JP6996714B2 - Method for Producing N-Succinyl-Hydroxy-D-Amino Acid and / or Hydroxy-D-Amino Acid - Google Patents

Description

Techniques related to the production of N-succinyl-hydroxy-D-amino acids and / or hydroxy-D-amino acids are disclosed.

Hydroxy amino acids are useful compounds in functional foods, cosmetics, pesticides, raw materials for pharmaceuticals and their intermediates, for example, 4-hydroxy-L-isoleucine, 3-hydroxy-L-lysine, cis-3-. Hydroxy-L-proline, cis-4-hydroxy-L-proline and the like are known (Non-Patent Documents 1 and 2, Patent Document 1). All of these compounds have been reported to be efficient production methods by microbial and enzymatic reactions, and all of them are L-form hydroxyamino acids (Non-Patent Documents 3 and 4 and Patent Document 2). A method for producing a D-form hydroxyamino acid has been reported in Patent Document 3 and the like, but there is room for improvement in production efficiency and the like.

JP 2006-199714 WO2014 / 129459 JP-A-2010-098975

Broca et al. , Am. J. Physiol. , 1999, 277, E617 Coulon et al. , Tetrahedron Lett. , 1998, 39, 6467 Shibasaki et al. , Apple. Environ. Microbial. , 1999, 65, 4028 Hibi et al. , Apple. Environ. Microbial. , 2011, 77, 6926 Odokonyero et al. , PNAS. , 2014,111,8535 Sakai A. et al. , Biochemistry, 2006, 45, 4455

Under the above circumstances, it is one of the challenges to provide a new method for producing N-succinyl-hydroxy-D-amino acid and / or hydroxy-D-amino acid.

As a result of intensive research to achieve the above problems, the following findings were obtained. That is, it has been previously known that the N-succinyl amino acid racemase has an activity of catalyzing a dehydration reaction in addition to the isomerization reaction of the N-succinyl amino acid (Non-Patent Documents 5 and 6). Specifically, in Non-Patent Document 5, all enzymes having N-succinyl amino acid racemase activity (hereinafter, also referred to as NSAR activity) derived from the genera Amycolatopsis, Deinococcus, Thermus, Enterococcus, and Listeria have NSAR activity. It has been reported to have higher dehydration activity (hereinafter, also referred to as OSBS activity). Further, Non-Patent Document 6 reports that the N-succinyl amino acid racemase derived from the genus Geobacillus has an OSBS activity equivalent to that of NSAR. Therefore, the N-succinyl amino acid racemase was considered unsuitable for the isomerization reaction of hydroxylated compounds. However, in practice, it has been found that the enzyme can efficiently convert N-succinyl-hydroxy-L-amino acid into D-form in a hydroxylated state. As a result of further research and improvement based on such findings, inventions represented by the following are provided.

Item 1.
A method for producing an N-succinyl-hydroxy-D-amino acid, which comprises the step (A) of allowing the N-succinyl amino acid racemase to act on the N-succinyl-hydroxy-L-amino acid. Item 2.
Item 2. The method according to Item 1, wherein the N-succinyl-hydroxy-D-amino acid is an N-succinyl-3-hydroxy-D-amino acid and / or an N-succinyl-4-hydroxy-D-amino acid.
Item 3.
A step (A) in which the N-succinyl amino acid racemase is allowed to act on the N-succinyl-hydroxy-L-amino acid, and a step (A) in which the N-succinyl-hydroxy-D-amino acid obtained in the step (A) is allowed to act on the D-succinylase. B)
A method for producing a hydroxy-D-amino acid, which comprises.
Item 4.
Item 3. The method according to Item 3, wherein the N-succinyl-hydroxy-D-amino acid is an N-succinyl-3-hydroxy-D-amino acid and / or an N-succinyl-4-hydroxy-D-amino acid.
Item 5.
Item 6. The method according to any one of Items 1 to 4, wherein the N-succinyl amino acid racemase is derived from a microorganism belonging to the genus Geobacillus or the genus Chloroflexota.
Item 6. Item 6. The method according to any one of Items 3 to 5, wherein the D-succinylase is derived from a microorganism belonging to the genus Cupriavidus or the genus Ralstonia.

N-succinyl-hydroxy-D-amino acids and / or hydroxy-D-amino acids can be efficiently produced.

It is shown that 3-hydroxy-D-isoleucine was produced in Example 2. It is shown that 3-hydroxy-D-leucine was produced in Example 3. It is shown that 3-hydroxy-D-valine was produced in Example 4. It is shown that 3-hydroxy-D-norleucine was produced in Example 5. It is shown that 3-hydroxy-D-norvaline was produced in Example 6. It is shown that 4-hydroxy-D-isoleucine was produced in Example 7.

Provided is a method for producing an N-succinyl-hydroxy-D-amino acid, which comprises the step (A) of allowing the N-succinyl amino acid racemase to act on the N-succinyl-hydroxy-L-amino acid.

The type of L-amino acid in N-succinyl-hydroxy-L-amino acid is arbitrary and can be appropriately selected depending on the intended purpose. According to the present invention, in principle, a hydroxy-D-amino acid containing a D-amino acid having the same side chain as the L-amino acid used for the N-succinyl-hydroxy-L-amino acid can be obtained.

Amino acids are compounds that have both amino and carboxyl groups. Specific amino acid types include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, treonine, tryptophan, tyrosine, and valine. Amino acids synthesized directly from genetic information such as; amino acids obtained by post-synthesis modification of proteins such as cystine, hydroxyproline, hydroxylysine, syloxin, O-phosphoserine, and desmosin; and β-alanine, sarcosine, ornithine, Examples thereof include amino acids that do not constitute proteins such as citrulin, creatine, γ-aminobutyric acid, opain, trimethylglycine, theanine, trichomic acid, cinic acid, doumoy acid, ibotenic acid, acromelic acid, norvaline, and norleucine. In one embodiment, preferred amino acids are isoleucine, leucine, norleucine, valine, norvaline, and phenylalanine.

