WO2001079503A1 - L-glutamate oxydase - Google Patents
L-glutamate oxydase Download PDFInfo
- Publication number
- WO2001079503A1 WO2001079503A1 PCT/JP2001/003345 JP0103345W WO0179503A1 WO 2001079503 A1 WO2001079503 A1 WO 2001079503A1 JP 0103345 W JP0103345 W JP 0103345W WO 0179503 A1 WO0179503 A1 WO 0179503A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glutamic acid
- acid oxidase
- molecular weight
- dna
- oxidase
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03011—L-Glutamate oxidase (1.4.3.11)
-
- 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.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
Definitions
- the present invention relates to a novel L-glutamic acid oxidase, a gene encoding the enzyme, and a method for producing the enzyme.
- L-glutamic acid oxidase is an enzyme that catalyzes the following reactions.
- Streptomyces produced using microorganisms belonging to the genus, (2) extremely high substrate specificity for L-glutamic acid, (3) relatively stable to temperature and pH fluctuations, (4) requiring FAD as a coenzyme Flavin enzyme.
- L-Glutamic acid is a major umami ingredient in foods, and is used in processed foods at about 1 million tons per year.
- L-glutamic acid in foods is L_dalminic acid
- L-glutamic acid oxidase is indispensable in the field of food component analysis because it can be easily measured using a kit or sensor using oxidase.
- L-dalminic acid oxidase is not always simple.
- L-glutamic acid oxidase derived from Streptomyces sp. X-119-6 is difficult to produce by a liquid culture method.
- the enzyme is produced by a solid culture method.
- L_glutamic acid oxidase can be easily prepared by recombinant DNA techniques, or a new property not provided by the native enzyme can be added. It is also possible to do so. However, the analysis of the gene encoding L-glutamic acid oxidase, and even the amino acid sequence of the enzyme have not been reported yet.
- the present inventors determined the N-terminal amino acid sequence of the L-glutamic acid oxidase subunit derived from Streptomyces sp. X-1 19-6, prepared a primer based on this sequence, and screened genomic DNA. As a result, a gene encoding a protein with an estimated molecular weight of about 76,000 was found, and the gene was introduced into Escherichia coli, and the enzyme obtained by culturing was purified.
- the present invention relates to a novel L-glutamic acid oxidase having the following physicochemical properties.
- (C) Molecular weight and subunit structure The molecular weight measured by SDS-polyacrylamide electrophoresis is 70,000 ⁇ 6,000, and the molecular weight measured by gel filtration is 140,000 ⁇ 10,000. It is a dimer of the same substance with a molecular weight of 70,000 ⁇ 6,000.
- the coenzyme is flavin adenine dinucleotide (FAD).
- FAD flavin adenine dinucleotide
- the present invention also relates to L-glutamic oxidase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added.
- the present invention also relates to an L-daltamic acid oxidase gene encoding the amino acid sequence.
- the present invention relates to an L-glutamic acid oxidase gene having a base sequence represented by SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted, inserted or added.
- the present invention provides an expression vector prepared by introducing any of the above DNA fragments into a plasmid, and a host microorganism using the expression vector and culturing the obtained transformant.
- the present invention relates to a method for producing L-dalminic acid oxidase, which comprises isolating and purifying L-dalminic acid oxidase.
- FIG. 1 shows the results of analyzing the base sequence and amino acid sequence of L-glutamic acid oxidase.
- the double line indicates the signal peptide
- the box indicates the FAD binding site.
- FIG. 2 shows the structure of plasmid pGS1.
- FIG. 3 shows the structure of plasmid pGOX-ma11.
- L-GOX in the figure indicates the L-glutamic acid oxidase gene.
- FIG. 4 shows the structure of plasmid pGAI1.
- GAO in the figure indicates the L-glutamic acid oxidase gene.
- FIG. 5 shows the optimum temperatures of L-glutamic acid oxidase of the present invention and L-glutamic acid oxidase derived from Streptomyces sp. Strain X-119-6.
