-sorbose for example, Darconobacter oxidans IF03254 strain
  • a fraction having SLDH activity is obtained from the resulting culture. Separate and collect (for example, in the case of Darconobacter oxydans I F03254 strain, since the S LDH activity is found in the cell membrane fraction, the culture is centrifuged to collect the cells, and then subjected to sonication or lysophil
  • the cells are disrupted by centrifugation and osmotic shock, etc., centrifuged at about 10,000 rpm to collect the supernatant, and then ultracentrifuged at about 30,000-40, OOO rpm to precipitate ( Membrane fraction) is obtained)).
  • an appropriate surfactant preferably a surfactant having a low denaturing power such as Triton X-100, is used from the obtained membrane fraction. Solubilize S LDH.
  • the target S LDH can be purified from the obtained solubilized fraction by appropriately combining separation techniques generally used for isolation and purification of enzyme proteins.
  • a method using a difference in solubility such as salting out and a solvent precipitation method a method using a difference in molecular weight such as dialysis, ultrafiltration, gel filtration, non-denaturing PAGE, SDS-PAGE, and ion Utilize differences in hydrophobicity, such as exchange chromatography, methods that use charges such as hydroxyapatite chromatography, methods that use specific affinity such as affinity chromatography, and reversed-phase high-performance liquid chromatography. And methods utilizing isoelectric point differences such as isoelectric focusing.
  • the production of the SLDH of the present invention by chemical synthesis is carried out, for example, by using all or a part of each sequence based on the amino acid sequence shown in SEQ ID NO: 1 and 3, preferably SEQ ID NO: 5, in a peptide synthesizer. And a complex is formed under appropriate reconstitution conditions for the resulting polypeptide.
  • the production of the SLDH of the present invention using the gene recombination technique is carried out by transforming a host cell transformed with an expression vector containing a DNA encoding each subunit of the SLDH of the present invention into a medium described below. And collecting the enzyme from the resulting culture.
  • the DNA encoding the large subunit, the small subunit and the cytochrome c-like polypeptide of the LDH of the present invention respectively encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or an equivalent thereof.
  • DNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or an equivalent thereof DNA and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5 DNA encoding tide or its equivalent.
  • the term “equivalent” means a polypeptide comprising 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 physicochemical properties of the SLDH of the present invention.
  • the DNA encoding the large subunit, small subunit and cytochrome c-like polypeptide of the S LDH of the present invention is a DNA substantially consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing, respectively.
  • a DNA consisting essentially of the base sequence represented by base numbers 124 to 591 in the base sequence represented by SEQ ID NO: 4 and a DNA consisting essentially of the base sequence represented by SEQ ID NO: 6 in the sequence listing.
  • the term “substantially DNA” refers to not only a DNA consisting of the above specific nucleotide sequence but also stringent conditions (in the present invention, a DNA having a homology of about 60% or more in the nucleotide sequence).
  • a peptide consisting of a base sequence capable of hybridizing with the DNA consisting of the above specific base sequence, and having the same physicochemical properties as the peptide encoded by the DNA consisting of the specific base sequence. Means the DNA that encodes.
  • the DNA encoding the small subunit of the SLDH of the present invention consists of the nucleotide sequence represented by nucleotide numbers 1 to 591 in the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing.
  • Such a DNA provides a small subunit precursor having a leader sequence at the N-terminus as an initial translation product. The leader sequence is cleaved and removed by intracellular protease to form a mature polypeptide.
  • the DNA encoding the large subunit, the small subunit and the cytochrome c-like polypeptide of the SLDH of the present invention is expressed polycistronically under the control of a single motor-gene. It exists in the form of a single gene group arranged in tandem so that it can be performed. In such an embodiment, the individual The open reading frames (ORFs) encoding subunits may partially overlap each other on the gene group. More preferably, as shown in SEQ ID NO: 7 in the Sequence Listing, the genes are arranged in the order of small subunit DNA, large subunit DNA, and cytochrome c-like polypeptide DNA from the 5 ′ upstream side.
  • the DNA of the present invention may be obtained by any method. For example, mR
  • CDNA prepared from NA DNA prepared from genomic DNA, DNA obtained by chemical synthesis, DNA obtained by amplifying the DNA or RNA or DNA by PCR using the PCR method, and these methods as appropriate These include DNAs constructed in combination.
  • the DNA of the present invention can be isolated, for example, by the following method. First,
  • the enzyme is completely or partially purified from cells or tissues that produce SLDH according to the method described above, and the N-terminal partial amino acid sequence of each subunit is determined by the Edman method.
  • the amino acid sequence of an oligopeptide obtained by partially decomposing each subunit of 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 this is used as a primer or a probe to perform PCR or colonization from RNA or DNA prepared from cells or tissues that produce SLDH.
  • One (or plaque) The DNA encoding each subunit is cloned by the hybridization method.
  • SLDH or a subunit thereof is prepared as an antigen using the whole or a part thereof as an antigen, and an antibody against the enzyme or a subunit thereof is prepared according to a conventional method, and is prepared from SLDH-producing cells or tissues. From the DNA or genomic DNA library, a DNA encoding each subunit of the enzyme can be cloned by antibody screening.