In the N-succinyl-hydroxy-L-amino acid, the hydroxy group may be bonded to any carbon atom of the L-amino acid. In one embodiment, the hydroxy group has the carboxyl carbon next to the carbon atom having the amino group of the amino acid at the 1-position, the carbon to which the amino group and the carboxyl group are bonded at the 2-position, the 3-position, the 4-position, and /. Or, it is preferably bonded to the carbon at the 5-position, and preferably bonded to the 3-position and / or the 4-position.

The N-succinyl-hydroxy-L-amino acid is known and can be obtained by any method. For example, the N-succinyl-hydroxy-L-amino acid can be obtained by hydroxylating the N-succinyl-L-amino acid with hydroxylase. The N-succinyl-L-amino acid is prepared by, for example, using succinic anhydride to bind a succinyl group to a nitrogen atom constituting the amino group of the L-amino acid under the conditions of pH 8 to 9, as shown in Examples described later. Can be obtained. The N-succinyl-hydroxy-L-amino acid can also be obtained by first hydroxylating the L-amino acid with hydroxylase to obtain the hydroxy-L-amino acid and then N-succinylating it.

Hydroxylase is a protein having an enzymatic activity (hereinafter referred to as “hydroxylase activity”) that hydroxylates any atom of a substrate molecule. The hydroxylase includes monooxygenase, which catalyzes the reaction of introducing one oxygen atom into a compound using an oxygen molecule as a substrate, and dioxygenase, which catalyzes the reaction of introducing both two oxygen atoms in an oxygen molecule into a compound.

Hydroxide is preferably an enzyme that catalyzes a reaction that regioselectively hydroxylates L-amino acids or N-succinyl-L-amino acids. Such enzymes include, for example, the genus Eschericia, the genus Pantoea, the genus Agrobacterium, the genus Borderella, the genus Streptomyces, the genus Photorbdus, the genus Gloeobacter, the genus Psychromonas, the genus Mesorhizobiruba. , Pseudomonas, hydroxylases from the genus Nostoc, and variants thereof. In one embodiment, the hydroxylase is preferably derived from the genus Burkholderia, Bacillus, or Nostoc, more preferably from the genus Burkholderiales.

As used herein, the term "variant" means an enzyme having an amino acid sequence having a certain degree of identity with respect to the amino acid sequence of the reference enzyme and having the same enzyme activity as the reference enzyme. Here, the identity of a certain level or more means, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more. The reference enzyme is arbitrary, and examples thereof include wild-type enzymes exemplified in this document and enzymes having the amino acid sequence of any one of SEQ ID NOs: 1 to 3.

Amino acid identity can be calculated using analytical tools available on the market or through the internet. For example, it can be calculated using the default parameters in the National Center for Biotechnology Information (NCBI) homology algorithm BLAST.

In the production of N-succinyl-hydroxy-L-amino acid, (1) hydroxylase that catalyzes a reaction in which N-succinyl-L-amino acid is used as a starting material and N-succinyl-L-amino acid is repositionably hydroxylated. (2) Using L-amino acid as a starting material, hydroxy-L-amino acid is synthesized and chemically synthesized using a hydroxylase that catalyzes the reaction of position-selectively hydroxylating L-amino acid. N-Succinyl-hydroxy-L-amino acid may be produced in. An example of (1) is a hydroxylase of the genus Burkholderiales (above, described in Patent Document 4) that specifically hydroxylates the 3-position of the N-succinyl-L-amino acid. As an example of (2), examples of the enzyme that hydroxylates the 4-position include, for example, Bacillus thuringiensis, Bacillus licheniformis, Bacillus sphereiformis, and hydroxylase derived from Bacillus sphaeriocus. (Described in Patent Document 4). Examples of the enzyme that hydroxylates the 5-position include hydroxylase derived from Nostoc punctiforme (described in WO2011 / 021717).

The conditions for hydroxylating the L-amino acid constituting the L-amino acid or the N-succinyl-L-amino acid with hydroxylase are not particularly limited and can be arbitrarily designed. For example, the above amino acid can be hydroxylated by mixing L-amino acid or N-succinyl-L-amino acid and hydroxylase in a reaction solution containing an arbitrary additive and a buffer under the following conditions.

The reaction temperature is not particularly limited as long as it is a temperature at which the hydroxylase used can function, and can be appropriately set according to the type of enzyme. The general temperature is 20 to 50 ° C, preferably 25 to 35 ° C. The pH at the time of reaction is not particularly limited as long as it is a pH at which the hydroxylase used functions, and can be appropriately set according to the type of enzyme. The general pH is pH 5-9, preferably pH 6-7.

The reaction solution preferably contains divalent iron ions, ascorbic acid, and / or 2-oxoglutaric acid as additives. The divalent iron ion is preferably contained, for example, at a final concentration of 0.1 mM to 1 M. Ascorbic acid is preferably contained, for example, at a final concentration of 0.1 mM to 1 M, preferably 1 mM to 10 mM. Ascorbic acid can also be replaced with tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl). The 2-oxoglutaric acid is preferably blended in the same molar amount or in the range of 2 times the molar amount with the reaction substrate N-succinyl-L-amino acid (or L-amino acid). This can significantly increase the activity of hydroxylase.

The buffer used for the reaction of hydroxylase is not particularly limited as long as the hydroxylase functions, and can be arbitrarily selected. In one embodiment, from the viewpoint of work efficiency, the buffer material is preferably the same as the buffer material used for the reaction of N-acylamino acid racemase and D-succinylase described later.

The reaction time for hydroxylation is not particularly limited as long as the desired hydroxyl L-amino acid or N-succinyl-hydroxy-L-amino acid can be obtained, and can be set arbitrarily. For example, the reaction time can be 3 to 72 hours, preferably 6 to 24 hours.

