JP5395395B2 - Production of cyclic amino acids by microorganisms - Google Patents

Description

  The present invention relates to a production method using an optically active cyclic amino acid microorganism, which is important as a synthetic raw material or intermediate in the creation of a pharmaceutical and agrochemical compound.

In the creation of a compound having pharmacological activity, a compound containing (S) -pipecolic acid or (S) -3-morpholine carboxylic acid, which is a cyclic amino acid, in the molecule, or a cyclic amino acid was used as a synthetic intermediate or a raw material. There are many examples (see Non-Patent Document 1 and Patent Documents 1 to 8). Therefore, (S) -pipecolic acid, (S) -3-morpholine carboxylic acid, (R) -3-thiomorpholine carboxylic acid, 5-hydroxy- (S) -pipecolic acid, which are optically active cyclic amino acids with high optical purity Etc. are compounds useful as raw materials for synthetic medical pesticides.

従来の光学活性（Ｓ）−3‐モルホリンカルボン酸を製造する方法としては、
光学活性アジリジン−２−カルボン酸誘導体を出発物質として、３段階の合成反応（２−クロロエタンの付加、ベンジル基の脱離およびトリエチルアミン環化剤として使用する環化反応）を経て製造する方法（非特許文献２、３参照）、
Ｌ−セリン誘導体から５段階の合成反応を経て製造する方法（非特許文献４参照）、
ジアミノ酸等からα位のアミノ基を脱アミノ化して１位に二重結合をもつ環状アミノ酸を生成する酵素（たとえばアミノ酸オキシダーゼ）と、シュードモナス属の微生物等から得られるＮ−メチル−Ｌ−アミノ酸デヒドロゲナーゼとを用いた酵素反応によって、Ｌ−４−オキサリジンから中間体の上記環状アミノ酸を経て（Ｓ）−３−モルホリンカルボン酸を製造する方法（特許文献９参照）が知られている。

As a conventional method for producing optically active (S) -3-morpholine carboxylic acid,
A method of producing an optically active aziridine-2-carboxylic acid derivative as a starting material through a three-step synthesis reaction (addition of 2-chloroethane, elimination of benzyl group and cyclization reaction used as a triethylamine cyclizing agent) Patent Documents 2 and 3),
A method of producing from an L-serine derivative through a five-step synthesis reaction (see Non-Patent Document 4),
An enzyme (for example, an amino acid oxidase) that generates a cyclic amino acid having a double bond at the 1-position by deaminating the amino group at the α-position from a diamino acid, etc., and an N-methyl-L-amino acid obtained from a microorganism of the genus Pseudomonas A method for producing (S) -3-morpholinecarboxylic acid from L-4-oxalidine through the cyclic amino acid as an intermediate by an enzymatic reaction using dehydrogenase (see Patent Document 9) is known.