  • the promoter gene of the present invention is a DNA comprising the base sequence represented by base numbers 1 to 680 in the base sequence shown in SEQ ID NO: 7 in the sequence listing, or DNA having a base sequence in which one or several bases have been deleted, substituted or added, and having promoter activity in a microorganism.
  • the term "microorganism” used herein is not particularly limited as long as it can function with a promoter originally belonging to a prokaryote such as a bacterium or an actinomycete, but is preferably a bacterium (eg, Escherichia coli, Bacillus subtilis, etc.), or an actinomycete. And some eukaryotes such as yeast.
  • the origin of the promoter gene of the present invention is not particularly limited as long as it has the above characteristics.
  • the promoter gene of the present invention is preferably a promoter that controls the transcription of DNA encoding each subunit of the SLDH of the present invention.
  • the promoter gene is preferably a bacterium belonging to the genus Darconobacter oxydance, more preferably a strain derived from the genus Darconobacter oxydans, more preferably from the strain Darconobacter oxydans IFO 3254, like the SLDH.
  • the promoter gene of the present invention can be obtained, for example, from a genomic DNA library prepared from a cell or tissue that produces an SLDH by the same method as the above-described method for isolating DNA encoding each subunit of SLDH. Plaques) can be cloned by hybridization or antibody screening.
  • 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 replicate and maintain or autonomously propagate in various host cells of prokaryotic cells and Z or eukaryotic cells, such as plasmid vector and phage vector. Is included.
  • the recombinant vector can be conveniently prepared by inserting any of the above DNAs into a closing vector or expression vector available in the art using an appropriate restriction enzyme site. it can.
  • the recombinant vector of the present invention comprises an expression vector containing a DNA functionally encoding each of the large and small subunits of S LDH, more preferably a cytochrome c-like polypeptide.
  • the expression vector functionally contains the DNA to be loaded.
  • “functionally contained” means that the DNA is arranged such that the DNA is transcribed in a host cell compatible with the vector and the polypeptide encoded thereby can be produced. I do.
  • it is a vector having an expression cassette in which a promoter region, an initiation codon, a DNA encoding any of the subunits, a stop codon, and a terminal region are continuously arranged.
  • the DNA encoding each subunit is arranged in tandem so that it can be transcribed and expressed polycistronically under the control of one promoter gene.
  • it is inserted downstream of the promoter gene.
  • the expression vector to be used includes a promoter region capable of functioning in various prokaryotic cells and various host cells such as Z or eukaryotic cells to express a gene arranged downstream thereof, and terminating the transcription of the gene.
  • a signal that is, 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.
  • the gene further contains a selection gene for transformant selection.
  • the expression vector may include a start codon and a stop codon, respectively, downstream of the promoter region and upstream of the terminator region.
  • the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell, in addition to the above promoter region and one terminator region.
  • the promoter region contains the promoter, operator and Shine-Dalgarno (SD) sequences.
  • SD Shine-Dalgarno
  • the promoter region is trp promoter, la. Promoter, rec A motor, I pp promoter, tac promoter, etc.
  • the host is Bacillus subtilis, SPO 1 promoter, SP 02 promoter, pen P promoter And the like.
  • the terminator region a commonly used natural or synthetic terminator can be used.
  • the selection marker genes include tetracycline, ampicillin, and kanamai.
  • a resistance gene to various drugs such as' can be used.
  • ATG is usually used as the initiation codon, but GTG can be used in some cases.
  • Conventional TGA, TAA and TAG are used as stop codons.
  • the expression vector of the present invention can be prepared by inserting the DNA into an appropriate site of one of known clone vectors capable of maintaining replication or autonomously replicating in a host cell to be transformed.
  • the host is a bacterium, Escherichia coli-derived pBR-based vectors, pUC-based vectors, etc., or B. subtilis-derived pUB110, pTP5, pC194, etc. Is exemplified.
  • the expression vector of the present invention is a vector in which a DNA encoding each of the large and small subunits of the LDH of the present invention is functionally arranged downstream of the promoter gene of the present invention, more preferably a cytochrome c-like.
  • the DNA encoding the polypeptide is the vector in which it is further functionally arranged.
  • each subunit DNA is a vector in which the small subunit DNA, the large subunit DNA, and the cytochrome c-like polypeptide DNA are arranged in this order from the promoter side.
  • the transformant of the present invention can be prepared by transforming a host cell with a recombinant vector containing DNA encoding each subunit of the SLDH of the present invention.
  • the host cell is not particularly limited as long as it is compatible with the recombinant vector to be used and can be transformed, and naturally-occurring cells or artificially produced mutant cells or cells commonly used in the art.
  • Various cells such as recombinant cells can be used.
  • Preferred are bacteria, especially Escherichia coli (for example, DH5a, HB101, etc.), Bacillus subtilis, and bacteria of the genus Darconobacter.
  • the introduction of the recombinant vector into a host cell can be carried out using a conventionally known method.
  • the host is a bacterium such as Escherichia coli or Bacillus subtilis
  • the method of Cohen et al. Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)]
  • the protoplast method [Mol. Gen. Genet., 168: 111 (1979)]
  • the combined method J. Mol. Biol., 56: 209 ( 1971)] and the like.
  • the transformant of the present invention contains an expression vector, preferably a cytochrome c-like polypeptide, functionally containing DNA encoding each of the large and small subnets encoding the SLDH of the present invention.