The amount of L-amino acid or N-succinyl-L-amino acid in the reaction solution is not particularly limited and can be appropriately set according to the purpose and the type of enzyme used. For example, the L-amino acid or N-succinyl-L-amino acid can be 0.01% to 50% (w / v), preferably 0.1% to 20% (w / v).

The amount of hydroxylase in the reaction solution is not particularly limited, and can be appropriately set according to the purpose, the type of enzyme used, and the like. For example, the concentration of hydroxylase is 0.1% to 50% (w / v), preferably 1% to 10% (w / v) as the wet cell weight.

A purified hydroxylase can be used, but when hydroxylase is produced by expressing it in a microorganism, it can be used in the state of the microorganism expressing it. In one embodiment, the hydroxylase is preferably in a state in which microorganisms are expressed from the viewpoint of stability. Therefore, the culture solution in which the microorganism is cultured for expressing hydroxylase can be used as it is as an enzyme solution.

The production of hydroxylase using a microorganism can be carried out by using a well-known method. For example, it can be carried out by transforming an appropriate host cell (for example, Escherichia coli) with an expression vector incorporating a DNA encoding hydroxylase and culturing it in the host under appropriate conditions.

Succinylation of L-amino acids can be performed by any method. For example, the N-succinyl-L-amino acid is obtained by reacting an amino acid and an equivalent amount or more of succinic anhydride in acetic acid, distilling off the solvent, and recrystallizing in ethyl acetate and / or methanol. Can be done. Further, N-succinyl-L-amino acid can also be obtained by reacting an amino acid and an equivalent amount or more of succinic anhydride in water while keeping the pH on the alkaline side, and then acidifying the pH to precipitate as crystals. ..

The N-succinyl amino acid racemase used in racemicizing the N-succinyl-hydroxy-L-amino acid to produce the N-succinyl-hydroxy-D-amino acid is not particularly limited as long as it has an activity capable of catalyzing such a reaction. .. The N-succinyl amino acid racemase that can be used is derived from Amycolatopsis sp., TS-1-60 (Japanese Patent Laid-Open No. 4-365482), Sebekia Bbenihana (hereinafter, simply referred to as Benihana). (Japanese Patent Laid-Open No. 2001-314191), Deinococcus radiodurans (Japanese Patent Laid-Open No. 2002-238581), Chloroflexus aurantiacus (Japanese Patent Laid-Open No. 2007-082534), Geobacillus. Geobacillus stearothermophilus derived from NCA1503 strain (Japanese Patent Laid-Open No. 2008-016942), Geobacillus kaustophilus derived from NBRC102445 strain (Japanese Patent Laid-Open No. 2015-91265), Thermus thermophilus derived from thermus thermophilus -91265) N-succinyl amino acid racemase and variants thereof and the like can be mentioned. In one embodiment, it is derived from Chloroflexus aurantiacus, Geobacillus stearomophilus NCA1503 strain, Geobacillus Kaustophilus (Geobacillus) from Geobacillus aurantiacus, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain, Geobacillus strain. ) N-succinyl amino acid racemase derived from the HB8 strain is preferable, and N-succinyl amino acid racemase derived from the genus Geobacillus or Chloroflexus is more preferable.

The reaction conditions for producing the N-succinyl-hydroxy-D-amino acid by allowing the N-succinyl amino acid racemase to act on the N-succinyl-hydroxy-L-amino acid are not particularly limited and can be arbitrarily designed. For example, racemization can be achieved by mixing N-succinyl-hydroxy-L-amino acid and N-succinyl amino acid racemase in a reaction solution containing any additive and buffer under the following conditions.

The reaction temperature is not particularly limited as long as it is a temperature at which the N-succinyl amino acid racemase to be used functions, and can be appropriately set according to the type of enzyme. The general temperature is 20 to 50 ° C, preferably 25 to 35 ° C. The pH at the time of reaction is not particularly limited as long as the pH at which the N-succinyl amino acid racemase used is functional, and can be appropriately set according to the type of enzyme. The general pH is pH 5-9, preferably pH 6-7.

The reaction solution preferably contains divalent cobalt ion, manganese ion, nickel ion, magnesium ion, zinc ion, iron ion, monovalent potassium ion, sodium ion, lithium ion and the like as additives, and cobalt ion is more preferable. preferable. The metal ion is preferably contained, for example, at a final concentration of 0.01 mM to 1 M, more preferably 0.1 mM to 1 mM.

The buffer used for the reaction of the N-succinyl amino acid racemase is not particularly limited as long as the N-succinyl amino acid racemase functions, and can be arbitrarily selected. For example, phosphate buffer solution, MES, ADA, PIPES, MOPS, HEPES, CHES, Tris-HCl, Bis-Tris-HCl, Tricine and the like can be mentioned.

The reaction time is not particularly limited as long as the desired N-succinyl-hydroxy-D-amino acid can be obtained, and can be set arbitrarily. For example, the reaction time can be 3 to 72 hours, preferably 6 to 24 hours.

The amount of N-succinyl-hydroxy-L-amino acid in the reaction solution is not particularly limited and can be appropriately set according to the purpose and the type of enzyme used. For example, the N-succinyl-hydroxy-L-amino acid can be 0.01% to 50% (w / v), preferably 0.1% to 20% (w / v).

The amount of the N-succinyl amino acid racemase in the reaction solution is not particularly limited, and can be appropriately set according to the purpose, the type of enzyme used, and the like. For example, the concentration of hydroxylase is 0.001 (g / L) to 10 (g / L), preferably 0.01 (g / L) to 1 (g / L) as the enzyme concentration.