Moreover, as a method for producing optically active (S) -pipecolic acid (sometimes referred to as L-pipecolic acid),
A method for producing (S) -pipecolic acid using L-lysine or a derivative thereof as a starting material and diazotization of an α-position amino group with sodium nitrite and a cyclization reaction using barium hydroxide or sodium hydroxide as a cyclizing agent (See Non-Patent Document 5 and Patent Document 10),
A method for producing (S) -pipecolic acid by reducing a compound having ethylene acetal at the ε-position of L-α-aminocaproic acid under acidic conditions (see Patent Document 11),
A method for producing (S) -pipecolic acid from L-lysine by an enzymatic reaction using N-methyl-L-amino acid dehydrogenase obtained from a microorganism of the genus Pseudomonas as described above (see Patent Document 9),
A method for producing (S) -pipecolic acid from L-lysine by a cell reaction using a microorganism belonging to any of the genus Alkagenes, Prodencia, Proteus, Bacillus, Agrobacterium, Morganella, and Planococcus (See Patent Document 12),
A gene (lat) encoding lysine-6-aminotransferase possessed by microorganisms belonging to the genus Flavobacterium and a gene encoding proline-5-carboxylate reductase (proC) possessed by Escherichia coli or coryneform bacteria were introduced. (S) -Pipecolic acid is produced from L-lysine via delta-1-piperidein-2-carboxylic acid by culture conversion using recombinant Escherichia coli or by using such a disruption solution of recombinant Escherichia coli. Method (see Patent Document 13),
The genes (ocd, pipA, rapL) encoding these enzymes were introduced by enzymatic reaction using ornithine cyclodeaminase possessed by Agrobacterium microorganisms or enzymes possessed by Streptomyces microorganisms homologous thereto. A method for producing (S) -pipecolic acid from L-lysine using a cell disruption solution of recombinant Escherichia coli (see Patent Document 14),
D-amino acid oxidase derived from microorganisms such as Neurospora, Pseudomonas, Escherichia coli and methanol-utilizing bacteria, or animal origin such as pig kidney and a reducing agent such as borohydride are converted into D- or DL-pipecolic acid. A method of producing (S) -pipecolic acid by a coupling reaction between selective oxidation of D-pipecolic acid to Δ ¹ -piperidein-2-carboxylic acid and a racemic formation reaction with a reducing agent (Patent Document) 15),
Racemate by enzymatic reaction using an enzyme (D-aminoacylase) capable of hydrolyzing an N-protected-D-pipecolic acid derivative, or by cell culture using an Arthrobacter microorganism containing the enzyme A method for producing (S) -pipecolic acid from a pipecolic acid derivative (see Patent Document 16),
Method for producing (S) -pipecolic acid by stereoselective hydrolysis of N-acetyl- (R, S) -pipecolic acid by an enzymatic reaction using N-acyl-pipecolic acid acylase possessed by a microorganism belonging to the genus Alkagenes (See Patent Document 17),
(S) -Pipecolic acid is converted from (R, S) -pipecolic acid amide by a cell reaction using Pseudomonas or Klebsiella microorganisms or by an enzymatic reaction using S-amino acid amidase contained in these microorganisms. A manufacturing method (see Patent Document 18),
(±) -Pipecolic acid is reacted with S-(−)-2-phenoxypropionic acid as an optical resolving agent in a solvent to form poorly water-soluble S-(−)-pipecolic acid · S-(−) A process for producing optically pure S-(-)-pipecolic acid by recovering 2-phenoxypropionate and metathesis with acid (see Patent Document 19),
An amino acid derivative having a halogen group at the end of the side chain and having an amino group protected with an acetyl group or the like (for example, N-acetyl-2-amino-6 obtained from a relatively inexpensive diethyl acetamidomalonate through a four-step synthesis reaction (S) -Pipecoline by enzymatic reaction and intramolecular cyclization in which L-aminoacylase possessed by microorganisms belonging to the genus Aspergillus is added to a racemic solution of (bromohexanoic acid) and deacetylation is performed by L-enantiomer specific action A method for producing an acid (see Patent Document 20) is known.

  As a method for producing optically active (R) -3-thiomorpholine carboxylic acid, a method by organic synthesis (see Non-Patent Documents 2 and 3), an enzyme reaction from L-4-thalidine, or a cell disruption solution. A method (see Patent Documents 9 and 14) is known.

As a method for producing 5-hydroxy- (S) -pipecolic acid, a method for producing 5-hydroxy-DL-lysine by enzymatic reaction or bacterial cell disruption solution (see Patent Documents 9 and 14) is known.
International Publication No. 02/83624 Pamphlet International Publication No. 02/85860 International Publication No. 2004/74291 International Publication No. 2004/92167 International Publication No. 2005/108359 US Pat. No. 7,169,780 US Publication No. 2002-099047 US Publication No. 2004-0152745 JP 2005-95167 A JP 2004-51606 A JP 2008-7480 A Japanese Patent No. 3266635 International Publication No. 01/48216 Japanese Patent No. 3934540 Japanese Patent No. 3135367 Special table 2002-509441 gazette JP-T 09-503669 JP 08-56552 A JP 2000-178253 A Japanese Patent Application Laid-Open No. 2004-261086 J. Med. Chem., 1995, Vol. 38, p.1853-1864 Bull. Chem. Soc. Jpn., 1987, Vol. 60, p.2963-2965 Peptide Chemistry, 1986, p.153-156 J. Org. Chem., 2007, Vol. 72, p.4254-4257 Chem. Pharm. Bull., 1976, Vol. 24, p.621-631 Antimicrobial Agents and Chemotherapy, 1967, p.401-406 Bull. Soc. Chim. Belg., 1982, p.713-723