  • This is a host cell transformed with an expression vector that further contains the DNA to be transferred. More preferably, it is a host cell transformed with the expression vector, wherein the promoter controlling the expression of the DNA is the promoter gene of the present invention.
  • the host cell When the transformant is produced for the purpose of producing 2KLGA from D-sorbitol, the host cell needs to be capable of converting L-sorbose into 2KLGA.
  • the host cell is a cell having SDH and SNDH activity.
  • Naturally occurring cells include, for example, bacteria belonging to the genus Dalconobacter genus Pacetopactor, and more specifically, the genus Darconobacter oxidans T100 and the like.
  • Such artificially produced cells may be transformed with an expression vector functionally containing DNA encoding SDH and SNDH isolated from the above-mentioned naturally occurring bacteria and the like. Cells. Specifically, E.
  • the SLDH of the present invention is obtained by culturing any transformant containing an expression vector functionally containing the DNA encoding the above-mentioned SLDH in an appropriate medium, and collecting SLDH from the obtained culture. It can be manufactured by the following.
  • the nutrient medium used is a medium containing saccharides such as glucose and fructoses as carbon sources, glycerol, preferably L-sorbose and D-sorbitol.
  • Inorganic or organic nitrogen sources eg, ammonium sulfate, ammonium chloride
  • other nutrient sources eg, inorganic salts (eg, sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, Vitamin Bl), antibiotics (eg, ampicillin, kanamycin, etc.) may be added to the medium.
  • D- sorbitol Ichiru yeast extract, C a C0 3, a medium a composition of glycerin opening one le.
  • concentration of sugar (D-sorbitol) in the medium is usually 1 to 50%, preferably 2 to 30%, more preferably 5 to 25%.
  • Culture of the transformant is usually performed at pH 5.5 to 8.5, preferably pH 7 to 7.5, 1
  • the reaction is carried out at 8 to 40 ° C, preferably at 20 to 30 ° C for 5 to 50 hours.
  • Purification of S LDH can be performed by appropriately combining various commonly used separation techniques according to the fraction in which S LDH activity is present.
  • the method for producing L_sorbose of the present invention comprises the steps of: culturing any of the transformants containing the current vector functionally containing the above-described DNA encoding SLDH in an appropriate medium; Alternatively, when S LDH activity is present in the intracellular fraction of the transformant, L-sorbose is produced by contacting the cell extract with D-sorbitol.
  • the method of contacting the culture with D-sorbitol also includes a method of culturing the transformant in a medium containing D-sorbitol.
  • a host cell capable of converting L-sorbose into 2KLGA which has been transformed with an expression vector functionally containing the above-described DNA encoding SLDH, can be transformed into an appropriate medium.
  • 2KLGA is produced by contacting the cell extract with D-sorbitol.
  • the method of bringing D-sorbitol into contact with the culture also includes a method of culturing the transformant in a medium containing D-sorbitol.
  • the culture medium and culture conditions used in the method for producing L-sorbose and the method for producing 2KLG of the present invention may be the same as or partially different from those used in the method for producing SLDH described above. .
  • the culture after completion of the culture is centrifuged or filtered to collect the cells, and the cells are collected in an appropriate buffer, for example, an acetate buffer (approx. The supernatant obtained by suspending the cells in PH5), crushing the cells by sonication or the like, and then centrifuging the cells may be used as the cell extract.
  • an acetate buffer approximately 0.1% The supernatant obtained by suspending the cells in PH5
  • crushing the cells by sonication or the like crushing the cells by sonication or the like, and then centrifuging the cells may be used as the cell extract.
  • the L-sorbose or 2KLGA thus produced is purified from a reaction solution (a culture supernatant when the transformant is cultured in a medium containing D-sorbitol) by a purification method generally used (for example, it can be purified using dialysis, gel filtration, column chromatography on a suitable adsorbent, high performance liquid chromatography, etc.).
  • Example 1 Purification of sorbitol dehydrogenase from Darconobacter oxydans I FO 3254
  • Darconobacta oxydans IF03254 strain was obtained from the Fermentation Research Institute (2-17-85, Jusanhoncho, Yodogawa-ku, Osaka, Japan, 5-32-0024).
  • a single colony of Gluconobacter oxydans IF ⁇ 3254 strain was placed in a 500-ml 1-volume Erlenmeyer flask containing 2.5% G ⁇ / course, 1.0% polypeptone, 0.5% yeast extract (Diff Laboratories) , USA) and 2.0 was inoculated in a medium consisting of 0% C a C 0 3 (each 1 0 Om 1 X 5).
  • the culture was performed at 30 ° C. and 250 rpm (5.0 cm—through) for 18 hours. After completion of the culture, the culture solution 500 ml) was centrifuged at 4 ° C and 6000 rpm for 10 minutes. The obtained cells were washed once with cold saline and centrifuged again under the same conditions as above.
  • the cells obtained in (2) were suspended in 20 ml of 10 mM acetate buffer (pH 5.0) and disrupted by sonication. The supernatant was recovered by centrifugation at 10,000 rpm for 10 minutes at 4 ° C, and ultracentrifuged at 32,000 rpm for 60 minutes at 4 ° C to obtain a cell membrane fraction (precipitate). .