A purified N-succinyl amino acid racemase can be used, but when the N-succinyl amino acid racemase is produced by expressing it in a microorganism, it can be used in the state of a microorganism expressing the enzyme. In one embodiment, the N-succinyl amino acid racemase is preferably in a state in which microorganisms are expressed from the viewpoint of stability. Therefore, in order to express the N-succinyl amino acid racemase, the culture solution in which the microorganism is cultured can be used as it is as an enzyme solution.

The production of the N-succinyl amino acid racemase using a microorganism can be carried out by using a well-known method. For example, it can be carried out by transforming an appropriate host cell (for example, Escherichia coli) with an expression vector incorporating DNA for N-succinyl amino acid racemase and culturing it in the host under appropriate conditions.

The hydroxy-D-amino acid can be obtained by allowing D-succinylase to act on the N-succinyl-hydroxy-D-amino acid obtained as described above (step B). The D-succinylase is not particularly limited as long as it has an activity of desuccinylating an N-succinyl-hydroxy-D-amino acid to produce a hydroxy-D-amino acid, and the type thereof is arbitrary. Specific examples of D-succinylase include Cupriavidus sp. P4-10-C strain, Cupriavidus metallicidurans, Cupriavidus necator, and Ralstonia pickettii (Ralstonia pickettii) derived from D-succinilas Japanese Patent Application Laid-Open No. 2012-205587) and the like can be mentioned. The preferred D-succinylase in one embodiment is a D-succinylase from the genus Cupriavidus or Ralstonia.

The reaction conditions for producing the hydroxy-D-amino acid by allowing D-succinylase to act on the N-succinyl-hydroxy-D-amino acid are not particularly limited and can be arbitrarily designed. For example, desuccinylization can be achieved by mixing N-succinyl-hydroxy-D-amino acid and D-succinylase in a reaction solution containing an arbitrary additive and a buffer under the following conditions.

The reaction temperature is not particularly limited as long as it is a temperature at which the D-succinylase to be used functions, and can be appropriately set according to the type of enzyme. The general temperature is 20 to 50 ° C, preferably 25 to 35 ° C. The pH at the time of reaction is not particularly limited as long as it is a pH at which the D-succinylase used functions, and can be appropriately set according to the type of enzyme. The general pH is pH 5-9, preferably pH 6-7.

The buffer used for the reaction of D-succinylase is not particularly limited as long as D-succinylase functions, and can be arbitrarily selected. For example, phosphate buffer solution, MES, ADA, PIPES, MOPS, HEPES, CHES, Tris-HCl, Bis-Tris-HCl, Tricine and the like can be mentioned.

The reaction time is not particularly limited as long as the desired hydroxy-D-amino acid can be obtained, and can be set arbitrarily. For example, the reaction time can be 3 to 72 hours, preferably 6 to 24 hours.

The amount of N-succinyl-hydroxy-D-amino acid in the reaction solution is not particularly limited and can be appropriately set according to the purpose and the type of enzyme used. For example, the N-succinyl-hydroxy-D-amino acid can be 0.01% to 50% (w / v), preferably 0.1% to 20% (w / v).

The amount of D-succinylase in the reaction solution is not particularly limited, and can be appropriately set according to the purpose, the type of enzyme used, and the like. For example, the concentration of D-succinylase is 0.0001 (g / L) to 1 (g / L), preferably 0.001 (g / L) to 0.1 (g / L) as the enzyme concentration.

Purified D-succinylase can be used, but when D-succinylase is produced by expressing it in a microorganism, it can be used in the state of a microorganism expressing the enzyme. In one embodiment, the D-succinylase is preferably in a state in which microorganisms are expressed from the viewpoint of stability. Therefore, the culture solution in which the microorganism is cultured for expressing D-succinylase can be used as it is as an enzyme solution.

The production of D-succinylase using a microorganism can be carried out by using a well-known method. For example, it can be carried out by transforming an appropriate host cell (for example, Escherichia coli) with an expression vector incorporating a DNA encoding D-succinylase and culturing it in the host under appropriate conditions.

In the hydroxy-D-amino acid thus obtained, the hydroxy group may be bonded to any carbon atom of the D-amino acid. In one embodiment, the hydroxy group is at the 3-position, 4-position, and / or with the carbon constituting the carboxyl group of the D-amino acid at the 1-position and the carbon to which the amino group and the carboxyl group are bonded at the 2-position. It is preferably bonded to the carbon at the 5-position, preferably to the 3-position and / or the 4-position.

In one embodiment, conversion of N-succinyl-hydroxy-L-amino acid to N-succinyl-hydroxy-D-amino acid and from N-succinyl-hydroxy-D-amino acid to hydroxyl in one reaction system (reaction solution). It is preferable from the viewpoint of efficiency that the conversion to -D-amino acid is performed simultaneously and / or continuously. In another embodiment, the N-succinyl-L-amino acid is converted to the N-succinyl-hydroxy-L-amino acid in the presence of hydroxylase, and the N-succinyl amino acids racemase and D-succinylase and other additions are added to the reaction solution. It is preferable from the viewpoint of efficiency to continuously produce hydroxy-D-amino acids by adding an agent.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[Reference Example 1] Preparation of Hydroxylase (SadA) Derived from Burkholderia ambifaria AMMD (DSM16078) Strain SEQ ID NO: 4 to amplify the total length of the hydroxylase (SadA) gene (SEQ ID NO: 1) derived from Burkholderia ambifaria AMMD (DSM16078) Strain. And a primer having the base sequence shown in 5 was synthesized. Next, the Burkholderia ambifaria AMMD (DSM16078) strain was cultured in a liquid culture, and genomic DNA was extracted from the obtained cells using a genomic extraction kit. PCR was performed using genomic DNA as a template and DNA polymerase KOD-Plus (manufactured by Toyobo) and the above primers. The amplified DNA fragment was inserted between the BamHI recognition site and the HindIII recognition site of the plasmid pQE80L to construct the expression vector pBaSadA. E. coli JM109 was transformed with this expression vector pBaSadA. The obtained recombinant Escherichia coli was cultured with shaking in a liquid LB medium containing 100 mg / L of ampicillin at 28 ° C., and when the OD (660 nm) value reached 0.8, IPTG was mediumd so that the final concentration was 1 mM. Was added to. After that, the cells were cultured with shaking at 28 ° C., and the cells were collected 20 hours after the start of the culture.

