JP5697327B2 - Method for producing cis-hydroxy-L-proline - Google Patents

Description

  The present invention relates to L-proline cis-hydroxylase derived from actinomycetes, a method for producing cis-hydroxy-L-proline using the hydroxylase, a recombinant vector comprising a polynucleotide encoding the hydroxylase, The present invention relates to a transformant containing the recombinant vector.

  Hydroxy-L-proline is a kind of modified amino acid having a structure in which a hydroxyl group is introduced into L-proline. There are four types of isomers depending on the difference in the site where the hydroxyl group is introduced (carbon atom at the 3rd or 4th position) and the spatial arrangement of the hydroxyl group (trans or cis configuration). Of the isomers of hydroxyproline, cis-4-hydroxy-L-proline is a substance useful as a synthetic intermediate material for pharmaceuticals such as carbapenem antibiotics and N-acetylhydroxyproline which is used as an anti-inflammatory agent.

As a method for producing cis-4-hydroxy-L-proline, a method of organic synthesis of a cis-4-hydroxy-L-proline derivative from a trans-4-hydroxy-L-proline derivative has been proposed (Patent Document 1). However, the trans-4-hydroxy-L-proline derivative itself, which is a raw material for synthesis, has a problem that it is expensive. A method for producing cis-4-hydroxy-L-proline from L-proline using a microorganism such as Helicoceras has also been proposed (Patent Documents 2 and 3), but the production amount is extremely 0.65 g / L. Low and not practical.
JP 2005-112761 A Japanese Patent No. 3005085 Japanese Patent No. 3005086

  There is a need to develop a process for economically and efficiently producing industrially useful cis-hydroxy-L-proline.

  The present invention provides L-proline cis-hydroxylase. The L-proline cis-hydroxylase of the present invention comprises (1) a protein comprising the amino acid sequence of SEQ ID NO: 1, and (2) one or several amino acids deleted or substituted in the amino acid sequence of SEQ ID NO: 1. Or a protein comprising an added amino acid sequence and having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity, and (3) 80% or more of the amino acid sequence of SEQ ID NO: 1 And a protein having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity, and (4) encoding the amino acid sequence of SEQ ID NO: 1. Encoding by a polynucleotide comprising a nucleotide sequence having a homology of 80% or more with the nucleotide sequence A protein comprising the encoded amino acid sequence and having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity; and (5) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. An amino acid sequence encoded by a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising L-proline cis-3-hydroxylase activity and L-proline cis-4 -At least one protein selected from the group consisting of a protein having hydroxylase activity and (6) a fusion protein in which a specific binding tag peptide is linked to any of the proteins (1) to (5) There is a case.

  The present invention provides a process for producing cis-hydroxy-L-proline. The method for producing cis-hydroxy-L-proline of the present invention comprises a step of preparing L-proline cis-hydroxylase of the present invention and L-proline, and (2) said L-proline cis-hydroxylase is prepared as described above. Acting on L-proline to obtain cis-3-hydroxy-L-proline and cis-4-hydroxy-L-proline.

  The present invention provides a recombinant vector comprising a polynucleotide encoding the L-proline cis-hydroxylase of the present invention.

  The present invention provides a transformant comprising the recombinant vector of the present invention.

  In the present specification, “protein”, “peptide”, “oligopeptide” or “polypeptide” is a compound in which two or more amino acids are linked by peptide bonds. “Proteins”, “peptides”, “oligopeptides” or “polypeptides” may include alkyl groups including methyl groups, phosphate groups, sugar chains, and / or ester bonds and other covalent modifications. . In addition, “protein”, “peptide”, “oligopeptide” or “polypeptide” is a metal ion, coenzyme, allosteric ligand or other atom, ion, atomic group, or other “protein”, “peptide”, “ When `` oligopeptides '' or `` polypeptides '', biopolymers such as sugars, lipids, nucleic acids, etc., polystyrene, polyethylene, polyvinyl, polyester or other synthetic polymers are bound or associated by covalent or non-covalent bonds There is.