In the method for producing optically active (S) -3-morpholine carboxylic acid or (R) -3-thiomorpholine carboxylic acid by chemical synthesis described in Non-Patent Documents 2 to 4, a step of removing an intermediate from the reaction solution at the end of each reaction Is necessary, and the operation is complicated. The production method of (S) -pipecolic acid by chemical synthesis described in Non-Patent Document 5 also has a large number of reaction steps and complicated operations, and the production method of Patent Document 11 is said that the raw material is not as inexpensive as L-lysine There's a problem. In the production method using the optical resolution agent described in Patent Document 19, the optical resolution agent is expensive, and only the unmodified pipecolic acid is targeted and cannot be used for the production of (S) -3-morpholinecarboxylic acid. is there.

In addition, as described in Patent Document 9, from an amino acid to (S) -3-morpholinecarboxylic acid, (S) -pipecolic acid, (R) -3-thiomorpholinecarboxylic acid, 5-hydroxy- (S ) -Pipecolic acid and other production methods increase the number of production steps, including low yields, expensive enzymes for enzyme reactions, and the need to prepare enzymes from cells・ There is a problem of increased manufacturing costs. The method for producing (S) -pipecolic acid, (R) -3-thiomorpholinecarboxylic acid, 5-hydroxy- (S) -pipecolic acid and the like using the enzyme reaction described in Patent Document 14 also performs the enzyme reaction. This enzyme is not necessarily satisfactory because it requires the preparation of the enzyme by disrupting the cells of genetically engineered Escherichia coli. The methods described in Patent Documents 15 to 17 for producing (S) -pipecolic acid from a racemate using an enzyme reaction also have the problem that the preparation of the enzyme is complicated and the production steps are many. The method described in Patent Document 20 also has a problem that the number of steps is large because a compound serving as a substrate for an enzyme reaction is prepared by organic synthesis.

Furthermore, in the method for producing (S) -pipecolic acid using a microorganism belonging to the genus Alcigenes isolated from the natural world described in Patent Document 12, the culture temperature during production of the microorganism used for the cell reaction Is a low temperature of 20 ° C., which causes an increase in cost when industrially produced. Moreover, the maximum production amount in this bacterial cell reaction was as low as about 4.2 g / L, which was not industrially satisfactory. Furthermore, since there is no description regarding the production of cyclic amino acids other than (S) -pipecolic acid using diamino acids other than L-lysine as a raw material, can it be applied to the production of (S) -3-morpholine carboxylic acid and the like? Whether it is unknown.

Regarding the method for producing (S) -pipecolic acid by culture conversion using genetically modified Escherichia coli described in Patent Document 13, a cyclic amino acid other than (S) -pipecolic acid is produced using a diamino acid other than L-lysine as a raw material. Since there is no description about what to do, it is unclear whether it can be applied to the production of (S) -3-morpholinecarboxylic acid and the like. The method for producing (S) -pipecolic acid by a cell reaction using a microorganism of the genus Pseudomonas or Klebsiella from racemic pipecol amide described in Patent Document 18 has a problem of low optical purity.

  That is, the object of the present invention is to provide a method for producing optically active cyclic L-amino acids from various diamino acids at low cost, efficiently and industrially by microbial conversion, using microorganisms isolated from nature or mutants thereof. It is to provide.

  The present inventor has intensively studied to achieve the above object. As a result, among various microorganisms isolated from various natural sources by the present inventor, microorganisms belonging to the genus Paracoccus, among them, the strain of Paracoccus sp. AB10292 (Paracoccus sp. AB10292) or a mutant thereof It was discovered that a certain Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain and Paracoccus reference strain produce optically active cyclic amino acids from various diamino acids, and the present invention has been completed.