[Reference Example 2] Measurement of activity of hydroxylase (SadA) derived from Burkholderia ambifaria AMMD (DSM16078) strain 50 μl (final concentration 50 mM) Bis-Tris / HCl buffer (pH 6.5), 15 μl (final concentration 15 mM) 2-oxoglutal Mix 10 μl (final concentration 10 mM) ascorbic acid, 50 μl (final concentration 5 mM) FeSO ₄ / _7H2O , 100 μl (final concentration 10 mM) N-succinyl-L-leucine, and 765 μl sterile distilled water. To this solution, 10 μl of a bacterial cell suspension prepared by washing the bacterial collection solution prepared by the method described in Reference Example 1 with physiological saline was added, the reaction was started at 30 ° C., and the reaction was stopped at an appropriate time. .. The succinic acid produced by this reaction was quantified with a succinic acid quantification kit (manufactured by Roche / R-Biopharm), and the enzyme activity was calculated. Enzyme activity was defined as 1 unit (U) when 1 μmol of succinic acid was produced from 2-oxoglutaric acid in 1 minute.

[Reference Example 3] Preparation of Hydroxylase (IDO) Derived from Bacillus thuringiensis 2-e-2 Strain Amplifies the total length of hydroxylase (IDO) (SEQ ID NO: 2) derived from Bacillus thuringiensis 2-e-2 strain described in Patent 5246639. Primers having the nucleotide sequences of SEQ ID NOs: 6 and 7 were synthesized for this purpose. Next, the Bacillus thuringiensis 2-e-2 strain was cultured in a liquid culture, and genomic DNA was extracted from the obtained cells using a genomic extraction kit. PCR was performed using the genomic DNA as a template with the DNA polymerase KOD-Plus (manufactured by Toyobo) and the above primers. The amplified DNA fragment was inserted between the BamHI recognition site and the HindIII recognition site of the plasmid pQE80L to construct the expression vector pBtIDO. E. coli JM109 was transformed with this expression vector pBtIDO. The obtained recombinant Escherichia coli was cultured with shaking in a liquid LB medium containing 100 mg / L of ampicillin at 28 ° C., and IPTG was added so that the final concentration was 1 mM when the OD (660 nm) value reached 0.8. did. After that, the cells were cultured with shaking at 28 ° C., and the cells were collected 20 hours after the start of the culture.

[Reference Example 4] Measurement of activity of hydroxylase (IDO) derived from Bacillus distilled water 2-e-2 strain 100 μl (final concentration 100 mM) HEEPS buffer (pH 7.0), 5 μl (final concentration 5 mM) 2-oxoglutaric acid, 5 μl (Final concentration 5 mM) ascorbic acid, 50 μl (final concentration 5 mM) FeSO ₄ / _7H2O , 50 μl (final concentration 5 mM) L-isoleucine, and 780 μl sterile distilled water were mixed, and this solution was mixed with Reference Example 3. The reaction was started at 30 ° C. and stopped at an appropriate time by adding 10 μl of the bacterial cell suspension prepared by washing the bacterial collection solution prepared by the method described in the above with physiological saline. 4-Hydroxyisoleucine produced by this reaction was quantified by HPLC, and the enzyme activity was calculated. Enzyme activity was defined as 1 unit (U) when 1 μmol of succinic acid was produced from 2-oxoglutaric acid in 1 minute.

[Reference Example 5] Preparation of N-succinyl amino acid racemase (NSAR) An expression vector pCaNSAR of NSAR derived from Chloroflexota aurantiacus was constructed by the method described in JP-A-2007-82534. E. coli JM109 was transformed with pCaNSAR. The obtained recombinant Escherichia coli was cultured with shaking at 28 ° C. using a liquid LB medium containing 100 mg / L of ampicillin so that the final concentration was 1 mM when the OD (660 nm) value reached 0.8. IPTG was added. After that, the cells were cultured with shaking at 28 ° C., and the cells were collected 20 hours after the start of the culture.

[Reference Example 6] Preparation of N-succinyl amino acid racemase (NSAR) An expression vector pGsNSAR of NSAR derived from Geobacillus stearothermophilus was constructed by the method described in JP-A-2008-061642. E. coli JM109 was transformed with pCaNSAR. The obtained recombinant Escherichia coli was cultured with shaking at 28 ° C. using a liquid LB medium containing 100 mg / L of ampicillin so that the final concentration was 1 mM when the OD (660 nm) value reached 0.8. IPTG was added. After that, the cells were cultured with shaking at 28 ° C., and the cells were collected 20 hours after the start of the culture.

[Reference Example 7] Measurement of activity of N-succinyl amino acid racemase 100 μl (final concentration 10 mM), 10 μl (final concentration 1 mM) chloride of N-succinyl-L-phenylalanine solution (substrate) as a substrate of 100 μl (final concentration 10 mM). Mix 10 μl (final concentration 1 mM) of cobalt aqueous solution, 100 μl (final concentration 25 mM) of 100 μl (final concentration 25 mM) Tris-HCl (0.25 M / pH 7.5), and 740 μl of 740 μl sterile distilled water. 50 μl of N-succinyl amino acid racemase solution having an appropriate concentration was added to the reaction solution, and the reaction was carried out at 30 ° C., and the reaction was stopped by boiling treatment. The produced N-succinyl-D-phenylalanine was quantified by high performance liquid chromatography, and the enzyme activity was calculated. Enzyme activity was defined as 1 unit (U) when N-succinyl-D-phenylalanine was produced from N-succinyl-L-phenylalanine in an amount of 1 μmole per minute.