  • the membrane fraction is suspended in 10 mM acetate buffer (pH 5.0, 6 ml), Triton X-100 is added to a final concentration of 1%, and the mixture is ice-cooled for 1.5 hours. It was left still. The obtained suspension was ultracentrifuged at 4 ° C. and 32,000 rpm for 1 hour to recover a supernatant (about 5 ml), which was used as a solubilized SLDH fraction.
  • T S Kge 1 DE was obtained by equilibrating the solubilized S LDH fraction (2 ml) with 25 mM Tris-HC 1 (pH 8.0) containing 0.1 ° / oT r ito ⁇ X—100.
  • the sample was subjected to ion exchange chromatography using an AE-5 PW column (7.5 mm ID x 75 mm, Toso-I) and a linear graph was drawn from the equilibrium buffer to an equilibrium buffer containing 0.25 M potassium chloride. And eluted with a gradient (flow rate: 1 ml, gradient time: 45 minutes). The eluate was fractionated every 1.5 minutes. Two experiments were performed in exactly the same way and the corresponding fractions were combined. Enzyme activity was measured by the phelicyanide method [Shinagawa et al., Agric. Biol. Chem., 46: 135-141 (1982); using the increase in absorbance at a wavelength of 660 nm as an index].
  • the amount of enzyme that catalyzes the oxidation of 1- / mo 1 of D-sorbitol per minute was defined as 1 unit.
  • the LDH activity of each fraction was measured, and the fractions in which the activity was observed (fractions 10 and 11) were combined.
  • the obtained fraction was dialyzed against 1 OmM acetate buffer (pH 5.0), and TS Kgel CM-5 PW equilibrated with the dialysate (7.5 mm ID x 75 mm, Tohoku Soichi) Ion using The mixture was subjected to exchange chromatography, and eluted with a linear gradient from the equilibration buffer to the equilibration buffer containing 0.2 M potassium chloride (flow rate: lmZ, gradient time: 40 minutes).
  • the eluate was fractionated every 1.5 minutes, and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [12.5% SDS-PAGE; Neochemical Chemistry Laboratory Course (Tokyo Kagaku Dojin), 1: 330 -387 (1990)] and enzyme activity.
  • SDS-PAGE analysis the SLDH activity corresponded to the 62 kDa and 20 kDa proteins, and it was inferred that the protein was a constituent of the desired SLDH (these values were This is in good agreement with the values reported in Shinagawa et al., Agric. Biol. Chem., 46: 135-141 (1982)).
  • the 62 kDa and 20 kDa proteins are referred to as a large subunit and a small subunit, respectively.
  • TSKgel el DEAE Concentrate the active fraction (2m 1) from 5 PW column chromatography to approximately 180 / il using a small ultrafiltration device (Mo 1 ecut II LGC, Millipore), and 12.5% SDS_PAGE Attached. After the proteins separated on the gel were plotted on a polyvinylidene difluoride (PVDF) membrane, the membrane was stained with 1% dross acid solution containing 0.1% Ponceso-S and visualized at 62 kDa. And the membrane containing the 20 kDa protein were cut out and washed with distilled water.
  • PVDF polyvinylidene difluoride
  • the membrane piece was directly subjected to an automated protein sequencer model 47 OA (Applied Biosystems) to determine the amino acid sequence of the N-terminal of each subunit.
  • the membrane was immersed in 10 OmM aqueous acetic acid containing 0.5% polybierpyrrolidone for 30 minutes, washed with distilled water, and then cut into 1 mm squares.
  • the cells were suspended in 25 mM Tris-HC1 (pH 8.0, 200 ⁇ l) containing 8% acetonitrile. The suspension was sonicated for 15 minutes, 1 ⁇ g of lysyl peptidase (Wako Pure Chemical Industries, Ltd.) was added, and the mixture was incubated at 37 ° C.
  • oligonucleotides consisting of a base sequence corresponding to the partial amino acid sequence of the large subunit were synthesized by the phosphoramidite method using DNA synthesizer model 392 (Applied Biosystems) (Table 2).
  • the synthesized oligonucleotides were desorbed from CPG polymer carrier (CPG: controlle dporeglass) with 28% aqueous ammonia and heated at 60 ° C for 9 hours to remove all protecting groups.
  • the solvent was distilled off from the reaction mixture under reduced pressure under reduced pressure, and the residue was dissolved in 200/1 TE buffer [10 mM Tris-HCl (pH 7.4), 1 mM EDTA].
  • the resulting solution was washed once with ether and precipitated with ethanol.
  • the oligonucleotide obtained by the precipitation was directly used for PCR.
  • Oligonucleotide primer consisting of a nucleotide sequence encoding an amino acid sequence nucleotide sequence 1 ) (SEQ ID NO:
  • H A, C or T
  • D A, G or D
  • B G, C or T
  • N A, C, G or T
  • L4-F (R) means a forward (reverse) primer having a base sequence corresponding to the amino acid sequence of L4 in Table 1 (otherwise similar)
  • PCR reaction was performed in which genomic DNA derived from the Darconobacter oxydans IF03254 strain was type III.
  • Reaction mixture [PCR buffer, genomic DNA: 180 ng, primer: 2.5 pmo1, dNTP: 200 each, and Taq DNA polymerase (2.5 units: Pa-Kin-Elma-Citus)]
  • the microtube containing the microtube was set in a high-temperature thermal reactor (Model HB-TR1; HighVide, UK).