  In the present specification, an amino acid may be represented by a compound name such as L-asparagine or L-glutamine, or may be represented by a conventional three-letter code such as Asn or Gln. When represented by a compound name, it is represented using a prefix (L- or D-) indicating the configuration of the α-carbon of the amino acid. When expressed in conventional three-letter code, the three-letter code represents an L-amino acid unless otherwise specified. In the present specification, an amino acid refers to any compound in which an amino group and a carboxyl group are bonded via at least one carbon atom and can be polymerized by a peptide bond. Amino acids in the present specification include, but are not limited to, 20 types of L-amino acids used for translation of proteins synthesized in ribosomes from messenger RNA in vivo and D-amino acids that are stereoisomers thereof. It may contain any natural or non-natural amino acid.

  In the present specification, regarding the isomer of hydroxyproline (hereinafter referred to as “Hyp”), the difference in the site where the hydroxyl group is introduced (carbon atom at the 3rd or 4th position) and the spatial arrangement of the hydroxyl group (trans Depending on the difference in the configuration or cis configuration, it may be represented as trans-3-Hyp, cis-3-Hyp, trans-4-Hyp, or cis-4-Hyp, respectively.

  The structure of cis-4-hydroxy-L-proline (cis-4-Hyp) is represented by Formula I.

  The L-proline cis-hydroxylase of the present invention is produced by expressing DNA consisting of a nucleotide sequence encoding its amino acid sequence 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 enzyme protein of the present invention is an inanimate expression system or an expression system using a host organism and an expression vector, provided that it has L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity. It may be used in the production method of cis-3-Hyp and / or cis-4-Hyp of the present invention in a state where other components are mixed. When the enzyme protein of the present invention is expressed in an expression system using the host organism and an expression vector, the host organism that expresses the enzyme protein, for example, the transformant of the present invention is alive and the cis- It may be used for the production of 3-Hyp and / or cis-4-Hyp. At this time, cis-3-Hyp and / or cis-4-Hyp of the present invention can be produced by a resting cell reaction system or fermentation method. Alternatively, the enzyme protein may be used in the method for producing cis-3-Hyp and / or cis-4-Hyp of the present invention in a purified state.

  The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of L-proline cis-hydroxylase derived from the actinomycetes Streptosporangium roseum Streptosporangium roseum NBRC 3776 strain. The amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 is registered under the accession number EEP13595 derived from the actinomycetes Streptosporangium roseum DSM 43021 strain and is annotated as 2-oxoglutarate-dependent hydroxylase It was. From the discovery that this L-proline cis-hydroxylase has L-proline cis-hydroxylase activity to produce cis-3-Hyp and cis-4-Hyp from proline as shown in the Examples of the present invention. The invention has been conceived.

  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.

  As used herein, “stringent conditions” refers to Sambrook, J. et al. And Russell, D .; W. This refers to the following experimental conditions in the Southern blot method described in Molecular Cloning A Laboratory Manual 3rd Edition, Cold Spring Harbor Laboratory Press (2001). 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 comparison target polynucleotide immobilized on the solid phase was subjected to 6 × SSC and 0.2 % Overnight in a solution consisting of SDS at 65 ° C. Thereafter, the solid phase was washed twice at 65 ° C. in a solution consisting of 1 × SSC and 0.1% SDS at 30 ° C. for 30 minutes each, and 65 ° C. 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 this specification, “hybridization under stringent conditions” means that the amount of the probe remaining on the solid phase immobilized with the polynucleotide comprising the nucleotide sequence to be compared is equal to the polynucleotide comprising 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.

  The L-proline cis-hydroxylase of the present invention comprises (1) a protein comprising the amino acid sequence of SEQ ID NO: 1, and (2) one or several amino acids deleted or substituted in the amino acid sequence of SEQ ID NO: 1. Or a protein comprising an added amino acid sequence and having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity, and (3) 80% or more of the amino acid sequence of SEQ ID NO: 1 And a protein having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity, and (4) encoding the amino acid sequence of SEQ ID NO: 1. Encoding by a polynucleotide comprising a nucleotide sequence having a homology of 80% or more with the nucleotide sequence A protein comprising the encoded amino acid sequence and having L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity; and (5) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. An amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide under stringent conditions, and has L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity The protein may be selected from the group consisting of (6) a specific binding tag peptide and a fusion protein linked to any one of the proteins (1) to (5).