[Reference Example 8] Preparation of D-succinylase (DSA) Cupriavidus sp. Primers having the nucleotide sequences of SEQ ID NOs: 8 and 9 were synthesized in order to amplify the total length of the D-succinylase gene (SEQ ID NO: 3) derived from the P4-10-C strain. Next, Cupriavidus sp. The P4-10-C strain was cultured in a liquid medium, and PCR was performed using the extracted genomic DNA as a template using the DNA polymerase KOD-Plus (manufactured by Toyobo) and the above-mentioned primers by the method described in Patent 5806664. The amplified DNA fragment was inserted between the NdeI recognition site and the BamHI recognition site of the plasmid pBluescript to construct the expression vector pCsDSA. Further, according to a conventional method, a site-specific mutation was introduced to obtain pCsDSA (L182E) in which the leucine residue at position 182 was replaced with a glutamic acid residue. E. coli JM109 was transformed with pCsDSA (L182E). The obtained recombinant Escherichia coli was cultured in a liquid LB medium containing 100 mg / L of ampicillin, and IPTG was added so that the final concentration was 1 mM when the OD (660 nm) value reached 0.8. After that, the cells were cultured with shaking at 28 ° C., and the cells were collected 20 hours after the start of the culture. The cells were collected by centrifugation.

[Reference Example 9] Measurement of D-succinylase activity Measurement was performed by an enzymatic method using the D-amino acid oxidase method.
<Reagent>
14 mM N-succinyl-D-valine 2.5 (U / mL) Peroxidase (Toyobo PEO-302)
1.5 mM 4-aminoantipyrine (manufactured by Daiichi Chemicals)
2.4 mM TOOS (manufactured by Doujin Kagaku Kenkyusho)
6.0 (U / mL) D-amino acid oxidase (DOX2 manufactured by Biozyme)
A 25 mM phosphate buffer solution containing the above is used as a reaction reagent.
<Measurement conditions>
Preheat 3.0 mL of the reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 mL of the enzyme solution and mixing gently, the absorbance change at 555 nm was recorded for 5 minutes with a spectrophotometer controlled at 37 ° C. using water as a control, and the absorbance change per minute (ΔODTEST) from the linear portion. To measure. In blinding, a solution that dissolves the enzyme instead of the enzyme solution is added to the reagent mixture, and the change in absorbance (ΔODBLANK) per minute is similarly measured. From these values, the D-succinylase activity is determined according to the following formula. Here, 1 unit (U) in D-succinylase activity is defined as the amount of enzyme that produces 1 micromol of D-amino acid per minute under the above conditions.
Activity (U / mL) =
{(ΔODTEST-ΔODBLANK) × 3.1 × dilution ratio} / {31.0 × 1/2 × 0.1 × 1.0}
In the formula, 3.1 is the amount of reaction reagent + enzyme solution (mL), 31.0 is the hydroxyl molecule absorption coefficient (cm2 / micromol) under the present activity measurement conditions, and 1/2 is generated by the enzyme reaction. The coefficient due to the fact that the Quinoneimine dye formed from one molecule of H2O2 is 1/2 molecule, 0.1 indicates the amount of the enzyme solution (mL), and 1.0 indicates the optical path length (cm) of the cell.

[Example 1] Production of (2R, 3R, 4S) -4-hydroxyisoleucine With DSA-expressing cells (100 ml of culture solution) and Reference Example 5 obtained in the same manner as in Reference Example 8. 10 ml of reaction solution (25 mM HEPES buffer (pH 7.0), 1 mM CoCl2, 15 mM N-succinyl- (2S, 3R, 4S)) containing cells expressing NSAR derived from Chloroflexus aurantiacus (100 ml of culture solution) obtained in the same manner. ) -4-Hydroxyisoleucine) and reacted at 28 ° C or 37 ° C for 20 hours. As for NSAR, instead of NSAR derived from Chloroflexota aurantiacus described in Reference Example 5, a cell (100 ml of culture solution) expressing NSAR derived from Geobacillus stearomophilus obtained in the same manner as in Reference Example 6 was used. Evaluation was also carried out.

To 10 μl of the obtained reaction solution, add 20 μl of 6% triethylamine and 20 μl of 1% 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC). After allowing to stand at room temperature for 10 minutes, chiral derivatization of amino acids was performed by adding 20 μl of 5% acetic acid. By separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions, amino acid reaction products ((2R, 3R, 4S) -4-hydroxyisoleucine (4-hydroxy-D-isoleucine)) and By-products ((2S, 3R, 4S) -4-hydroxyisoleucine (4-hydroxy-L-isoleucine)) were measured.
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: UV absorption (254 nm)

As a result, the reaction product ((2R, 3R, 4S) -4-hydroxyisoleucine (4-hydroxy-D-isoleucine)) could be confirmed regardless of which NSAR was used (Table 1). ). Since the reaction efficiency was higher when NSAR (CaNSAR) derived from Chloroflexus aurantiacus was used than when NSAR (GsNSA) derived from Geobacillus stearomophilus was used, Chloroflexota-derived NSAR (GsNSA) was used in the following examples. used. When any NSAR was used, the reaction efficiency was higher when the reaction temperature was 37 ° C than when it was 28 ° C.