  • One cycle 95 ° C, 0.5 minute (denaturation), 55 ° C, 1 minute
  • the PCR reaction consisting of (ringing) and 72 ° C for 2 minutes (extension) was performed for 30 cycles.
  • the PCR reaction using LN-F and L4-R primers was subjected to 2.0% agarose gel electrophoresis, and a specific amplified fragment band of about 500 bp was detected.
  • the gel portion containing the amplified fragment was cut out, and the DNA fragment was purified using a purification column.
  • the obtained DNA and pGEM-T (Promega) were ligated using T4 DNA ligase (Takara Shuzo), and Maniatis et al. [Molecular Cloning, 2nd ed., Vol. 1, Cold Spring Harbor Laboratory Press, 1989, USA] The ligation was used to transform coliDH1OB according to the method described in (1).
  • the desired plasmid pSLD-1 was obtained from one of the transformants, its properties were examined by restriction enzyme mapping, and then the nucleotide sequence of the inserted DNA was determined.
  • the DNA was composed of 531 bp (corresponding to base numbers 24 to 554 in SEQ ID NO: 2 in the Sequence Listing), and the sequences of the primers used in PCR were found at the 3 and 5 ′ ends.
  • the amino acid sequence encoded in the internal sequence leading to the primer was identical to the partial amino acid sequence of the large subunit of the purified SLDH.
  • pSLD-1 was digested with SphI and PstI (Nitsubion), and the insert DNA was separated and recovered by 0.5% agarose gel electrophoresis.
  • Use the DIG labeling kit (Boehringer Mannheim) to attach the obtained DNA fragment to the attached DIG labeling was performed according to the mouth protocol.
  • the dialysate was extracted with 20 ml of phenol and dialyzed twice with 2 L of TE buffer to obtain a desired chromosomal DNA solution (20 ml, 56 xg / ml).
  • the chromosomal DNA was partially digested with Sau3AI, and the obtained fragments were separated on a sucrose density gradient to obtain a fragment centered at 8 to 20 kbp in size.
  • —3 Promega was cloned into the BamHI site.
  • the size of the library one by Taitachietsu click to indicator strain E. coli LE 3 9 2 was. 5 X 1 0 5 clones.
  • Phage DNA was extracted from the positive clone (# 1), digested with BamHI and Sa1I (two bonbons), and subjected to 0.8% agarose gel electrophoresis. The DNA fragment separated on the gel was transferred onto a Nitrocell orifice filter by electroblotting, and a Southern blot was performed using the above-mentioned DG-labeled probe. As a result, a DNA fragment of about 1 kbp hybridized with the DIG-labeled probe.
  • DNA sequence analysis of type I DNA was carried out using the 370A DNA Sequencer (Applied Biosystems) according to the attached protocol by the didoxita minification method. .
  • Initial sequencing was performed using the Ml3 sequencing primer, Universal Primer and Reverse Primer (New EnglndBio1abs, USA). Further analysis was performed using the synthetic oligonucleotides listed in Table 3.
  • Lambda phage # 1 insert DNA primer for sequencing-Primer Single nucleotide sequence (Sequence number in Sequence Listing)
  • ORF 1 As a result of determining the nucleotide sequence of about 3780 bp, two ORFs were found.
  • the first ORF (ORF 1; base number 681 to 1274 in SEQ ID NO: 7) consists of 594 bp, and corresponds to the N-terminal and internal partial amino acid sequences of the small submit of SLDH. Contained an array.
  • ORF 1 is a small subunit precursor containing a 41 amino acid leader sequence. (197 amino acids; SEQ ID NO: 3).
  • the second ORF (ORF 2; nucleotide numbers 1293 to 2930 in SEQ ID NO: 7 in the Sequence Listing) consists of 1638 bp and encodes a large subunit [545 amino acids (including the initiation methionine); SEQ ID NO: 1 in Sequence Listing].
  • ORF2 nucleotide sequence presumed to encode a part of the cytochrome c-like polypeptide did not include the full-length coding region in which it was found.
  • Lambda phage # 1 Construction of plasmid for DNA sequence Therefore, lambda DNA was extracted from another positive clone (# 7) in the same manner, and this DNA was digested with Sa1I. The 0 kbp and 7.6 kbp S a I I / Sail fragments were obtained. The former was subcloned into pHSG298 and the latter into pBIueScriptIIKS (+), respectively, to obtain pSD37R and pBL7Sa17 (FIG. 2).
  • DNA sequence analysis of type I DNA was carried out using the 37 OA DNA Sequencer (Applied Biosystems) according to the attached protocol according to the attached protocol.
  • Initial sequencing was performed using the Ml 3 -Queen Sinda primers, Universal Primer and Reverse Primer (New Englnd Biolabs, USA). Further analysis was performed using the synthetic oligonucleotides listed in Table 4.
  • the third ORF (ORF 3; base numbers 2923 to 4359 in SEQ ID NO: 7) consists of 1437 bp and has a cytochrome c-like polypeptide [478 amino acids; SEQ ID NO: 5 in Sequence Listing. ] was coded.
