WO2010047188A1 - Procédé de production d'hydroxydes d'acides aminés aliphatique l et d - Google Patents

Procédé de production d'hydroxydes d'acides aminés aliphatique l et d Download PDF

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WO2010047188A1
WO2010047188A1 PCT/JP2009/065737 JP2009065737W WO2010047188A1 WO 2010047188 A1 WO2010047188 A1 WO 2010047188A1 JP 2009065737 W JP2009065737 W JP 2009065737W WO 2010047188 A1 WO2010047188 A1 WO 2010047188A1
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amino acid
aliphatic amino
protein
seq
acid sequence
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Japanese (ja)
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邦器 木野
良太郎 原
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学校法人 早稲田大学
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    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • the present invention relates to a method for producing L- and D-aliphatic amino acid hydroxides, and more specifically to a method for producing aliphatic amino acid hydroxides using aliphatic amino acid hydroxylase.
  • 4-hydroxy-L-isoleucine have medicinal properties as therapeutic agents for type 2 diabetes.
  • the most famous of these is 4-hydroxy-L-isoleucine purified from the leguminous plant Trigonella foenum-graecum L. known as the herbal medicine Koroha or the spice fenugreek. (Hereinafter referred to as “4-HI”) (Non-patent Document 1).
  • 4-HI exhibits activity to promote insulin secretion in rat pancreas and human islets. Broca, C.I. Et al., Am. J. et al. Physiol. 277: E617-E623 (1999).
  • 4-HI can be produced using isoleucine hydroxylase in the fenugreek extract (Non-patent Document 2).
  • the enzyme has not been identified, and the enzyme can only be obtained from fenugreek extract and cannot be obtained in large quantities.
  • the enzyme is unstable, the method for producing 4-HI using isoleucine hydroxylase in the fenugreek extract is insufficient as an industrial production method. Haefele et al., Phytochemistry, 44: 563-566 (1997).
  • L-isoleucine 4-hydroxylase isoleucine dioxygenase derived from Bacillus thuringiensis can synthesize 4-HI using L? Isoleucine as a substrate, but this enzyme has strict substrate specificity.
  • Patent Document 3 There is a report (Patent Document 3) that it does not react with amino acids other than L-isoleucine (L-leucine, L-valine, L-glutamic acid, L-lysine and D-isoleucine).
  • ⁇ -hydroxynorvaline and some stereoisomers of ⁇ -hydroxyvaline, compounds having a structure similar to 4-HI, have also been shown to have an activity of promoting insulin secretion in rat islets (non- Patent Document 5).
  • the present invention provides L- and D-aliphatic amino acid dioxygenases.
  • the aliphatic amino acid dioxygenase of the present invention catalyzes the hydroxylation reaction of L- and D-forms of an aliphatic amino acid in the presence of 2-oxoglutarate and divalent iron ions.
  • the L- and D-aliphatic amino acid dioxygenase of the present invention includes (1) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6, and (2) 1 in the amino acid sequence of SEQ ID NO: 2, 4 or 6.
  • 2-oxoglutarate-dependent dioxygenase activity comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and catalyzing the hydroxylation of L- and D-forms of aliphatic amino acids
  • a protein having 2-oxoglutarate-dependent dioxygenase activity and (4) a nucleotide having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 1, 3, or 5
  • the present invention provides a method for producing a hydroxide of an aliphatic amino acid.
  • the method for producing a hydroxide of an aliphatic amino acid according to the present invention comprises L- and C-catalyzing the hydroxylation reaction of an L-form and a D-form of an aliphatic amino acid in the presence of 2-oxoglutaric acid and divalent iron ions.
  • Preparing a D-aliphatic amino acid dioxygenase and an aliphatic amino acid and allowing the L- and D-aliphatic amino acid dioxygenases to act on the aliphatic amino acid to produce a hydroxide of the aliphatic amino acid.
  • the 2-oxoglutarate-dependent dioxygenase includes (1) a protein comprising any one amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and (2) of SEQ ID NO: 2, 4, 6 or 8. It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to any one of the amino acid sequences described above, and catalyzes the hydroxylation reaction of L- and D-forms of aliphatic amino acids A protein having 2-oxoglutarate-dependent dioxygenase activity, and (3) an amino acid sequence having 80% or more homology with any one of SEQ ID NOs: 2, 4, 6 or 8; A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of the L-form and D-form of an aliphatic amino acid, and (4) SEQ ID NOs: 1, 3 An amino acid sequence encoded by a polynucleotide consisting of a nucleotide sequence having a homology of
  • the present invention provides a recombinant vector comprising a polynucleotide encoding the L- and D-aliphatic amino acid dioxygenase of the present invention.