- A is E. coli; L-glutamic acid oxidase (MBP-LG0X fusion protein from E. coli JM109 / pGOX mall) fused with maltose binding protein (MBP) from iM109 / pG0X mall;
- B is factor Xa. ! ⁇ L-glutamic acid oxidase (after LGOX (factor Xa treatment) derived from E.
- FIG. 6 shows the optimum pH of L-glutamic acid oxidase of the present invention and L-glutamic acid oxidase derived from Streptomyces sp. Strain X-119-6.
- A Escherichia coli ⁇ MBP-LGOX fusion protein derived from M109 / pGOX mall
- B LGOX (after factor Xa treatment) derived from E. coli 109 / pG0X mall
- C E. coli
- FIG. 7 shows the thermal stability of L-glutamic acid oxidase of the present invention and L-glutamic acid oxidase derived from Streptomyces sp. Strain X-119-6.
- diamonds indicate LGOX from Streptomyces sp.X-119-6 strain
- triangles indicate LGOX from E.
- novel L-glutamic acid oxidase of the present invention has the following physicochemical properties.
- the coenzyme is flavin adenine dinucleotide (FAD).
- the L-dalminic acid oxidase of the present invention has an amino acid sequence represented by SEQ ID NO: 1.
- the amino acid sequence is not limited to the amino acid sequence represented by SEQ ID NO: 1, as long as the L-glutamic acid oxidase activity is maintained.
- One or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 may be deleted, substituted, modified or added. Specific examples of such an amino acid sequence include those obtained by converting N-terminal alanine to methionine, those obtained by further adding methionine to the N-terminal side of alanine, and the like.
- the L-glutamic acid oxidase of the present invention also includes an enzyme having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 as long as L-glutamic acid oxidase activity is maintained.
- Such an enzyme of the present invention can be obtained by obtaining a gene encoding the L_glutamic acid oxidase from Streptomyces sp. X-119-6, using this to produce a recombinant vector, The obtained transformant is obtained by transforming a host cell using a vector, and culturing the transformant and collecting L-glutamic acid oxidase from the culture.
- the host is not particularly limited, but Escherichia coli is preferred.
- the gene encoding L-glutamic acid oxidase of the present invention may be any one that encodes SEQ ID NO: 1 or a mutant in which an amino acid is deleted, substituted, inserted or added in the amino acid sequence thereof. However, those having a base sequence represented by SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted, inserted or added are preferable.
- the gene of the present invention also includes a gene having 90% or more homology with the base sequence shown in SEQ ID NO: 2.
- L-glutamic acid oxidase of the present invention is represented by nucleotide numbers 235 to 2295 in FIG. 1, or a DNA fragment that hybridizes with a DNA fragment thereof under stringent conditions can also be used.
- the hybridization under the stringent condition is 5 XSS C (1 XS
- SC is 8.76 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water), 0.1% wZv N-lauroyl sarcosine 'sodium salt, 0.02% w / v
- a hybridization reaction was carried out at 60 ° C for about 20 hours using a solution containing SDS and 0.5% wZv blocking reagent, Means to hybridize.
- a DNA fragment further comprising a sequence encoding a sidanal peptide and a Z or SD (Shine-Dalgarno) sequence upstream of the gene encoding L-glutamic acid oxidase can also be used.
- a DNA fragment By using such a DNA fragment, it is possible to increase the production amount of the enzyme or to simplify the purification of the enzyme depending on the host used. If such a DNA fragment is specifically described based on FIG. 1, a DNA fragment having a sequence represented by at least nucleotides 183 to 2295 in FIG. 1 can be exemplified. It contains an SD sequence and a signal peptide consisting of 14 amino acid residues.
- chromosomal DNA is extracted from actinomycetes belonging to the genus Streptomyces by a known method, and the obtained DNA is incorporated into a plasmid or phage vector, preferably a plasmid, for example, E. coli or actinomycete.
- a plasmid for example, E. coli or actinomycete.
- Prepare library one. Any plasmid can be used as a plasmid incorporating the DNA, as long as it can be replicated and maintained in a host. Examples of the plasmid include plasmids of Escherichia coli and actinomycetes. Specifically, plasmids for E.