  • a homologous search was performed on the base sequence of ORF 3 and the amino acid sequence encoded by the same on a database (GenBank, SWISS-PROT). 51.6% of the cytochrome c-553 gene derived from 8 strains, and 36.2% homology with the cytochrome c subunit precursor of alcohol dehydrogenase derived from acetobacter.polyoxogenes in amino acid sequence .
  • cytochrome c-like polypeptide conserved the three-site heme binding site consensus sequence Cys Xaa Xaa CysHis (SEQ ID NO: 11) reported in acetopactor 'polyoxogenes (SEQ ID NO: 11). Amino acid numbers 50 to 54, 197 to 2 ° 1 and 340 to 344 in SEQ ID NO.
  • the insert DNA of lambda phage # 7 is composed of a region encoding the small subunit precursor of SLDH, a large subunit, and a cytochrome c-like polypeptide, respectively, and a region of the promoter that expresses them polycistronically. And a complete gene group expression unit consisting of a terminator region. Table 5 summarizes the composition of the SL DH gene group.
  • PSD37R prepared in Example 5 was digested with Sa1I and HindIII, and a 2.4 kb fragment was isolated. Also, pBL7Sa17 was digested with SaII and BamHI, and a 2.4 kb fragment was isolated. Both fragments were ligated with a 2.8 kb fragment of pUC18 previously digested with HindIII and BamHI to obtain an expression vector PSLDH3 containing the entire gene expression unit of SLDH (see above construction). A schematic diagram is shown in Fig. 3). Escherichia coli DH10B is transformed with the expression vector according to a conventional method. did.
  • a single colony of the transformant (, co1DH10B / pSLDH3) obtained in (1) was added to 100 ml of L-Amp medium [1 ⁇ 1 ⁇ 2Bactotripton (Diff Laboratories), 0.5% yeast extract, 0.5% sodium chloride and 50 ⁇ g / m 1 ampicillin (pH 7.2)] and cultured at 37 ° C for 18 hours. 5 ml was taken from the obtained culture, transferred to 500 ml of L-Amp medium, and cultured at 37 ° C for 7 hours. The culture was centrifuged at 6,000 rpm for 10 minutes to collect the cells.
  • L-Amp medium 1 ⁇ 1 ⁇ 2Bactotripton (Diff Laboratories), 0.5% yeast extract, 0.5% sodium chloride and 50 ⁇ g / m 1 ampicillin (pH 7.2)
  • the obtained cells were washed with 0.85% saline, suspended in 2 Oml of 10 mM acetate buffer (pH 5.0), and each 1 minute at 30-second intervals for a total of more than 3 times. The cells were disrupted by sonication. The obtained cell lysate was centrifuged at 10, OO O pm for 10 minutes to recover the supernatant.
  • Plasmid fraction Enzyme activity (UZml) Specific activity (AtUZmg) pUC18 cell lysate 43 51
  • Oligonucleotides designed to serve as forma primers for amplifying the partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol dehydrogenase.
  • Oligonucleotides designed to act as reverse primers to amplify the partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol dehydrogenase.
  • D-oligonucleotide designed to act as a forward primer to amplify a partial nucleotide sequence of DNA encoding the large subunit of sorbitol dehydrogenase.
  • Oligonucleotides designed to act as reverse primers to amplify the partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol dehydrogenase.
  • Oligonucleotides designed to serve as forma primers for amplifying the partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol dehydrogenase.
  • Oligonucleotides designed to act as a fourth primer for amplifying the partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol-dehydrogenase.
  • Oligonucleotides designed to act as reverse primers to amplify nucleotide sequences.
  • Oligonucleotides designed to act as a formal primer for amplifying a partial nucleotide sequence of DNA that encodes a large subunit of D-sorbitol dehydrogenase.
  • Oligonucleotides designed to act as reverse primers to amplify a partial nucleotide sequence of DNA encoding the large subunit of D-sorbitol dehydrogenase.
  • Oligonucleotides designed to serve as a for-primed primer for amplifying the partial nucleotide sequence of DNA encoding the large subunit of D-so / levitol dehydrogenase.
  • Oligonucleotides designed to act as primers for lambda phage # 1 insert DNA sequencing.
  • SEQ ID NO: 3 6 Oligonucleotides designed to serve as primers for sequencing the lambda phage # 1 insert DNA.
  • Oligonucleotides designed to act as primers for lambda phage # 1 insert DNA sequencing.
  • Oligonucleotide designed to serve as a primer for sequencing of lambda phage # 1 insert DNA.
  • Oligonucleotides designed to act as primers for lambda phage # 1 insert DNA sequencing.
  • Oligonucleotides designed to act as primers for lambda phage # 1 insert DNA sequencing.
  • Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 1 insert DNA Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 1 insert DNA.
  • Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 1 insert DNA Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 1 insert DNA.
  • Oligonucleotide designed to serve as a primer for sequencing the insert DNA of lambda phage # 1. SEQ ID NO: 4 5
  • Oligonucleotides designed to act as primers for lambda phage # 1 insert DNA sequencing.
  • Oligonucleotides designed to act as primers for the lambda phage # 1 insert DNA sequence.
  • Oligonucleotides designed to act as primers for the sequencing of lambda phage # 7 insert DNA Oligonucleotides designed to act as primers for the sequencing of lambda phage # 7 insert DNA.