  • the present invention provides a transformant containing the recombinant vector of the present invention.
  • the protein of the present invention is produced by expressing a DNA comprising a nucleotide sequence encoding the amino acid sequence of the protein of the present invention in an inanimate expression system or an expression system using a host organism and an expression vector.
  • the host organisms include prokaryotes such as E. coli and Bacillus subtilis and eukaryotes such as yeast, fungi, plants and animals.
  • An expression system using the host organism and expression vector of the present invention may be a part of an organism such as a cell or tissue, or an entire individual of the organism.
  • the protein of the present invention may be prepared by subjecting L- and D of the present invention to a mixture of an inanimate expression system or a host organism and other components of an expression system using an expression vector, provided that it has hydroxylase activity for aliphatic amino acids.
  • -It may be used in a method for producing a hydroxide of an aliphatic amino acid, or may be used in a method for producing a hydroxide of an L- and D-aliphatic amino acid of the present invention in a purified state.
  • dioxygenase or hydroxylase refers to any protein having an enzyme activity that hydroxylates any atom of a substrate molecule (hereinafter referred to as “hydroxylase activity”).
  • the protein having hydroxylase activity for the L- and D-aliphatic amino acids of the present invention may be a protein derived from any species.
  • Proteins having hydroxylase activity for L- and D-aliphatic amino acids of the present invention include genus Greobacter such as Gleobacter violaceus, and Pseudomonas syringae sapiera syringae.
  • Pseudomonas genus such as Phaseolicola, Chromobacterium violaceum such as Chromobacterium violaceum, Bacillus cerium B. ⁇ Bacillis genus (Bacillus licheniformis), Bacillus sphaericus (Bacillus sphaericus), Escherichia coli, other Escherichia ter, It may be derived from any organism including bacteria containing the genus (Arthrobacter), filamentous fungi or fungi containing the genus Aspergillus, archaea, plants and animals.
  • the protein having hydroxylase activity of the present invention includes a protein of GenBank accession number glr2602 (hereinafter referred to as “glr2602 protein”) derived from Gloeobacter violaceus PCC 7421, Pseudomonas syringae pasoba phaseolicola (on-site). pv.
  • phaseolicola 1448A derived from the GenBank accession number PSPPH_3986 of protein (hereinafter referred to as "PSPPH_3986 protein”.) and, Chromobacterium violaceum (Chromobacterium violaceum) NBRC 12614 T derived from the GenBank accession number CV_3308 of protein (hereinafter referred to as "CV And 3308 protein "called.), Bacillus cereus (Bacillus cereus) ATCC14579 of GenBank accession number BC_1061 derived from a protein (hereinafter referred to, including that.) And” BC_1061 protein "is not limited to this.
  • the nucleotide sequence consisting of 654 bases encoding the glr2602 protein is listed in SEQ ID NO: 1, and the amino acid sequence consisting of 217 amino acids of the glr2602 protein is listed in SEQ ID NO: 2.
  • the nucleotide sequence consisting of 720 bases encoding the PSPPH — 3986 protein is listed in SEQ ID NO: 3, and the amino acid sequence consisting of 239 amino acids of the PSPPH — 3986 protein is listed in SEQ ID NO: 4.
  • the nucleotide sequence consisting of 924 bases encoding the CV — 3308 protein is listed in SEQ ID NO: 5, and the amino acid sequence consisting of 307 amino acids of the CV — 3308 protein is listed in SEQ ID NO: 6.
  • the nucleotide sequence consisting of 753 bases encoding the BC — 1061 protein is listed in SEQ ID NO: 7, and the amino acid sequence consisting of 250 amino acids of the BC — 1061 protein is listed in SEQ ID NO:
  • the homology of nucleotide sequences means that the nucleotide sequences of the present invention and the nucleotide sequence to be compared are aligned so that the number of nucleotide sequences that match is the largest. Expressed as a percentage of the quotient divided by the total number of nucleotides of the nucleotide sequence of the present invention.