- coli include pBR322 [Gene, 2, 95 (1977)], pBR325 (Gene, 4, 121 (1978)), pUC13 [Gene, 19, 259 (1982)] and the like.
- actinomycete plasmids include pi J 61 CGene, 20, 51 (1982)), pi J 702 [J. Gen. Microbiol., 129, 2703 (1983)] and the like.
- the thus obtained plasmid vector is introduced into a host.
- the host include Escherichia coli and actinomycetes.
- Escherichia coli When Escherichia coli is used as a host, the productivity of the novel L-glutamic acid oxidase of the present invention is also good and preferred.
- Escherichia coli include Escherichia coli K12DH1 [Pro., Natl. Acad. Sci., USA 60, 160 (1968)] and JM103 [Nucl. Acids. Res., 9, 309 (1981)].
- actinomycetes include Streptomyces' lividans (Streptomyces 1 ividans) TK64 and its derivative [Genetics Manipulation of Streptomyces, A Laboratory Manual].
- a method for transforming a host with a plasmid may be a method known per se.
- Escherichia coli the calcium chloride method described in Molecular Cloning [Cold Spring Harbor Laboratory, 239 (1982)] or the like, or the calcium chloride / rubidium chloride method can be used.
- actinomycetes the method is performed by a protoplast method described in Genetics Manipulation of Streptomyces, A Laboratory Manual.
- the L-glutamate oxidase gene is cloned from the E. coli or actinomycete DNA library obtained as described above.
- a cloning method a method known per se, for example, a method using 4-aminoantipyrine, phenol, and peroxidase, a method using a functional expression to develop color in the presence of hydrogen peroxide, and a chemical synthesis based on an amino acid sequence
- a colony hybridization method using an oligonucleotide as a probe [Molecular Cloning, Cold Spring Harbor Laboratory, (1982)].
- the gene encoding glutamate oxidase cloned in this way may optionally be a plasmid, such as pBR322, pUC12, pUC13, pUC18, pUC19, pUC118, pUC119, pIJ702. , P IJ It may be subcloned into 61, pi J101, pIJ486 or pIJ425.
- the nucleotide sequence of the obtained DNA can be determined by a method known per se, for example, the Maxam-Gilbert method [Pro. Natl. Acad. Sci., USA 74,560 (1977)], the dideoxy method [Nucl. Acids. Res., 9, 309 ( 1981)] or the dither method [Nucl. Acids. Res., 14, 1319 (1986)].
- the amino acid sequence deduced from the nucleotide sequence of the obtained DNA is compared with the amino acid sequence at the N-terminal of the known L-glutamic acid oxidase which has been analyzed in advance, and the L-glutamic acid oxidase is compared. Confirm the presence of a DNA fragment encoding As a result, when the entire region of the L-glutamic acid oxidase gene is not covered, the DNA is re-cloned by a colony hybridization method, a PCR method, etc. using the previously cloned DNA fragment as a probe. The DNA encoding the entire region of the L-glutamic acid oxidase gene is obtained.
- L-glutamic acid oxidase is performed by preparing an expression vector by inserting the cloned DNA fragment into a known plasmid having a promoter or the like, and transforming it using a microorganism (such as actinomycetes or Escherichia coli). ) Is cultured according to a method known per se to produce L-glutamic acid oxidase in a culture solution or cells. Furthermore, it can be produced in the form of a fusion protein with maltose binding protein (MBP) by a conventional method. The transformant may be cultured according to a method known per se. For example, glucose, glycerin, dextrin, sucrose, starch, molasses, etc.
- MBP maltose binding protein
- Culture temperature is usually about 10-5 0 ° C, preferably about 20-40 ° C.
- the culturing time is about 1 to 96 hours, preferably about 10 to 72 hours. Culture may be performed by adding aeration or stirring as necessary.
- the expression of the gene is induced by a method commonly used in the promoter used.
- a method commonly used in the promoter used For example, when a lac promoter, a tac promoter, a Taq promoter, or the like is used, an appropriate amount of an expression inducer, isopropyl- ⁇ -D-thiogalactopyranoside (hereinafter abbreviated as IPTG), is added in the middle stage of the culture.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- the cells are recovered from the culture thus prepared by membrane separation or centrifugation.
- the recovered cells are suspended in an appropriate buffer and physically disrupted by ultrasonic treatment or French press treatment, or enzymatically lysed by lysozyme treatment, etc. Is removed by centrifugation to prepare a cell-free extract. If necessary, heat treatment, ammonium sulfate precipitation, dialysis, solvent treatment with ethanol, etc., and various chromatographic treatments can be used to combine L-daltamine. Acid oxidase can be isolated and purified.
- L-glutamate oxidase from Streptomyces sp. X-119--6 (ATC C 39343) is composed of three subunits, a and A, which were analyzed in advance. Designed from the N-terminal amino acid sequence (CK subunit: Ala-Asn-Glu-Met-Thr-Tyr-Glu ' ⁇ , Asubunit: Ala-Ile-Vat Thr_Ile-Pro-Phe' '') The following probes cu and a were used.
- Probe 5 '-GC (CorG) ATCGT (CorG) AC (Cor.G) ATCCC (CorG) TT-3'
- Chromosomal DNA derived from Streptomyces sp. X-119-6 was prepared by a conventional method, cut with BamHI, and subjected to agarose gel electrophoresis. DNA was recovered. Using this DNA as a vector, pUC19 (Takara Shuzo) and Escherichia coli MV1184 (Takara Shuzo) as a host were used to create a DNA library, and the recombinant was used for colony hybridization using a probe. Provided. Hybridization was performed at 55 ° C, and washing was performed sequentially at 58 ° C, 62 ° C, and 64 ° C for 10 minutes each. The resulting membrane was subjected to autoradiography, and about 5000 strains were screened. As a result, 12 positive colonies were obtained.
- Plasmids were extracted from the obtained 12 strains of positive colonies, the plasmid was excised with BamHI to cut out the inserted DNA portion, and subjected to Southern hybridization.As a result, 5 strains of plasmids having strong signals were obtained. Was. All of the above insert DNAs were confirmed to have a size of 2.3 kb, and a restriction enzyme map of the insert DNA was prepared. HI (0 kb), Sma I (0.15 kb), Sty I (1.05 kb), Sph I (1.5 kb), Kpnl (1.76 kb), Sail (2. 2 kb)> BamHI (2.3 kb).
- restriction enzymes other than Sa1I and SmaI on the restriction map were used for the obtained plasmid. And subjected to Southern hybridization.
- the ⁇ -subunit of L-glutamic acid oxidase derived from Streptomyces sp. X—119—6 has a molecular weight of about 40,000 and a base sequence of about 1 kb. This has been clarified by prior analysis. However, it was revealed that the above 2.3 kb DNA contained only 582 b ⁇ , ie, about 60% of the a subunit, counted from the valine residue of GTG, which is considered to be the translation initiation site.
- the chromosomal DNA was cut with SacI, and then subjected to agarose gel electrophoresis to recover DNA.
- SacI 6 kb DNA digested with SacI is used as a vector ⁇ ⁇
- a library was prepared by ligating to AP E press phage DNA (Toyobo) and packaging. Using this, E. Co 1 i XL 1 blue MR Fl (Toyobo) was infected to form plaques. Blotting was performed directly from this plaque, and a plaque that had a stronger signal due to plaque hybridization using a KpnI_BamHI fragment of about 0.5 kb in a 2.3 kb BamHI fragment as a probe Were obtained.
- the vector portion was converted into pBK-CMV by the Single-clone Excition method (Nucleic Acids Res., 15, 7583-7600 (1988)), and plasmid extraction was further performed.
- the resulting plasmid was designated as pGS1 (FIG. 2), and the inserted fragment was confirmed by restriction enzyme digestion and agarose gel electrophoresis, and subjected to Southern hybridization analysis using a probe and ⁇ .
- SacI (0 kb), BamHI (0.1 kb), and SmaI (0.3 kb) were sequentially arranged from the upstream of the insert.
- the ⁇ -probe also binds downstream of the probe ⁇ , and is likely to contain the full length of the L-glutamic acid oxidase gene.
- the base sequence was determined to analyze the frame ( ⁇ RF).
- the molecular weight of the protein calculated from the gene side is 76, 359 excluding the signal peptide, which is the value of the previously reported Streptomyces sp. 9-6 (ATCC 39 34 3) Total molecular weight of each subunit of L-glutamic acid oxidase derived from about 70,000 ( ⁇ subunit: about 44,000, / 3 subunit about 16 , 000, sub-unit: about 9,000).
- the plasmid pGS1 containing the full length L-glutamic acid oxidase gene was used as a DNA sample, and the following primers (A) and (B) were purchased from Pharmacia, and the L-dalminic acid oxidase gene 0 was obtained by PCR. RF amplification was performed.
- Amplification of L one glutamate Okishida Ichize gene by PCR uses the LA Tag DNA Polymerase (Takara Shuzo Co., Ltd.), 1 0 XP CR buffer (5 1), 2 5 mM Mg C l 2 (5 l), dNTP ( 8 I), Primer DNA (A) and (B) (each l l O pmol) and DNA sample (approx.0.5 g) to make final volume to 501, GeneAmp PCR from Perkin-Elmer Cetus Instrument Heat denaturation (96 ° C, 10 seconds), annealing (55 ° C, 30 seconds), and polymerization (60 ° C, 120 seconds) were performed using the System 2400. This was done by repeating 25 times.
- p GOX-ma 11 has a Taq promoter overnight and a maltose binding protein (MBP) gene introduced upstream of the multiple cloning site, and Streptomyces sp.
- MBP maltose binding protein
- Escherichia coli JM109 was transformed with the plasmid pGOX-ma11, and the resulting transformant was inoculated into 5 ml of 2 XTY medium containing 50 g / m1 of ampicillin, and incubated at 30 ° C. After shaking culture for about an hour, the cells were inoculated into 2 XTY medium (1 liter) containing 20 g / l darcos, and cultured with shaking at 24 ° C for about 24 hours. Further, IPTG was added to the culture solution, and the culture was continued at the same temperature for about 24 hours. After completion of the culture, L-glutamic acid oxidase was isolated and purified according to the following purification method.
- the L-glutamic acid oxidase gene was amplified using LA-P kit (Takara Shuzo) using chromosome DNA derived from (ATCC 39343) as a template.
- Amplification of L-glutamate oxidase gene by LA-PCR is performed by dNTP,
- the reaction solution was treated with a mixture of phenol z-form (1: 1), and ethanol was added to the water-soluble fraction to precipitate DNA.
- the DNA collected by precipitation was separated by agarose gel electrophoresis, and a DNA fragment corresponding to about 2 kb was purified.
- the DNA was cleaved with restriction enzymes NcoI and HindIII and ligated with plasmid pTrc99A (Pharmacia Biotech), also digested with restriction enzymes NcoI and HindIII, using T4 DNA ligase.
- Escherichia coli JM109 (Takara Shuzo) was transformed using the ligation reaction solution, and plasmid pGAI1 was isolated from the resulting ampicillin-resistant transformant.
- pGAI1 is located at the NcoI-HindIII cleavage site downstream of the rc promoter at the NcI-HindIII cleavage site.
- Tables 3 and 4 show the results obtained by setting the concentrations of various amino acids to 8.2 mM and 32.8 mM, respectively, and allowing the above-mentioned ⁇ to ⁇ ⁇ to act on L-dalminic acid oxidase.
- the L-glutamic acid oxidase of the present invention has high substrate specificity for L-dalminic acid.
- Optimum temperature Reaction at each temperature of 20 ° C to 80 ° C (20 ° C to 60 ° C for MBP-LG0X fusion protein derived from Escherichia coli JM109 / pGOX mall).
- Optimum PH As buffer, acetate buffer (pH 3.5-6.0), potassium phosphate buffer (PH 6.0-8.0), borate buffer (pH 8.0-: 10) . 0) use.
- the L-glutamic acid oxidase of the present invention is a novel enzyme consisting of two identical subunits having a molecular weight of about 70 kDa, which is different from those previously reported.
- the L-glutamic acid oxidase can be easily and inexpensively produced using a transformant such as Escherichia coli by the recombinant gene technique.
- L-Dallamic acid oxidase having the following physicochemical properties:
- Coenzyme is flavin adenine dinucleotide (FAD).
- L-daltamate oxidase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted, inserted or added.
- L-Dal duminic acid oxidase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted, inserted or added.
- L-glutamic acid oxidase gene according to claim 3, wherein the gene has a base sequence shown in SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted, inserted or added. L-glutamate oxidase gene.
- An expression vector prepared by introducing a gene encoding the L-glutamic acid oxidase according to claim 3 or 4 into a plasmid.
- a host microorganism is transformed with the expression vector according to claim 8, and the resulting transformant is cultured to produce L-glutamic acid oxidase.
- a method for producing L-glutamic acid oxidase comprising isolating and purifying lipase.
- L-glutamic acid oxidase obtained by the method according to claim 9 or 10, having the following physicochemical properties.
- Coenzyme is flavin adenine dinucleotide (FAD).
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU48791/01A AU4879101A (en) | 2000-04-19 | 2001-04-19 | L-glutamate oxidase |
CA002406130A CA2406130C (en) | 2000-04-19 | 2001-04-19 | L-glutamate oxidase |
US10/257,398 US7109008B2 (en) | 2000-04-19 | 2001-04-19 | L-glutamate oxidase |
EP01921904A EP1277839B1 (en) | 2000-04-19 | 2001-04-19 | L-glutamate oxidase |
DE60132989T DE60132989T2 (de) | 2000-04-19 | 2001-04-19 | L-glutamatoxidase |
JP2001577486A JP4002765B2 (ja) | 2000-04-19 | 2001-04-19 | L−グルタミン酸オキシダーゼ |
US11/399,446 US20060228790A1 (en) | 2000-04-19 | 2006-04-07 | L-glutamate oxidase |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000-117749 | 2000-04-19 | ||
JP2000117749 | 2000-04-19 | ||
JP2001-57848 | 2001-03-02 | ||
JP2001057848 | 2001-03-02 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/399,446 Division US20060228790A1 (en) | 2000-04-19 | 2006-04-07 | L-glutamate oxidase |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001079503A1 true WO2001079503A1 (fr) | 2001-10-25 |
Family
ID=26590377
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2001/003345 WO2001079503A1 (fr) | 2000-04-19 | 2001-04-19 | L-glutamate oxydase |
Country Status (8)
Country | Link |
---|---|
US (2) | US7109008B2 (ja) |
EP (1) | EP1277839B1 (ja) |
JP (1) | JP4002765B2 (ja) |
AT (1) | ATE387501T1 (ja) |
AU (1) | AU4879101A (ja) |
CA (1) | CA2406130C (ja) |
DE (1) | DE60132989T2 (ja) |
WO (1) | WO2001079503A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106282205A (zh) * | 2015-06-12 | 2017-01-04 | 上海市农业科学院 | 一种高比活l-谷氨酸氧化酶基因多位点突变体及其制备方法和应用 |
CN107686850A (zh) * | 2016-08-04 | 2018-02-13 | 中国科学院天津工业生物技术研究所 | 一种利用共表达重组菌株转化生产α‑酮戊二酸的方法 |
EP3492599A1 (en) | 2017-11-29 | 2019-06-05 | Yamasa Corporation | Dried l-glutamate oxidase composition |
WO2021193598A1 (ja) | 2020-03-24 | 2021-09-30 | 味の素株式会社 | L-グルタミン酸オキシダーゼ変異体 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60132989T2 (de) * | 2000-04-19 | 2009-02-19 | Yamasa Corp., Choshi | L-glutamatoxidase |
US9249450B2 (en) | 2013-03-15 | 2016-02-02 | Abbott Point Of Care Inc. | Glutamate oxidase mutagenesis for diagnostic testing |
CN103388002A (zh) * | 2013-07-23 | 2013-11-13 | 江南大学 | 一种产l-谷氨酸氧化酶重组菌株的构建方法及其应用 |
CN105331642B (zh) * | 2015-11-30 | 2020-06-19 | 浙江汇宁生物科技有限公司 | 一种利用L-谷氨酸氧化酶催化生产α-酮戊二酸的方法 |
CN110283837A (zh) * | 2019-04-19 | 2019-09-27 | 中国科学院天津工业生物技术研究所 | 一种高酶活性l-谷氨酸氧化酶突变体及其制备方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0097949A2 (en) * | 1982-06-29 | 1984-01-11 | Yamasa Shoyu Kabushiki Kaisha | L-glutamic acid oxidase, its production, and its use |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58149677A (ja) * | 1982-03-02 | 1983-09-06 | Toyo Jozo Co Ltd | L−グルタミン酸オキシダ−ゼ(h↓2o↓2・ジエネレイテイング)およびその製造法、ならびに分析方法 |
DE60132989T2 (de) * | 2000-04-19 | 2009-02-19 | Yamasa Corp., Choshi | L-glutamatoxidase |
-
2001
- 2001-04-19 DE DE60132989T patent/DE60132989T2/de not_active Expired - Lifetime
- 2001-04-19 AU AU48791/01A patent/AU4879101A/en not_active Abandoned
- 2001-04-19 WO PCT/JP2001/003345 patent/WO2001079503A1/ja active IP Right Grant
- 2001-04-19 JP JP2001577486A patent/JP4002765B2/ja not_active Expired - Lifetime
- 2001-04-19 EP EP01921904A patent/EP1277839B1/en not_active Expired - Lifetime
- 2001-04-19 CA CA002406130A patent/CA2406130C/en not_active Expired - Lifetime
- 2001-04-19 AT AT01921904T patent/ATE387501T1/de not_active IP Right Cessation
- 2001-04-19 US US10/257,398 patent/US7109008B2/en not_active Expired - Lifetime
-
2006
- 2006-04-07 US US11/399,446 patent/US20060228790A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0097949A2 (en) * | 1982-06-29 | 1984-01-11 | Yamasa Shoyu Kabushiki Kaisha | L-glutamic acid oxidase, its production, and its use |
Non-Patent Citations (3)
Title |
---|
ANNETTE BOHMER ET AL.: "A novel L-glutamate oxidase from streptomyces endus", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 182, no. 2, 1989, pages 327 - 332, XP002941760 * |
CHIEN-YUAN CHEN ET AL.: "A common precursor for the three subunits of L-glutamate oxidase encoded by gox gene from streptomyces platensis NTU 3304", CANADIAN JOURNAL OF MICROBIOLOGY, vol. 47, no. 3, March 2001 (2001-03-01), pages 269 - 275, XP002941759 * |
HITOSHI KUSAKABE ET AL.: "Purification and properties of a new enzyme, L-glutamate oxidase, from streptomyces sp. X-119-6 grown on wheat bran", AGRICULTURAL AND BIOLOGICAL CHEMISTRY, vol. 47, no. 6, 1983, pages 1323 - 1328, XP002941777 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106282205A (zh) * | 2015-06-12 | 2017-01-04 | 上海市农业科学院 | 一种高比活l-谷氨酸氧化酶基因多位点突变体及其制备方法和应用 |
CN106282205B (zh) * | 2015-06-12 | 2021-06-01 | 上海市农业科学院 | 一种高比活l-谷氨酸氧化酶基因多位点突变体及其制备方法和应用 |
CN107686850A (zh) * | 2016-08-04 | 2018-02-13 | 中国科学院天津工业生物技术研究所 | 一种利用共表达重组菌株转化生产α‑酮戊二酸的方法 |
CN107686850B (zh) * | 2016-08-04 | 2022-08-09 | 中国科学院天津工业生物技术研究所 | 一种利用共表达重组菌株转化生产α-酮戊二酸的方法 |
EP3492599A1 (en) | 2017-11-29 | 2019-06-05 | Yamasa Corporation | Dried l-glutamate oxidase composition |
WO2021193598A1 (ja) | 2020-03-24 | 2021-09-30 | 味の素株式会社 | L-グルタミン酸オキシダーゼ変異体 |
Also Published As
Publication number | Publication date |
---|---|
US20040091989A1 (en) | 2004-05-13 |
US7109008B2 (en) | 2006-09-19 |
ATE387501T1 (de) | 2008-03-15 |
DE60132989D1 (de) | 2008-04-10 |
US20060228790A1 (en) | 2006-10-12 |
CA2406130A1 (en) | 2001-10-25 |
AU4879101A (en) | 2001-10-30 |
CA2406130C (en) | 2009-07-07 |
JP4002765B2 (ja) | 2007-11-07 |
EP1277839B1 (en) | 2008-02-27 |
EP1277839A4 (en) | 2004-05-12 |
EP1277839A1 (en) | 2003-01-22 |
DE60132989T2 (de) | 2009-02-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9453206B2 (en) | Modified leucine dehydrogenase | |
US7670822B2 (en) | Aldolase and production process of 4-hydroxy-L-isoleucine | |
US20060228790A1 (en) | L-glutamate oxidase | |
Wong et al. | Molecular properties of pyruvate formate-lyase activating enzyme | |
US11667897B2 (en) | D-amino acid oxidative enzyme mutant and application thereof | |
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 | |
JP6690529B2 (ja) | γ−グルタミルバリン合成酵素、及び、γ−グルタミルバリルグリシンの製造法 | |
CN113969268A (zh) | Glu/Leu/Phe/Val脱氢酶突变体及其在制备L-草铵膦中的应用 | |
US7115735B2 (en) | Dehydrogenase and a gene encoding the same | |
US20220235333A1 (en) | Modified Phenylalanine Dehydrogenase | |
Chatani et al. | Structural and Functional Changes in Bovine Pancreatic Ribonuclease A by the Replacement of Phel20 with Other Hydrophobic Residues | |
JP7493335B2 (ja) | 変異型ヒスチジン脱炭酸酵素およびその用途 | |
EP0984066B1 (en) | Activator for methanol dehydrogenase and gene thereof | |
JP2011223896A (ja) | ペプチドの製造方法 | |
JP7386616B2 (ja) | L体環状アミノ酸の製造方法 | |
WO2021039611A1 (ja) | ペントシジンの測定方法及び測定用キット | |
JP5703455B2 (ja) | キヌクリジノン還元酵素及びそれを用いた光学活性3−キヌクリジノールの製造方法 | |
JP4415247B2 (ja) | 新規なグリセロールキナーゼ、該遺伝子及び該遺伝子を用いたグリセロールキナーゼの製造法 | |
JP2008022766A (ja) | ウリカーゼの比活性を向上させる方法、および比活性の向上した改変型ウリカーゼ | |
JP2005218342A (ja) | 新規甘味タンパク質及びその製法 | |
JPH11299491A (ja) | 改変セリンアセチルトランスフェラーゼ、改変セリンアセチルトランスフェラーゼ遺伝子、新規な組み換え体dna及び改変セリンアセチルトランスフェラーゼの製造法 | |
JP2003339384A (ja) | 新規プリンヌクレオシドホスホリラーゼ、該遺伝子及び新規プリンヌクレオシドホスホリラーゼの製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2001 577486 Country of ref document: JP Kind code of ref document: A |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001921904 Country of ref document: EP Ref document number: 2406130 Country of ref document: CA |
|
WWP | Wipo information: published in national office |
Ref document number: 2001921904 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10257398 Country of ref document: US |
|
WWG | Wipo information: grant in national office |
Ref document number: 2001921904 Country of ref document: EP |