  • Oligonucleotides designed to act as primers for the lambda phage # 7 insert DNA sequence.
  • Oligonucleotide designed to serve as a primer for sequencing insert lambda phage # 7 DNA.
  • Oligonucleotide designed to serve as a primer for sequencing DNA lambda phage # 7 insert DNA.
  • Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 7 insert DNA Oligonucleotides designed to serve as primers for the sequencing of lambda phage # 7 insert DNA.
  • Oligonucleotide designed to serve as a primer for sequencing DNA lambda phage # 7 insert DNA.
  • Oligonucleotides designed to act as primers for lambda phage # 7 insert DNA sequencing.
  • SEQ ID NO: 6 2 Oligonucleotide designed to act as a primer for sequencing insert DNA of lambda phage # 7.
  • Oligonucleotides designed to serve as primers for lambda phage # 7 insert DNA sequencing.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne trois sous-unités constituant la D-sorbitol déshydrogénase, des gènes codant celles-ci, un procédé de production de cette D-sorbitol déshydrogénase, consistant à cultiver des cellules transformées par des vecteurs d'expression contenant des gènes promoteurs appropriés à l'expression des gènes ci-dessus; elle concerne également les gènes ci-dessus mentionnés, ainsi qu'un procédé de production de L-sorbose ou d'acide 2-céto-L-gulonique, dans lequel on utilise la culture ci-dessus. Ce procédé permet de produire facilement en grande quantité de l'acide 2-céto-L-gulonique en tant que précurseur de l'acide L-ascorbique.
PCT/JP1998/004612 1997-10-17 1998-10-13 D-sorbitol deshydrogenase, genes et utilisation de celle-ci WO1999020763A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP28528097 1997-10-17
JP9/285280 1997-10-17

Publications (1)

Publication Number Publication Date
WO1999020763A1 true WO1999020763A1 (fr) 1999-04-29

Family

ID=17689476

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/004612 WO1999020763A1 (fr) 1997-10-17 1998-10-13 D-sorbitol deshydrogenase, genes et utilisation de celle-ci

Country Status (1)

Country Link
WO (1) WO1999020763A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000055329A1 (fr) * 1999-03-17 2000-09-21 Fujisawa Pharmaceutical Co., Ltd. Sorbitol deshydrogenase, gene codant pour celle-ci et leur utilisation
WO2001048214A2 (fr) * 1999-12-24 2001-07-05 Südzucker Aktiengesellschaft Mannheim/Ochsenfurt Procede de preparation de 6,0-$g(a)-d-glucopyranosyle-d-sorbite
WO2014171635A1 (fr) * 2013-04-17 2014-10-23 건국대학교 산학협력단 D-sorbitol déshydrogénase inédite et procédé de production de l-sorbose au moyen de celle-ci
EP2906695A1 (fr) * 2012-10-10 2015-08-19 Biocon Limited Séquence nucléotidique et procédé associé

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0549480A (ja) * 1990-06-14 1993-03-02 Asahi Chem Ind Co Ltd Ｃ型チトクロム遺伝子及び酸化発酵法
JPH06189790A (ja) * 1991-06-10 1994-07-12 Toyobo Co Ltd ソルビトールの測定法およびそのための試薬

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0549480A (ja) * 1990-06-14 1993-03-02 Asahi Chem Ind Co Ltd Ｃ型チトクロム遺伝子及び酸化発酵法
JPH06189790A (ja) * 1991-06-10 1994-07-12 Toyobo Co Ltd ソルビトールの測定法およびそのための試薬

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHOI E.-S., LEE E.-H., RHEE S.-K.: "PURIFICATION OF A MEMBRANE-BOUND SORBITOL DEHYDROGENASE FROM GLUCONOBACTER SUBOXYDANS.", FEMS MICROBIOLOGY LETTERS, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 125., no. 01., 1 January 1995 (1995-01-01), GB, pages 45 - 49., XP002914609, ISSN: 0378-1097, DOI: 10.1016/0378-1097(94)00470-C *
KONDO K., HORINOUCHI S.: "CHARACTERIZATION OF THE GENES ENCODING THE THREE-COMPONENT MEMBRANE-BOUND ALCOHOL DEHYDROGENASE FROM GLUCONOBACTER SUBOXYDANS AND THEIR EXPRESSION IN ACETOBACTER PASTEURIANUS.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 63., no. 03., 1 March 1994 (1994-03-01), US, pages 1131 - 1138., XP002914612, ISSN: 0099-2240 *