  • amino acid sequence homology is determined by aligning the amino acid sequences of the present invention and the amino acid sequence to be compared so that the number of amino acid residues having the same sequence is the largest.
  • nucleotide sequence and amino acid sequence of the present invention can be calculated by using the sequence alignment program CLUSTALW well known to those skilled in the art.
  • stringent conditions refers to Sambrook, J. et al. And Russell, D .; W. , Molecular Cloning A Laboratory Manual 3rd Edition, Cold Spring Harbor Laboratory Press (2001), and the Southern blot method described in the following experimental conditions.
  • a polynucleotide having a nucleotide sequence to be compared is band-formed by agarose electrophoresis and then immobilized on a nitrocellulose filter or other solid phase by capillary action or electrophoresis. Pre-wash with a solution consisting of 6x SSC and 0.2% SDS.
  • a hybridization reaction between a probe obtained by labeling a polynucleotide comprising the nucleotide sequence of the present invention with a radioisotope or other labeling substance and a polynucleotide to be compared immobilized on the solid phase is performed with 6 ⁇ SSC and 0.2. % In SDS solution at 65 ° C overnight. Thereafter, the solid phase was washed twice at 65 ° C. for 30 minutes each in a solution consisting of 1 ⁇ SSC and 0.1% SDS, and 65 ° in a solution consisting of 0.2 ⁇ SSC and 0.1% SDS. C. Wash twice for 30 minutes each. Finally, the amount of the probe remaining on the solid phase is determined by quantifying the labeling substance.
  • hybridization under “stringent conditions” means that the amount of the probe remaining on the solid phase immobilized with the polynucleotide consisting of the nucleotide sequence to be compared is equal to the polynucleotide consisting of the nucleotide sequence of the present invention. It refers to at least 25%, preferably at least 50%, more preferably at least 75% or more of the amount of probe remaining in the immobilized solid phase of the positive control experiment.
  • hydroxylase activity refers to the ability to act on a substrate molecule and perform a reaction to hydroxylate any carbon atom of the substrate molecule.
  • aliphatic amino acid refers to an amino acid containing an aliphatic hydrocarbon in the side chain.
  • the aliphatic amino acids in the present specification are included in 20 kinds of L-amino acids used for translation of proteins synthesized from messenger RNA by ribosome in vivo such as L-leucine, L-isoleucine and L-valine.
  • L-amino acids such as L-norvaline and L-norleucine
  • optical isomers thereof such as D-leucine, D-isoleucine, D-valine, D-norvaline and D-norleucine
  • the L- and D-aliphatic amino acid dioxygenases of the present invention are 2-oxoglutarates that catalyze the hydroxylation reaction of L- and D-forms of aliphatic amino acids in the presence of 2-oxoglutarate and divalent iron ions. It is an acid-dependent dioxygenase.
  • the L- and D-aliphatic amino acid dioxygenases of the present invention perform hydroxylation of L- and D-forms of aliphatic amino acids in the presence of 2-oxoglutarate, L-ascorbic acid and divalent iron ions. Preferably it is done.
  • the aliphatic amino acid hydroxide obtained by the production method of the present invention is based on specific adsorption on an ion exchange resin or other carrier, thin layer chromatography, high performance liquid chromatography (HPLC), other chromatography, or organic solvent. It is recovered by methods well known to those skilled in the art such as extraction and crystallization.
  • the peptides are also evaluated for production or purity using analytical techniques well known to those skilled in the art, such as HPLC or mass spectrometry (MS) methods.
  • the chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of D-norleucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation reaction product of L-valine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of L-leucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of L-isoleucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of L-norvaline by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of L-norleucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of D-valine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation reaction product of D-leucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of D-isoleucine by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation reaction product of D-norvaline by glr2602 protein.
  • the MS chart which shows the MS analysis result of the hydroxylation product of D-norleucine by glr2602 protein.
  • Table 1 shows the aliphatic amino acid hydroxylases used in the examples.
  • Amplification of the target L- and D-aliphatic amino acid dioxygenase genes was carried out using Expand High Fidelity PCR System (Roche) with polymerase chain reaction (PCR) using the chromosomal DNA of each microorganism as a template.
  • PCR consists of 1 cycle at 94 ° C for 180 seconds, 15 seconds at 94 ° C, 10 seconds at 50 ° C and 25 seconds at 72 ° C, 25 cycles, and 420 seconds at 72 ° C.
  • the reaction was performed under the reaction conditions (Table 2).
  • Table 3 shows the cloning and expression conditions for each of the aliphatic amino acid hydroxylases.
  • the base sequences of primers used for isolation of the genes of the respective enzymes are listed in SEQ ID NOs: 9-16.
  • E. coli derived from a single colony is cultured in 5 mL of LB-A liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 100 ⁇ g / mL ampicillin) at 37 ° C. for 16 hours, and QIAprep Spin Plasmids were extracted using Miniprep Kit (Qiagen). The DNA inserted into the extracted plasmid was analyzed by a DNA sequencer (Applied Biosystems), and it was confirmed that the target aliphatic amino acid hydroxylase gene was inserted.
  • E. coli Rosetta2 (DE3) was transformed with the recombinant plasmid obtained in Example 1.
  • the Escherichia coli was cultured using LB-AC agar medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar, 50 ⁇ g / mL ampicillin and 34 ⁇ g / mL chloramphenicol). The culture was stationary overnight at ° C.
  • the cells recovered from the liquid medium by centrifugation at 4 ° C. and 5000 ⁇ g for 10 minutes are suspended in 5 mL of 20 mM HEPES / NaOH buffer (pH 7.5), subjected to ultrasonic disruption for 3 minutes, and then subjected to 4 ° C. And centrifuged at 20000 ⁇ g for 30 minutes.
  • the collected supernatant (cell-free extract) was subjected to affinity chromatography using a His trap HP column (GE Healthcare) to separate aliphatic amino acid hydroxylase. Thereafter, the buffer was exchanged using a PD-10 column (GE Healthcare) to prepare a purified enzyme.
  • Hydroxylation reaction to various amino acids Hydroxylation reaction to various amino acids was performed using the purified enzyme obtained in Example 2. Since the purified enzyme was predicted to be 2-oxoglutarate-dependent dioxygenase, a standard reaction solution containing 1-oxoglutarate (1 mM amino acid, 5 mM 2-oxoglutarate, 1 mM L-ascorbic acid, 0.1 mM sulfuric acid sulfate 1 The enzyme reaction was performed using iron, 50 mM potassium phosphate buffer, 0.5 mg / mL various purified enzymes). The composition of the standard reaction solution is shown in Table 4 below.
  • Standard reaction solutions shown in Table 4 were prepared, and after enzyme addition, the mixture was incubated for 20 hours while stirring at 25 ° C. and 170 rpm. Thereafter, the reaction solution was filtered using a 0.45 ⁇ m filter and subjected to LC / MS analysis. As a negative control test, a 2-oxoglutarate non-addition system test and an enzyme non-addition system test were also performed.
  • HPLC analysis HPLC analysis was performed by a high performance liquid chromatograph L2000 series (Hitachi) using a post-column analysis method.
  • the configuration of the HPLC analyzer is shown in FIG.
  • the HPLC analysis conditions are shown in Table 5.
  • an eluent and a reaction liquid are connected to each of the pump A and the pump B, and the sample injected by the autosampler and the eluent are applied to an analysis column to be separated, and the eluate and the reaction liquid are separated.
  • the reaction was performed by measuring with a fluorescence detector.
  • the composition of the pump A eluent is 20 mM phosphate buffer, 5 mM 1-heptanesulfonic acid, 10% or 20% methanol (pH 2.5), and the composition of the pump B reaction liquid is 22 g / L boric acid, 12 g / L sodium hydroxide, 0.8 g / L orthophthalaldehyde, 2 g / L N-acetylcysteine.
  • the analytical column used was a Cosmo Seal 5C18-MS-II 4.6 mm ⁇ 150 mm analytical column (Nacalai Tesque), and the column temperature was 40 ° C. (Table 5-a).
  • the HPLC analysis pump program was 20 minutes at 0.7 mL / min eluent and 20 minutes at 0.3 mL / min reaction (Table 5-b). Fluorescence was measured at an excitation wavelength of 340 nm and a detection wavelength of 450 nm.
  • FIG. 3-12 is a chromatographic chart showing the HPLC analysis results after the reaction of the reaction solution containing these substrates.
  • the numbers in the figure are the elution time of each substrate peak and the maximum peak elution time of the product.
  • peaks of unknown compounds are detected when L- and D-leucine, L-isoleucine, L- and D-norvaline, and L- and D-norleucine are used as substrates. It was.
  • peaks of unknown compounds were detected when L- and D-leucine, L-isoleucine, L-norvaline, and L- and D-norleucine were used as substrates.
  • MS analysis A sample in which an unknown compound peak was detected in HPLC analysis was purified by a solid-phase extraction column according to the procedure shown in FIG. 2, and MS analysis was performed using LCQ Deca (Thermo Quest). Table 7 shows the MS analysis conditions.
  • the ionization method was an electrospray method, and the sample was directly injected by a syringe.
  • Sheath Gas Flow Rate is 20 arb
  • Aux Gas Flow Rate is 20 arb
  • Spray Voltage is 5 kV
  • Capillary Temp is 200 ° C
  • Capillary Voltage is 17 V
  • Tube Lensoffs is set to 5 Tubes.
  • FIG. 13-22 is an MS chart showing the MS analysis results after the reaction of the reaction solution containing these substrates. The numbers in the figure are the mass-to-charge ratios of the hydroxides of the respective substrates.
  • the aliphatic amino acid dioxygenase of the present invention is unique in that it catalyzes the hydroxylation reaction of not only the L-form of an aliphatic amino acid but also the D-form.
  • the present invention is useful for industrial production of hydroxides of L? And D-aliphatic amino acids.

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Abstract

La présente invention concerne : une hydroxylase d'acide aminé aliphatique présentant une faible spécificité au substrat ; et un procédé de fabrication d'un hydroxyde d'un acide aminé aliphatique à l'aide de l'enzyme, qui permet de produire un hydroxyde d'un acide aminé aliphatique parmi plusieurs acides aminés aliphatiques en modifiant une molécule de substrat pour l'enzyme. La présente invention concerne spécifiquement : une dioxygénase d'acide aminé aliphatique L ou D qui peut catalyser la réaction d'hydroxylation d'une forme L ou D d'un acide aminé aliphatique en présence d'acide 2-oxoglutarique ou d'un ion fer bivalent ; et un procédé de production d'un hydroxyde d'un acide aminé L ou D aliphatique. La dioxygénase d'acide aminé aliphatique L ou D peut être une protéine comprenant une séquence d'acides aminés décrite dans la SEQ ID N°2, la SEQ ID N°4, la SEQ ID N°6 ou la SEQ ID N°8. Le procédé de production d'un hydroxyde d'un acide aminé aliphatique L ou D comprend une étape consistant à permettre à la dioxygénase d'acide aminé aliphatique L ou D d'agir sur un acide aminé aliphatique pour produire un hydroxyde de l'acide aminé aliphatique.
PCT/JP2009/065737 2008-10-22 2009-09-09 Procédé de production d'hydroxydes d'acides aminés aliphatique l et d WO2010047188A1 (fr)

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CN105566136A (zh) * 2016-01-19 2016-05-11 天津科技大学 一种从发酵液中分离提取4-羟基异亮氨酸的方法

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WO2021111503A1 (fr) * 2019-12-02 2021-06-10 花王株式会社 Couche d'absorption de lumière et son procédé de fabrication, dispersion liquide, élément de conversion photoélectrique et cellule solaire

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HAEFELE, C. ET AL.: "Characterization of a dioxygenase from Trigonella foenum-graecum involved in 4-hydroxyisoleucine biosynthesis", PHYTOCHEMISTRY, vol. 44, no. 4, 1997, pages 563 - 6 *
JOARDAR, V. ET AL.: "Accession: YP-276119 [gi: 71735316], Definition: conserved hypothetical protein [Pseudomonas syringae pv. phaseolicola 1448A].", NCBI PROTEIN, 3 August 2005 (2005-08-03), Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/sviewer/viewer.fcgi?71735316:OLD04:12068379> [retrieved on 20091007] *
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VASCONCELOS, A.T.R. ET AL.: "Accession: NP- 902978 [gi: 34498763], Definition: conserved hypothetical protein [Chromobacterium violaceum ATCC 12472].", NCBI PROTEIN, 8 September 2003 (2003-09-08), Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/sviewer/viewer.fcgi?34498763:OLD03:4736031> [retrieved on 20091007] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105566136A (zh) * 2016-01-19 2016-05-11 天津科技大学 一种从发酵液中分离提取4-羟基异亮氨酸的方法

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