[Example 2] Production of (2R, 3R) -3-hydroxyisoleucine L-isoleucine (6.5 g) was dissolved in 100 ml of water, and the pH was adjusted to 8 to 9 with a sodium hydroxide solution. 5 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, freeze-drying gave 13 g of a disodium salt of N-succinyl-L-isoleucine. Next, 10 ml of the reaction solution (50 mM Bis-Tris / HCl buffer (pH 6.5), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid) was added to the cells expressing SadA (100 ml of the culture solution) obtained in the same manner as in Reference Example 1. Acid, 0.5 mM FeSO ₄ / _7H2 O, 30 mM N-succinyl-L-isoleucine) was suspended and reacted at 30 ° C. for 20 hours. Subsequently, CoCl ₂ was added to the reaction solution so as to have a final concentration of 1 mM, and the NSAR-expressing cells (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the same as in Reference Example 8 were obtained. The cells expressing the DSA (L182E) (100 ml of the culture solution) were suspended and reacted at 30 ° C. for 20 hours.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. Amino acid reaction products ((2R, 3R) -3-hydroxyisoleucine (3-hydroxy-D-isoleucine)) were measured by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions. ..
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode)

As shown in FIG. 1, since a new peak showing a molecular weight of 318 was detected on HPLC, the formation of (2R, 3R) -3-hydroxyisoleucine was confirmed. The peak with a molecular weight of 300, which is considered to be a dehydrated product, is a by-product generated during the hydroxylation reaction with SadA, and no increase in the dehydrated product was observed in the N-succinyl amino acid racemase and D-succinylase reactions.

[Example 3] Production of (2R, 3R) -3-hydroxyleucine L-leucine (6.5 g) was dissolved in 100 ml of water, and the pH was adjusted to 8 to 9 with a sodium hydroxide solution. 5 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, freeze-drying was performed to obtain 13 g of N-succinyl-L-leucine. Then
Bacteria expressing SadA (100 ml of culture solution) obtained in the same manner as in Reference Example 1 were mixed with 10 ml of a reaction solution (50 mM Bis-Tris / HCl buffer (pH 6.5), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid, It was suspended in 0.5 mM FeSO ₄ / _7H2 O, 30 mM N-succinyl-L-leucine) and reacted at 30 ° C. for 20 hours. Subsequently, CoCl ₂ was added to the reaction solution so as to have a final concentration of 1 mM, and the NSAR-expressing cells (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the same as in Reference Example 8 were obtained. The cells expressing the DSA (L182E) (100 ml of the culture solution) were suspended and reacted at 30 ° C. for 20 hours.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. Amino acid reaction products ((2R, 3R) -3-hydroxyleucine (3-hydroxy-D-leucine)) were measured by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions. ..
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode)

As shown in FIG. 2, since a new peak showing a molecular weight of 318 was detected on HPLC, the formation of (2R, 3R) -3-hydroxyleucine was confirmed. On the other hand, no peak formation with a molecular weight of 300, which is considered to be a dehydrated product, was observed.

[Example 4] Production of (2R) -3-hydroxyvaline L-valine (5.8 g) was dissolved in 100 ml of water, and the pH was adjusted to 8 to 9 with a sodium hydroxide solution. 5 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, lyophilization was performed to obtain 12 g of N-succinyl-L-valine. Next, 10 ml of the reaction solution (50 mM Bis-Tris / HCl buffer (pH 6.5), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid) was added to the cells expressing SadA (100 ml of the culture solution) obtained in the same manner as in Reference Example 1. The cells were suspended in acid (0.5 mM FeSO ₄ / _7H2 O, 30 mM N-succinyl-L-valine) and reacted at 30 ° C. for 20 hours. Subsequently, CoCl ₂ was added to the reaction solution so as to have a final concentration of 1 mM, and the NSAR-expressing cells (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the same as in Reference Example 8 were obtained. The cells expressing the DSA (L182E) (100 ml of the culture solution) were suspended and reacted at 30 ° C. for 20 hours.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. The amino acid reaction product ((2R) -3-hydroxyvaline (3-hydroxy-D-valine)) was measured by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions.
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode)

As shown in FIG. 3, the formation of (2R) -3-hydroxyvaline was confirmed by detecting a new peak showing a molecular weight of 304 on HPLC. The peak of molecular weight 286 considered to be dehydrated product is a by-product generated during the hydroxylation reaction by SadA, and no increase in dehydrated product was observed in the N-succinyl amino acid racemase and D-succinylase reactions.

[Example 5] Production of (2R, 3R) -3-hydroxynorleucine L-norleucine (6.5 g) was dissolved in 100 ml of water, and the pH was adjusted to 8 to 9 with a sodium hydroxide solution. 5 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, freeze-drying was performed to obtain 13 g of N-succinyl-L-norleucine. Next, 10 ml of the reaction solution (50 mM Bis-Tris / HCl buffer (pH 6.5), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid) was added to the cells expressing SadA (100 ml of the culture solution) obtained in the same manner as in Reference Example 1. Acid, 0.5 mM FeSO ₄ / _7H2 O, 30 mM N-succinyl-L-norleucine) was suspended and reacted at 30 ° C. for 20 hours. Subsequently, CoCl ₂ was added to the reaction solution so as to have a final concentration of 1 mM, and the NSAR-expressing cells (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the same as in Reference Example 8 were obtained. The cells expressing the DSA (L182E) (100 ml of the culture solution) were suspended and reacted at 30 ° C. for 20 hours.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. Amino acid reaction products ((2R, 3R) -3-hydroxynorleucine (3-hydroxy-D-norleucine)) were measured by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions. did.
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode)

As shown in FIG. 4, a new peak showing a molecular weight of 318 was detected on HPLC, confirming the production of (2R, 3R) -3-hydroxynorleucine. On the other hand, no peak formation with a molecular weight of 300, which is considered to be a dehydrated product, was observed.

[Example 6] Production of (2R, 3R) -3-hydroxynorvaline L-norvaline (5.8 g) was dissolved in 100 ml of water, and the pH was adjusted to 8 to 9 with a sodium hydroxide solution. 5 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, freeze-drying was performed to obtain 12 g of N-succinyl-L-norvaline. Next, 10 ml of the reaction solution (50 mM Bis-Tris / HCl buffer (pH 6.5), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid) was added to the cells expressing SadA (100 ml of the culture solution) obtained in the same manner as in Reference Example 1. The cells were suspended in acid (0.5 mM FeSO ₄ / _7H2 O, 30 mM N-succinyl-L-norvaline) and reacted at 30 ° C. for 20 hours. Subsequently, CoCl ₂ was added to the reaction solution so as to have a final concentration of 1 mM, and the NSAR-expressing cells (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the same as in Reference Example 8 were obtained. The cells expressing the DSA (L182E) (100 ml of the culture solution) were suspended and reacted at 30 ° C. for 20 hours.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. Amino acid reaction products ((2R, 3R) -3-hydroxynorvaline (3-hydroxy-D-norvaline)) were measured by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions. did.
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode)

As shown in FIG. 5, a new peak showing a molecular weight of 304 was detected on HPLC, confirming the formation of (2R, 3R) -3-hydroxynorvaline. On the other hand, no peak formation with a molecular weight of 286, which is considered to be a dehydrated product, was observed.

[Example 7] Production of (2R, 3R, 4S) -4-hydroxyisoleucine 10 ml (100 ml of culture solution) of cells expressing IDO obtained in the same manner as in Reference Example 3 (100 ml of culture solution) was added. Suspended in 50 mM Bis-Tris / HCl buffer (pH 6.0), 45 mM 2-oxoglutaric acid, 10 mM ascorbic acid, 0.5 mM FeSO ₄ / _7H2 O, 30 mM L-isoleucine) and reacted at 25 ° C. for 20 hours. rice field. After adjusting the pH of the reaction solution to 2, the supernatant was recovered by centrifugation. The supernatant was added to a strong acid cation exchange resin (DOWNEX 50W × 8), washed 3 times with distilled water, and eluted with 1M NH ₄ OH. Further, the eluate was added to a strong basic anion exchange resin (DOWNEX 1 × 8), washed 3 times with distilled water, and eluted with 1M CH ₃ COOH. The eluate was neutralized with NH ₄ OH and then freeze-dried to obtain (2S, 3R, 4S) -4-hydroxyisoleucine.
(2S, 3R, 4S) -4-hydroxyisoleucine (7.3 g) was dissolved in 100 ml of water and adjusted to pH 8-9 with sodium hydroxide solution. 5.6 g of succinic anhydride was added and completely dissolved in sodium hydroxide solution while maintaining pH 8-9. A further small amount of succinic anhydride was added in the same manner, and this operation was repeated until the ninhydrin color development on the TLC plate disappeared. After neutralization with a hydrochloric acid solution, freeze-drying gave 14.6 g of N-succinyl- (2S, 3R, 4S) -4-hydroxyisoleucine. Subsequently, the cells expressing NSAR (100 ml of culture solution) obtained in the same manner as in Reference Example 5 and the cells expressing DSA (L182E) obtained in the same manner as in Reference Example 8 (100 ml of culture solution) were subsequently obtained. Was suspended in 10 ml of the reaction solution (25 mM HEPES buffer (pH 7.0), 1 mM CoCl ₂ , 30 mM N-succinyl- (2S, 3R, 4S) -4-hydroxyisoleucine) and reacted at 30 ° C. for 20 hours. rice field.

10 μl of the obtained reaction solution was derivatized using the AccQ / Tag method of Waters. Amino acid reaction products ((2R, 3R, 4S) -4-hydroxyisoleucine (4-hydroxy-D-isoleucine)) were obtained by separating and analyzing this sample by high performance liquid chromatography (HPLC) under the following conditions. It was measured.
Column: XBridge C18 5 μm (2.1 × 150 mm, manufactured by Waters) Eluent A: Ammonium acetate (10 mmol / L, pH 5)
Eluent B: Methanol (0 to 15 min (20 → 60%), 15 to 15.1 min (20% → 80%), 15.1 to 20 min (20%))
Flow velocity: 0.3 ml / min
Temperature: 30 ° C
Detection: Mass spectrometer (positive ion mode), fluorescence detector (excitation wavelength 250 nm, detection wavelength 395 nm)

As shown in FIG. 6, the major peak of the amino acid produced in the reaction solution measured by the fluorescence detector on HPLC was (2R, 3R, 4S) -4-hydroxyisoleucine showing a molecular weight of 318. In addition, a peak of (2S, 3R, 4S) -4-hydroxyisoleucine having a molecular weight of 318 and a peak considered to be a dehydrated product having a molecular weight of 300 were also measured, but the amount produced was small.

Claims (5)

The production of N-succinyl-hydroxy-D-amino acid comprising the step (A) of reacting N-succinyl-hydroxy-L-amino acid with N-succinyl amino acid racemase.
The N-succinyl amino acid racemase is derived from a microorganism belonging to the genus Geobacillus or the genus Chloroflexota.
The amino acids constituting the N-succinyl-hydroxy-D-amino acid are isoleucine, leucine, norleucine, valine or norvaline .
Method. The method of claim 1, wherein the N-succinyl-hydroxy-D-amino acid is an N-succinyl-3-hydroxy-D-amino acid and / or an N-succinyl-4-hydroxy-D-amino acid. A step (A) in which the N-succinyl amino acid racemase is allowed to act on the N-succinyl-hydroxy-L-amino acid, and a step (A) in which the N-succinyl-hydroxy-D-amino acid obtained in the step (A) is allowed to act on the D-succinylase. B)
Including
The N-succinyl amino acid racemase is derived from a microorganism belonging to the genus Geobacillus or the genus Chloroflexota.
The amino acids constituting the N-succinyl-hydroxy-D-amino acid are isoleucine, leucine, norleucine, valine or norvaline.
A method for producing a hydroxy-D-amino acid. The method of claim 3, wherein the N-succinyl-hydroxy-D-amino acid is an N-succinyl-3-hydroxy-D-amino acid and / or an N-succinyl-4-hydroxy-D-amino acid. The method according to claim 3 or 4, wherein the D-succinylase is derived from a microorganism belonging to the genus Cupriavidus or the genus Ralstonia.

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