SCHAUDER S., SCHNEIDER K.-H., GIFFHORN F.: "POLYOL METABOLISM OF RHODOBACTER SPHAEROIDES: BIOCHEMICAL CHARACTERIZATION OF A SHORT-CHAIN SORBITOL DEHYDROGENASE.", MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, GB, vol. 141., 1 January 1995 (1995-01-01), GB, pages 1857 - 1863., XP002914613, ISSN: 1350-0872 *
SHINAGAWA E, ET AL.: "PURIFICATION AND CHARACTERIZATION OF D-SORBITOL DEHYDROGENASE FROM MEMBRANE OF GLUCONOBACTER SUBOXYDANS VAR. ALPHA", AGRICULTURAL AND BIOLOGICAL CHEMISTRY, AGRICULTURAL CHEMICAL SOCIETY OF JAPAN, JP, vol. 46, no. 01, 1 January 1982 (1982-01-01), JP, pages 135 - 141, XP002914610, ISSN: 0002-1369 *
STEIN M. A., SCHAEFER A., GIFFHORN F.: "CLONING, NUCLEOTIDE SEQUENCE, AND OVEREXPRESSION OF SMOS, A COMPONENT OF A NOVEL OPERON ENCODING AN ABC TRANSPORTER AND POLYOL DEHYDROGENASES OF RHODOBACTER SPHAEROIDES SI4.", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 179., no. 20., 13 October 1997 (1997-10-13), US, pages 6335 - 6340., XP002914611, ISSN: 0021-9193 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000055329A1 (fr) * 1999-03-17 2000-09-21 Fujisawa Pharmaceutical Co., Ltd. Sorbitol deshydrogenase, gene codant pour celle-ci et leur utilisation
US6825018B1 (en) 1999-03-17 2004-11-30 Fujisawa Pharmaceutical Co. Ltd. Sorbitol dehydrogenase, gene encoding the same and use thereof
WO2001048214A2 (fr) * 1999-12-24 2001-07-05 Südzucker Aktiengesellschaft Mannheim/Ochsenfurt Procede de preparation de 6,0-$g(a)-d-glucopyranosyle-d-sorbite
DE19963126A1 (de) * 1999-12-24 2001-07-12 Suedzucker Ag Verfahren zur Herstellung von 6-O-alpha-D-Glucopyranosyl-D-sorbit
WO2001048214A3 (fr) * 1999-12-24 2002-04-25 Markwart Kunz Procede de preparation de 6,0-$g(a)-d-glucopyranosyle-d-sorbite
US7208301B2 (en) 1999-12-24 2007-04-24 Südzucker Aktiengesellschaft Mannheim/Ochsenfurt Method for producing 6-0-α-D-glucopyranosyl-D-sorbitol
EP2906695A1 (fr) * 2012-10-10 2015-08-19 Biocon Limited Séquence nucléotidique et procédé associé
EP2906695A4 (fr) * 2012-10-10 2016-05-18 Biocon Ltd Séquence nucléotidique et procédé associé
WO2014171635A1 (fr) * 2013-04-17 2014-10-23 건국대학교 산학협력단 D-sorbitol déshydrogénase inédite et procédé de production de l-sorbose au moyen de celle-ci

Similar Documents

Publication Publication Date Title
JP4012257B2 (ja) 新規カルボニル還元酵素、およびこれをコードする遺伝子、ならびにこれらの利用方法
JP4173571B2 (ja) 新規アルコールアルデヒド脱水素酵素
JP4528624B2 (ja) アルデヒドデヒドロゲナーゼ遺伝子
JP5224572B2 (ja) デキストラン生成酵素遺伝子、デキストラン生成酵素およびその製造方法、デキストランの製造方法
JP4394761B2 (ja) Ｄ−ソルビトール・デヒドロゲナーゼ遺伝子
JP3880624B2 (ja) （２．５―ｄｋｇ）リダクターゼの各種改良型変異体
US6825018B1 (en) Sorbitol dehydrogenase, gene encoding the same and use thereof
WO2003091430A1 (fr) SOUS-UNITE ss DE LA GLUCOSE DESHYDROGENASE ET ADN LA CODANT
WO1999020763A1 (fr) D-sorbitol deshydrogenase, genes et utilisation de celle-ci
JP4880859B2 (ja) 新規カルボニル還元酵素、その遺伝子、およびその利用法
WO2005123921A1 (fr) Nouvelle glycerol déhydrogènase, gène relatif et procédé de leur utilisation
JP4102138B2 (ja) グルコース脱水素酵素の製造方法
JP2003159079A (ja) アルコール／アルデヒドデヒドロゲナーゼ、その製造方法およびその用途
KR100227960B1 (ko) 글루코노박터 서브옥시단스의 솔비톨 탈수소효소 및 이의 유전자
JP2000032988A (ja) 新規遺伝子及びその遺伝子を保有する形質転換細胞
JP4318473B2 (ja) グルコース脱水素酵素の製造法
JPH06303981A (ja) ホルムアルデヒド脱水素酵素活性を有する蛋白質の遺伝情報を有するｄｎａ並びにホルムアルデヒド脱水素酵素の製造法
WO2000008170A1 (fr) Gene participant a la production d'acide homoglutamique, et utilisation associee
JPH11318438A (ja) 組換え微生物３―ヒドロキシ酪酸デヒドロゲナ―ゼ、その製造方法およびその使用
JP3330670B2 (ja) アルケンモノオキシゲナーゼ、これをコードする遺伝子及び形質転換微生物並びにアルケンのエポキシ化方法
JP4221075B2 (ja) シトクロムｃおよびその遺伝子
JP2004041062A (ja) 補酵素依存型を改変したギ酸脱水素酵素
JP4066203B2 (ja) 新規なクレアチニンアミドヒドロラーゼ
JP2001161375A (ja) コレステロール・エステラーゼ遺伝子、組み換え体ｄｎａ及びコレステロール・エステラーゼの製造法
JP2000125856A (ja) 新規なＬ−α−グリセロホスフェートオキシダーゼおよびその製造方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase