WO2010047188A1

WO2010047188A1 - Process for producing l- and d-aliphatic amino acid hydroxides

Info

Publication number: WO2010047188A1
Application number: PCT/JP2009/065737
Authority: WO
Inventors: 邦器木野; 良太郎原
Original assignee: 学校法人早稲田大学
Priority date: 2008-10-22
Filing date: 2009-09-09
Publication date: 2010-04-29
Also published as: JP2010098975A

Abstract

Disclosed are: an aliphatic amino acid hydroxylase having low substrate specificity; and a process for producing a hydroxide of an aliphatic amino acid by using the enzyme, which can produce a hydroxide of any one of various aliphatic amino acids by changing a substrate molecule for the enzyme. Specifically disclosed are: an L- or D-aliphatic amino acid dioxygenase which can catalyze the hydroxylation reaction of an L- or D-form of an aliphatic amino acid in the presence of 2-oxoglutaric acid and a bivalent iron ion; and a process for producing a hydroxide of an L- or D-aliphatic amino acid. The L- or D-aliphatic amino acid dioxygenase may be a protein comprising an amino acid sequence depicted in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. The process for producing a hydroxide of an L- or D-aliphatic amino acid comprises a step of allowing the L- or D-aliphatic amino acid dioxygenase to act on an aliphatic amino acid to produce a hydroxide of the aliphatic amino acid.

Description

Process for producing L- and D-aliphatic amino acid hydroxides

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.

It is known that aliphatic amino acid hydroxides such as 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). However, the enzyme has not been identified, and the enzyme can only be obtained from fenugreek extract and cannot be obtained in large quantities. In addition, since 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).

Another method reported for the production of 4-HI is that 4-hydroxy-3-methyl-2-ketopentanoic acid aldolase and a branched chain amino acid aminotransferase are coupled to form 4-HI from 4-acetoaldehyde and 2-oxobutanoic acid. There is a method of synthesizing 4-HI via hydroxy-3-methyl-2-ketopentanoic acid (Patent Document 1, Non-Patent Documents 3 and 4). Furthermore, a method for supplying 2-oxobutanoic acid as a raw material from threonine by threonine dehydratase is also disclosed (Patent Document 2). However, since these enzymes have low optical specificity of the substrate, in addition to 4-hydroxyisoleucine: 2S, 3R, 4S, which is useful as a pharmaceutical, 2S, 3R, 4R, which has no physiological activity, is produced as a by-product. There are problems such as low reaction efficiency due to the step reaction.
JP 2008-109924 A Ogawa, J .; Et al., Biosci. Biotechnol. Biochem. 71: 1607-1615 (2007). Smirnov, S .; V. Et al., FEMS Microbiol. Lett. , 273: 70-77 (2007) JP 2008-073037 A

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. 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). Γ-hydroxynorvaline and some stereoisomers of γ-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). In the future, in order to investigate the biological activities of hydroxides of aliphatic amino acids extensively, we developed an aliphatic amino acid hydroxylase with low substrate specificity and used it to change various fat molecules by simply changing the substrate molecule. It is necessary to develop a method for producing an aliphatic amino acid hydroxide capable of producing an aliphatic amino acid hydroxide.
Broca, C.I. Et al., Eur. J. et al. Pharmacol. 390: 339-45 (2000). JP 2008-173116 A

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 And (3) an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 2, 4 or 6, 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 A protein having an amino acid sequence encoded by a polynucleotide consisting of a sequence and having a 2-oxoglutarate-dependent dioxygenase activity that catalyzes a hydroxylation reaction of L- and D-forms of an aliphatic amino acid; ) Consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 3 or 5 and comprising L- and D-forms of aliphatic amino acids A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes a hydroxylation reaction, and (6) a fusion protein in which a specific binding tag peptide is linked to any one of the above-mentioned (1) to (5) May be selected from the group.

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. Obtaining a step.

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 80% or more with any one nucleotide sequence of 5 or 7, and an aliphatic amino acid L-form and D-form water A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes an oxidation reaction, and (5) a polynucleotide comprising any one nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 under stringent conditions A protein having an amino acid sequence encoded by a hybridizing polynucleotide and having a 2-oxoglutarate-dependent dioxygenase activity that catalyzes a hydroxylation reaction of L- and D-forms of an aliphatic amino acid; ) The specific binding tag peptide is the above (1) to (5) It may be selected from the group consisting of a fusion protein linked to the protein of Zureka.