  The fusion protein of the present invention is obtained by linking a specific binding tag peptide to the amino terminus or carboxyl terminus of any one of the above-mentioned (1) to (5) L-proline cis-hydroxylase.

  When preparing the L-proline cis-hydroxylase of any one of (1) to (5) above, the specific binding tag peptide of the present invention can more easily detect, separate or purify the expressed protein. In order to enable the above, it is a polypeptide that specifically binds to other proteins, polysaccharides, glycolipids, nucleic acids and their derivatives, resins and the like. The ligand that binds to the specific binding tag may be immobilized in a solid support, either in a free state dissolved in an aqueous solution. Therefore, since the fusion protein of the present invention specifically binds to the ligand immobilized on the solid support, other components of the expression system can be washed away. Thereafter, the fusion protein can be separated and recovered from the solid support by adding a ligand in a free state or changing pH, ionic strength and other conditions. Specific binding tags of the present invention include, but are not limited to, His tags, myc tags, HA tags, intein tags, MBP, GST and similar polypeptides. The specific binding tag of the present invention may have any amino acid sequence provided that the fusion protein retains L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity. .

  The L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity of the protein of the present invention are the same as the protein of the present invention, L-proline, 2-oxoglutarate, divalent iron ion, In some cases, cis-3-Hyp and cis-4-Hyp produced by reacting the protein with L-proline in a reaction solution containing L-ascorbic acid may be evaluated.

  Quantification of cis-3-Hyp and cis-4-Hyp may be performed by use of analytical instruments well known to those skilled in the art, such as LC / MS.

  Step (2) of the production method of the present invention is carried out using a reaction solution containing a buffer component for pH adjustment in addition to L-proline cis-hydroxylase and L-proline of the present invention. There is. Preferably, HEPES is used as the buffer component, and the pH may be adjusted to 7.0 to 7.5. Preferably, the reaction solution may further contain 2-oxoglutaric acid involved as an electron donor in the hydroxylation reaction by the protein of the present invention. The reaction solution may further contain divalent iron ions, L-ascorbic acid and the like.

  In order to make the L-proline cis-hydroxylase of the present invention act on L-proline in step (2) of the production method of the present invention, the reaction solution is incubated at a predetermined reaction time and reaction temperature. In the production method of the present invention, the concentration of the L-proline cis-hydroxylase, L-proline, divalent iron ion, 2-oxoglutaric acid, etc. of the present invention in the reaction solution, the amount of the reaction solution, the reaction time, the reaction Whether it is temperature or other reaction conditions depends on the target production amount and yield of cis-3-Hyp and cis-4-Hyp, the time, cost, equipment, etc. required for production, and other conditions. May be determined by one skilled in the art. The reaction temperature of the L-proline cis-hydroxylase of the present invention is preferably set to 15 ° C, and the reaction pH condition of the L-proline cis-hydroxylase is preferably set to pH 6.0.

  The cis-4-Hyp obtained by the production method of the present invention may be recovered by a combination of operations well known to those skilled in the art such as centrifugation, column chromatography, lyophilization and the like. In addition, when cis-3-Hyp and cis-4-Hyp obtained by the production method of the present invention are evaluated for production amount or purity using analytical techniques well known to those skilled in the art, such as LC / MS. There is.

  As used herein, the term “recombinant vector” refers to a vector that incorporates a polynucleotide encoding a protein having a desired function, which is used to express a protein having a desired function in a host organism.

  As used herein, the term “vector” is used to replicate and express a protein having a desired function in the host organism by introducing a polynucleotide encoding the protein having the desired function into the host organism. Genetic factors, including but not limited to plasmids, viruses, phages, cosmids and the like. Preferably the vector may be a plasmid. More preferably, the vector may be a pET-21a (+) plasmid.

  The recombinant vector of the present invention is prepared by ligating a polynucleotide encoding the protein of the present invention with any vector using genetic engineering techniques well known to those skilled in the art using restriction enzymes, DNA ligation enzymes, etc. May be.

  In the present specification, the term “transformant” refers to a desired trait associated with a protein having a desired function by introducing a recombinant vector into which a polynucleotide encoding a protein having a desired function is incorporated. It is a creature that has become able to.

  In the present specification, the “host organism” is an organism into which a recombinant vector incorporating a polynucleotide encoding a protein having a desired function is introduced in the production of a transformant. The host organisms include prokaryotes such as E. coli and Bacillus subtilis and eukaryotes such as yeast, fungi, plants and animals. The host organism may be E. coli.

  The transformant of the present invention is produced by introducing the recombinant vector of the present invention into any appropriate host organism. The introduction of the recombinant vector may be carried out by various techniques well known to those skilled in the art, including an electroporation method for creating a void in a cell membrane by electrical stimulation, a heat shock method performed in combination with calcium ion treatment, and the like.

The map of the recombinant vector of this invention. The electropherogram of L-proline cis-hydroxylase of this invention. The conceptual diagram which shows the procedure of the reaction stop about the hydroxylation reaction test, and the derivatization of an amino acid. The chromatogram of the HPLC analysis result which shows that the peak of two types of products of L-proline cis-hydroxylase of this invention can be isolate | separated. The graph which shows 2-oxoglutarate dependence of L-proline cis-hydroxylase activity of this invention. The graph which shows the reaction temperature dependence of L-proline cis-hydroxylase of this invention. The graph which shows the reaction pH dependence of L-proline cis-hydroxylase of this invention. The graph which shows the reaction inhibitor specificity of L-proline cis-hydroxylase of this invention. The graph which shows that L-proline cis-hydroxylase of this invention produces | generates two types of products simultaneously.

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

1. 1. Cloning, introduction and expression of gene encoding L-proline cis-hydroxylase 1-1. Method (Extraction of microbial chromosomal DNA as template for gene amplification)
The actinomycetes Streptosporangium roseum Streptosporangium roseum NBRC 3776 strain was obtained from National Institute of Technology and Evaluation, and this chromosomal DNA was used as a template for gene amplification.

  The actinomycetes were added to 5 mL of ISP Medium no. In 2 (1% Bacto Malt Extract, 0.4% Bacto Yeast extract, 0.4% glucose), liquid shaking culture was performed at 28 ° C. for 2 days. After incubation, the cells were collected by centrifugation (4 ° C., 5000 × g, 10 minutes), and chromosomal DNA was extracted from each microorganism according to a conventional method.

(Amplification of target gene)
The gene encoding the L-proline cis-hydroxylase of the present invention was amplified by polymerase chain reaction (PCR) using the chromosomal DNA of actinomycetes of Streptosporangium roseum NBRC 3776 strain as a template. A GC-RICH PCR system (Roche) was used for the PCR. The reaction conditions are shown in Table 1. The cloning and expression conditions are shown in Table 2. The sense primer and antisense primer using the actinomycetes Streptosporangium roseum Streptosporangium roseum NBRC 3776 as a template are shown in SEQ ID NOs: 2 and 3, respectively.

(Acquisition of recombinant plasmid)
The target DNA amplified by PCR is used as insert DNA, and 1 μg of each insert DNA and 1 μg of vector pET-21a (+) are digested by a reaction at 37 ° C. for 16 hours using restriction enzymes NdeI and XhoI. did. After purifying the cleavage product with GFX PCR Purification Kit (GE Healthcare), each insert DNA and vector were ligated by reaction at 16 ° C for 3 hours using DNA Ligation Kit <Mighty Mix> (Takara). did. The ligation product was introduced into Escherichia coli JM109 strain treated with calcium chloride by the heat shock method. Escherichia coli JM109 strain carrying each recombinant plasmid was cultured on LB-A agar medium (1% Bacto Trypton, 0.5% Bacto Yeast extract, 1% NaCl, 1.5% Bacto Agar, 100 μg / mL ampicillin). Incubate for 16 hours at ° C, then in LB-A liquid medium (1% Bacto Trypton, 0.5% Bacto Yeast extract, 1% NaCl, 100 μg / mL ampicillin) at 37 ° C for 16 hours, then Plasmids were extracted using QIAprep Spin Miniprep Kit (QIAGEN). The internal base sequence of the extracted plasmid was analyzed with a DNA sequencer, and it was confirmed that the desired DNA was inserted. A plasmid map of the prepared recombinant plasmid is shown in FIG.

(Expression of target gene)
A recombinant plasmid (pESrP4H) in which insertion of DNA encoding L-proline cis-hydroxylase of the present invention was confirmed was introduced into Escherichia coli Rosetta2 (DE3) treated with calcium chloride by the heat shock method, and the procedure shown in Table 3 It was expressed in. That is, the E. coli was cultured on LB-AC agar medium (1% Bacto Trypton, 0.5% Bacto Yeast extract, 1% NaCl, 1.5% Bacto Agar, 100 μg / mL ampicillin, 34 μg / mL chloramphenicol). Incubated overnight at ° C. The grown single colony was inoculated into 5 mL of LB-AC liquid medium (1% Bacto Trypton, 0.5% Bacto Yeast extract, 1% NaCl, 100 μg / mL ampicillin, 34 μg / mL chloramphenicol), 37 ° C., The shaking culture was performed at 200 rpm for 16 hours. Thereafter, 1 mL of the liquid medium was inoculated into 100 mL of a fresh LB-AC liquid medium, and cultured with shaking at 37 ° C. and 200 rpm. O. D. _{When 660} = 0.5 is reached, isopropyl-β-d-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and the gene expression is performed by culturing at 25 ° C. and 100 rpm. Induced. After 6 hours, the cultured cells were collected by centrifugation (4 ° C., 5000 × g, 10 minutes) and suspended in 5 mL of 20 mM HEPES / NaOH buffer (pH 7.5). The suspension was sonicated (3 minutes) and then centrifuged (4 ° C., 20000 × g, 30 minutes) to recover the supernatant (cell-free extract).

1-2. Results The gene encoding the L-proline cis-hydroxylase of the present invention was successfully cloned by the above-described extraction and amplification operations. A recombinant plasmid containing the gene could be prepared by the above-described recombination operation or the like. The plasmid map of the obtained recombinant plasmid is shown in FIG. By introducing the recombinant plasmid, a transformant having the recombinant plasmid could be prepared. Cell-free extracts containing the L-proline cis-hydroxylase of the present invention could be obtained by expressing the gene with the transformant. The obtained cell-free extract is purified by nickel affinity chromatography and gel filtration chromatography, and the protein obtained as a single band by SDS-PAGE is shown in FIG. The protein was used as a purified enzyme and used in the following experiments.

2. 2. Hydroxylation reaction of L-proline by L-proline cis-hydroxylase of the present invention 2-1. Method The hydroxylation reaction of L-proline by the L-proline cis-hydroxylase of the present invention obtained in Example 1 was carried out. A reaction solution having the composition shown in Table 3 (hereinafter referred to as “standard reaction solution”) was prepared, and the reaction was performed at 25 ° C. for 1 hour. After the reaction, the reaction was stopped and derivatization of all amino acids contained in the reaction solution was performed using Murphy reagent (1-fluoro-2,4-dinitrophenyl-5-L-alaninamide) according to the procedure of FIG. . Thereafter, the reaction solution was filtered using a 0.45 μm filter and subjected to high performance liquid chromatography (HPLC) analysis. Various conditions for the HPLC analysis are shown in Tables 4 (a) and (b).

2-2. Results When HPLC analysis was performed on a standard sample of L-proline and four isomers of Hyp, all the isomers of Hyp were detected as separated single peaks (FIG. 4). Therefore, the peak substance was identified based on the retention time.

FIG. 5 shows the results of examining the 2-oxoglutarate dependence of the reaction for the purified enzyme. From the composition of the reaction mixture shown in Table 3, the reaction activity was completely lost even if any of proline, 2-oxoglutarate (2-OG), FeSO ₄ and the enzyme was omitted. Thus, it was shown that the reaction of the L-proline cis-hydroxylase of the present invention is 2-oxoglutarate dependent.

  FIG. 6 shows the results of examining the optimum reaction temperature for the purified enzyme. The enzyme activity was highest at 15 ° C and was completely lost at 50 ° C.

  FIG. 7 shows the results of examining the optimum reaction pH for the purified enzyme. The enzyme activity was highest at pH 6.0, decreased to 50% to 30% at pH 7-10, and completely lost at pH 5.5.

FIG. 8 shows the results of examining the reaction inhibitor specificity of the purified enzyme. Enzymatic activity was not inhibited by MgSO ₄ . Some inhibition was observed with hydroxylamine, MnCl ₂ and FeCl ₃ . _EDTA, the reaction was strongly inhibited by CoCl _2, ZnSO _4, CuSO 4 and NiSO _4.

  FIG. 9 shows that the L-proline cis-hydroxylase of the present invention produces two types of products simultaneously. The L-proline cis-hydroxylase of the present invention preferentially hydroxylates L-proline from the 3rd position to the 4th position, and produces water about 3 times as much as cis-3-Hyp. It was shown to be an oxidase.

  From these results, since cis-3-Hyp and cis-4-Hyp are produced in the presence of L-proline cis-hydroxylase and L-proline of the present invention, the protein positions L-proline. It was confirmed to be a hydroxylase that preferentially and stereoselectively hydroxylates to produce cis-3-Hyp and cis-4-Hyp. The putative protein function disclosed in databases such as Entrez Protein was “2-oxoglutarate-dependent hydroxylase”. However, the results of this experiment confirmed that the functions of the protein were L-proline cis-3-hydroxylase and L-proline cis-4-hydroxylase. Further, in the reaction catalyzed by the L-proline cis-hydroxylase of the present invention, cis-3-Hyp and cis-4-Hyp are produced in the presence of 2-OG. -Hydroxylase was confirmed to be a 2-OG-dependent dioxygenase in which an oxygen atom is added between the 3- and 4-position carbon atoms of Pro and a hydrogen atom bonded thereto in the presence of 2-OG. .

  Table 5 shows the composition of the reaction solution of the resting cells of Escherichia coli, which is a transformant expressing L-proline cis-hydroxylase of the present invention.

  Even when the resting cell reaction solution shown in Table 5 was used, the L-proline cis-hydroxylase of the present invention exhibited L-proline cis-3-hydroxylase activity and L-proline cis-4-hydroxylase activity. (Not shown).

Claims (4)

Hydroxy-L, characterized in that L-proline is obtained by allowing L-proline to act on the enzyme protein of the following [1] or [2] or a host organism that expresses the enzyme protein. -Proline production method.
[1] an enzyme protein having the amino acid sequence represented by SEQ ID NO: 1 [2] consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and enzyme protein having L- proline cis-3-hydroxylase activity and L- proline cis-4-hydroxylase activity The host organism that expresses the enzyme protein according to [1] or [2] of claim 1 is a transformant in which the polynucleotide according to [ 3] below is incorporated into the host organism. Manufacturing method.
[3] polynucleotide encoding the enzyme protein according to [1] or [2] according to claim 1 A host organism that expresses the enzyme protein according to either [1] or [2] of claim 1 or a transformant in which the polynucleotide of [ 3] of claim 2 is incorporated into the host is cultured. A method for producing hydroxy-L-proline, wherein hydroxy-L-proline is obtained by a fermentation method.   The production method according to any one of claims 1 to 3, wherein the host organism is a prokaryote.

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