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.

In the present specification, 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: 8.

In the present specification, 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. Similarly, in the present specification, 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. Is expressed as a percentage of the quotient obtained by dividing the sum of the number of amino acid residues that match by the total number of amino acid residues of the amino acid sequence of the present invention. The homology of the 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.

In this specification, “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. In the present specification, 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.

As used herein, “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.

As used herein, “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. Including aliphatic amino acids, L-amino acids such as L-norvaline and L-norleucine, and optical isomers thereof such as D-leucine, D-isoleucine, D-valine, D-norvaline and D-norleucine However, it is not limited to these.

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.

Schematic diagram of the HPLC configuration. The schematic diagram which shows the sample refinement | purification procedure by an ion exchange column. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of L-valine by glr2602 protein. The chromato chart which shows the HPLC analysis result of the hydroxylation reaction product of L-leucine by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of L-isoleucine by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of L-norvaline by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of L-norleucine by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of D-valine by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of D-leucine by glr2602 protein. The chromatogram which shows the HPLC analysis result of the hydroxylation reaction product of D-isoleucine by glr2602 protein. The chromato chart which shows the HPLC analysis result of the hydroxylation reaction product of D-norvaline by glr2602 protein. 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.

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

Cloning of Gene Encoding Aliphatic Amino Acid Hydroxylase 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 below 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.

1 μg of PCR-amplified DNA of the gene for the desired aliphatic amino acid hydroxylase and 1 μg of the vector pET-21a (+) were digested with restriction enzymes NdeI and XhoI at 37 ° C. for 16 hours. Thereafter, the DNA purified by GFX PCR Purification Kit (GE Healthcare) was ligated by reacting with DNA Ligation Kit <Mighty Mix> (Takara) at 16 ° C for 3 hours and introduced into E. coli strain JM109. . The Escherichia coli is cultured at 37 ° C. for 16 hours on LB-A agar medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar and 100 μg / mL ampicillin). A single colony of E. coli harboring the recombinant plasmid was isolated. 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 μg / 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.

Expression and Purification of Aliphatic Amino Acid Hydroxylase 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 μg / mL ampicillin and 34 μg / mL chloramphenicol). The culture was stationary overnight at ° C. Growing single colonies are inoculated into 5 mL of LB-AC liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 100 μg / mL ampicillin and 34 μg / mL chloramphenicol), 37 ° C, shaking culture was performed at 200 rpm for 16 hours. Thereafter, 1 mL of the liquid medium was inoculated into 100 mL of fresh LB-AC liquid medium and cultured with shaking at 37 ° C. and 200 rpm. O. D. _{When 660} reaches 0.5, isopropyl-β-d-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and shaking culture is performed at 25 ° C. and 100 rpm for 9 hours. Induced gene expression. 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 μm 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.

In the HPLC analysis, 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.

As a result of HPLC analysis of the reaction solution, it was detected only when the reaction system contained all of the substrate amino acid, 2-oxoglutaric acid, L-ascorbic acid, ferrous sulfate, purified enzyme and the like by comparison with the negative control. The peak was judged as the product peak. In the enzyme reaction system using glr2602 protein, L- and D-valine, L- and D-leucine, L- and D-isoleucine, L- and D-norvaline, and L- and D-norleucine are used as substrates. Product was detected. 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. In the enzyme reaction system using PSPPH_3986 protein, 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. In the enzyme reaction system using CV_3308 protein, peaks of unknown compounds were detected when L- and D-leucine, L-isoleucine, L-norvaline, and L- and D-norleucine were used as substrates. In the enzyme reaction system using BC — 1061 protein, peaks of unknown compounds were detected when L-leucine, L-isoleucine, L-norvaline, and L- and D-norleucine were used as substrates. Table 6 summarizes the elution times of the maximum peaks of unknown compounds detected for the four types of proteins.

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, and Tube Lensoffs is set to 5 Tubes.

As a result of MS analysis, in an enzyme reaction system using glr2602 protein, L- and D-valine, L- and D-leucine, L- and D-isoleucine, L- and D-norvaline, L- and D- When norleucine was used as a substrate, proton addition ions having a mass number of 16 were detected. 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. Similarly, in the enzyme reaction system using PSPPH_3986 protein, when L- and D-leucine, L-isoleucine, L- and D-norvaline, and L- and D-norleucine are used as substrates, CV_3308 protein is used. In the enzyme reaction system, when L- and D-leucine, L-isoleucine, L-norvaline, and L- and D-norleucine are used as substrates, L_leucine is used in the enzyme reaction system using BC_1061 protein. When L-isoleucine, L-norvaline, and L- and D-norleucine were used as substrates, proton-added ions each having a mass number of 16 were detected. Table 8 summarizes the mass-to-charge ratios of the detected protonated ions of the product. From this result, it can be said that the unknown compound identified by HPLC analysis is a compound in which a hydroxyl group is introduced into the substrate.

The synthesis of 4-HI from L-isoleucine was evaluated by comparing chromatographic charts and MS charts of commercially available 4-HI reagents and reaction products. As a result, it was concluded that the reaction product obtained from L-isoleucine by the purified enzyme obtained in Example 2 was 4-HI.

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. Thus, the present invention is useful for industrial production of hydroxides of L? And D-aliphatic amino acids.

Claims

L- and D-aliphatic amino acid dioxygenase, which catalyzes the hydroxylation reaction of L- and D-forms of aliphatic amino acids in the presence of 2-oxoglutarate and divalent iron ions.
The L- and D-aliphatic amino acid dioxygenases are:
(1) a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 6,
(2) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, and an L-form and D-form of an aliphatic amino acid. A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of
(3) 2-oxoglutar comprising an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 2, 4 or 6, and catalyzing the hydroxylation reaction of L- and D-forms of aliphatic amino acids A protein having acid-dependent dioxygenase activity;
(4) An amino acid sequence encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1, 3, or 5 and having a nucleotide sequence showing 80% or more homology, and an L-form and a D-form of an aliphatic amino acid A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of
(5) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5, and an L-form and D- A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation of the body,
(6) The L- of claim 1, wherein the specific binding tag peptide is selected from the group consisting of a fusion protein linked to any one of the proteins (1) to (5). And D-aliphatic amino acid dioxygenase.
L- and D-aliphatic amino acid dioxygenases catalyzing the hydroxylation of L- and D-forms of aliphatic amino acids in the presence of 2-oxoglutaric acid and divalent iron ions, and aliphatic amino acids are prepared And steps to
And reacting the L- and D-aliphatic amino acid dioxygenase with the aliphatic amino acid to obtain a hydroxide of the aliphatic amino acid. A method for producing a hydroxide of an amino acid.
The L- and D-aliphatic amino acid dioxygenases are:
(1) a protein consisting of any one amino acid sequence of SEQ ID NOs: 2, 4, 6 or 8;
(2) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6 or 8, and an aliphatic amino acid A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of L-form and D-form of
(3) It consists of an amino acid sequence showing 80% or more homology with any one amino acid sequence of SEQ ID NOs: 2, 4, 6 or 8, and the hydroxylated L- and D-forms of aliphatic amino acids A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the reaction;
(4) consisting of an amino acid sequence encoded by a polynucleotide consisting of a nucleotide sequence having a homology of 80% or more with any one nucleotide sequence of SEQ ID NOs: 1, 3, 5 or 7, and an aliphatic amino acid A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of L-form and D-form;
(5) an aliphatic amino acid comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising any one nucleotide sequence of SEQ ID NOs: 1, 3, 5 or 7; A protein having 2-oxoglutarate-dependent dioxygenase activity that catalyzes the hydroxylation reaction of L-form and D-form of
(6) The L- of claim 3, wherein the specific binding tag peptide is selected from the group consisting of a fusion protein linked to any one of the proteins (1) to (5). And a method for producing a hydroxide of a D-aliphatic amino acid.
A recombinant vector comprising a polynucleotide encoding the L- and D-aliphatic amino acid dioxygenase according to claim 1.
A transformant comprising the recombinant vector according to claim 5.