That is, the present invention
Formula (I)

［式（Ｉ）中、Ｘは、飽和のまたは部分的に若しくは全体的に不飽和の、直鎖または分枝鎖のＣ₁−Ｃ₆炭化水素鎖を表し、該炭化水素鎖は、その内部および／または末端に、Ｏ、Ｓ、Ｐ、Ｎの内から選択する一個または数個のヘテロ原子またはヘテロ原子団を含んでいてもよく、あるいは、−Ｒ、−ＯＲ、−ＳＲ、＝Ｏ、−Ｃ(Ｏ)ＯＲ、−Ｃ(Ｓ)ＯＲ、−Ｃ(Ｏ)ＮＲ'Ｒ"、−Ｃ(Ｓ)ＮＲ'ＲＲ"、−ＣＮ、−ＮＯ₂、−Ｘ、−ＭｇＸ、−ＮＲ'Ｒ"、
−ＮＲ'Ｃ(Ｏ)Ｒ、−ＳｉＲ及び−ＳｉＯＲ（Ｒ、Ｒ'及びＲ"は、同一のまたは相違する
、水素または２〜２０の炭素原子を有する直鎖若しくは分枝鎖の飽和のまたは全体的に若しくは部分的に不飽和の炭化水素基を表す。）の内から選択する一個または数個の同じまたは異なる基によって置換されていてもよく、Ｒ'とＲ"は、それらを有する原子と共に環を形成していてもよい。］で示されるＬ−ジアミノ酸および／またはその塩類を、式（ＩＩ）

[In the formula (I), X represents a saturated or partially or wholly unsaturated, linear or branched C ₁ -C ₆ hydrocarbon chain, And / or the terminal may include one or several heteroatoms or heteroatom groups selected from O, S, P, N, or -R, -OR, -SR, = O, -C (O) OR, -C ( S) OR, -C (O) NR'R ", - C (S) NR'RR", - CN, -NO 2, -X, -MgX, -NR ' R ",
-NR'C (O) R, -SiR and -SiOR (R, R 'and R "are the same or different, hydrogen or linear or branched saturated with 2-20 carbon atoms or Which represents a hydrocarbon group which is unsaturated in whole or in part.) R ′ and R ″ may be substituted by one or several of the same or different groups selected from And may form a ring. An L-diamino acid and / or a salt thereof represented by the formula (II)

［式（ＩＩ）中、Ｘは式（Ｉ）中で規定した通りである。］で示される環状アミノ酸および／またはその塩類に変換する活性を有するパラコッカス属に属する微生物を用い、培養変換或いは菌体反応変換によって、式（Ｉ）で示されるＬ−ジアミノ酸および／またはその塩類、または式（Ｉ）で示されるＬ−ジアミノ酸および対応するＤ−ジアミノ酸、それらの塩類を様々な割合で含む鏡像体混合物から、式（ＩＩ）で示される環状アミノ酸および／またはその塩類を効率よく生産する製造方法に関するものである。

[In formula (II), X is as defined in formula (I). L-diamino acid and / or a salt thereof represented by the formula (I) by using a microorganism belonging to the genus Paracoccus having an activity to convert to a cyclic amino acid and / or a salt thereof represented by the following formula: Or a cyclic amino acid of formula (II) and / or a salt thereof from an enantiomeric mixture containing L-diamino acid of formula (I) and the corresponding D-diamino acid and salts thereof in various proportions. The present invention relates to a manufacturing method for efficient production.

  The method for producing a cyclic amino acid using a microorganism of the present invention is a stable production / supply from an inexpensive diamino acid to various optically active cyclic L-amino acids using a microorganism belonging to the genus Paracoccus or a mutant thereof that can be easily cultured. It is possible to improve the production efficiency of optically active cyclic amino acids and reduce the production cost.

  The L-diamino acid represented by the formula (I) and / or its salt used in the present invention, or the L-diamino acid represented by the formula (I) and the corresponding D-diamino acid, and their salts in various proportions. The enantiomeric mixture to be contained can be used by purchasing a commercially available product, or can be produced from a fermentation product by a microorganism by a known method.

For example, in the case of L-4-oxalidine, L-4-oxalidine monohydrochloride (manufactured by Wako Pure Chemical Industries and Aldrich) is commercially available, and as described in Non-Patent Document 6, Streptomyces chartreuse or It is also possible to culture actinomycetes belonging to Streptomyces erythrochromogenes and to separate and purify L-4-oxalidine and / or its salts from the culture solution. Moreover, you may isolate | separate newly L-4- oxalidine production microorganisms from the natural world, and may use it for manufacture. By producing L-4-oxalidine by fermentation production, L-4-oxalidine as a starting material can be easily obtained at low cost.

L-lysine hydrochloride or a mixture of L- and D-lysine hydrochloride, L-4-thialysine hydrochloride and 5- (R, S) hydroxy-L- and 5- (R, S) hydroxy-D-lysine hydrochloride As the mixture, a commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.) can be purchased and used.

The bacterium producing optically active cyclic amino acid used in the method of the present invention may be any microorganism that belongs to the genus Paracoccus and has the ability to produce an optically active cyclic amino acid. Examples of the microorganism belonging to the genus Paracoccus include, for example, Paracoccus verustus NBRC14567 (Paracoccus versutus), Paracoccus thiocyanata N
BRC14569 (Paracoccus thiocyanatus), Paracoccus aminophilus NBRC16710 (Paracoccus aminophilus), Paracoccus aminoborans NBRC1
6711 (Paracoccus aminovorans), Paracoccus cocriii NBRC16713 (Paracoccus kocurii), Paracoccus alkaliphilus NBRC16719 (Paracoccus alcaliphilus), Paracoccus cerinifilus NBRC10078 (Paracoccus)
seriniphilus), Paracoccus coriensis NBRC102292 (Paracoccus koreensis), Paracoccus pantotrophus NBRC102493 (Paracoccus pantotrophus), Paracoccus denitrificans NBRC102528 (Paracoccus denitr
ificans), Paracoccus sp. AB10292 (Paracoccus sp. AB10292), Paracoccus sp. AB10302 (Paracoccus sp. AB10302) and the like.

Among the above microorganisms belonging to the genus Paracoccus, Paracoccus sp AB10292
(Paracoccus sp. AB10292) strain is a strain newly isolated from soil collected by the present inventor from Isehara City, Kanagawa Prefecture. The mycological properties of this AB10292 strain are described below.

(A) Morphological features (1) Cell morphology: Neisseria gonorrhoeae and size is 0.1-1.1 × 1.1-2.0 μm
(2) Growth on soy bean, casein, digest agar medium: Growth is good. The colony shape is circular, the raised state is hemispherical, the periphery of the colony is an entire edge, and the color tone is a glossy cream color.

(B) Physiological and biochemical properties (1) Gram staining: −
(2) OF test: −
(3) Growth under aerobic conditions: +
(4) Growth under anaerobic conditions: −
(5) Growth temperature:
10 ° C +
27 ° C +
37 ° C-
(6) Catalase: +
(7) Oxidase: +
(8) Urease: −
(9) Nitrate reducibility: +
(10) Denitrification reaction: −
(11) 6% salt tolerance: −
(12) β-galactosidase: −
(13) Arginine dihydrolase: −
(14) L-lysine decarboxylase: −
(15) L-ornithine decarboxylase: −
(16) Availability of citric acid: −
(17) Hydrogen sulfide production: −
(18) Tryptophaminase: −
(19) Indole production: −
(20) Acetoin production: −
(21) Gelatinase: −
(22) Acid production from various sugars:
D-glucose −
D-mannitol-
D-sorbitol-
D-melibiose-
L-rhamnose-
L-arabinose-
Sucrose-
Myo-Inositol −
D-amidagrine −
(23) Utilization of carbon sources
D-glucose +
D-xylose +
D-mannitol-
D-fructose-
Sucrose-
Myo-Inositol −
As described above, the main characteristics of the AB10292 strain were gram-negative bacilli that grew under aerobic conditions, were positive for catalase, oxidase, and nitrate reduction, and were negative for urease and denitrification.

  Furthermore, from the results of the base sequence analysis of 16S ribosome, this strain was considered to be a bacterium closely related to the genus Paracoccus. However, as shown in Table 1, since the homology coincidence with the Paracoccus genus reference strain is low, it was difficult to specify the species from the result of the base sequence analysis of 16S ribosome.

そのため、Bergey's manual of Systematic Bacteriology (1984) 及び International
Journal of Systematic Bacteriology 誌を参考にして、上記５菌株とＡＢ１０２９２株との生理・生化学的比較試験を行った。

Therefore, Bergey's manual of Systematic Bacteriology (1984) and International
With reference to Journal of Systematic Bacteriology, a comparative physiological and biochemical test was conducted between the above five strains and AB10292 strain.

  As a result, it was different from Paracoccus denitrificans NBRC102528 (Paracoccus denitrificans) and Paracoccus aminoborans NBRC16711 (Paracoccus aminovorans) in that the AB10292 strain does not have denitrification activity and cannot grow at 37 ° C.

  It differs from Paracoccus alkalifilus NBRC16719 (Paracoccus alcaliphilus) in that AB10292 strain does not have urease activity and cannot grow at 37 ° C. This was clearly different from the point that Paracoccus seriniphilus NBRC100808 (Paracoccus seriniphilus) cannot grow on the casein digest agar medium.

It differs from Paracoccus aminophilus NBRC16710 (Paracoccus aminophilus) in that the AB10292 strain has nitrate reducing activity. From the above results, it was difficult to identify the species even if the physiological and biochemical properties were compared.

  Therefore, the present inventor identified this strain as Paracoccus sp. And named it Paracoccus sp. AB10292. The strain was applied for deposit at the National Institute of Advanced Industrial Science and Technology Biodeposition Center and was commissioned as FERM P-21605 on July 16, 2008.

So far optically active cyclic amino acid producing bacteria Paracoccus sp AB10292 (Parac
occus sp. AB10292) strains have been described, but in general the fungiological properties of fungi are very variable and are not constant. Fungi are natural or commonly used UV irradiation, X-ray irradiation, mutagenesis agents (for example, N-methyl-N-nitro-N-nitrosoguanidine, ethylmethanesulfonate, etc.) or artificial recombination using genetic recombination. It is a well-known fact that mutation is performed by means of mutation. All strains belonging to the genus Paracoccus and capable of producing optically active cyclic amino acids, including such natural mutants and artificial mutants, can be used in the present invention.

Specific examples thereof include the Paracoccus sp. AB10302 strain, which is a high-concentration L-lysine hydrochloride-resistant mutant strain of the Paracoccus sp. AB10292 (Paracoccus sp. AB10292) strain. This mutant strain was applied for deposit at the National Institute of Advanced Industrial Science and Technology Biodeposit Center and was commissioned as FERM P-21606 on July 16, 2008.

  During culturing of these microorganisms, diamino acid is added directly to the culture solution, and culturing is carried out until most of the diamino acid is converted to optically active amino acid. After culturing these microorganisms to obtain microbial cells, the microbial cells may be suspended in a reaction solution containing a diamino acid and reacted. In addition, mutants or gene recombinants of these microorganisms or processed microbial cells thereof can be used in the present invention.

  Medium components other than diamino acids used in the production of cells used for the culture conversion and cell reaction of the present invention include carbon sources, nitrogen sources, inorganic salts, growth factors and nutrients required by the microorganisms used. Any medium may be used as long as it contains. As the carbon source, those which can use optically active cyclic amino acid producing bacteria such as glucose, xylose, maltose, glycerin, starch, dextrin, starch syrup, molasses, animal / vegetable oil and the like can be used. As the nitrogen source, use can be made of soybean powder, wheat germ, fish meal, corn steep liquor, yeast extract, amino acids, sodium nitrate, ammonium nitrate and other optically active cyclic amino acid producing bacteria. If necessary, a trace metal salt such as cobalt, iron, nickel, manganese or the like can be added. In addition, inorganic and organic substances that promote the production of optically active cyclic amino acids such as 6-aminohexanoic acid can be appropriately added. Moreover, even if it uses the culture medium which does not add an inorganic nitrogen source like ammonium sulfate and does not add a diamino acid, the microbial cell which can be used for a microbial cell reaction can be produced.

  The culture in the culture conversion of the present invention is carried out under aerobic conditions, such as stationary culture, shaking culture, and stirring culture. Usually, the culture temperature is in the range of 15 to 40 ° C, but in many cases the culture is performed at 24 to 30 ° C. The pH during the culture is preferably in the range of 3.0 to 8.0, and the pH in the culture solution may be adjusted as necessary, or a pH adjusting agent such as calcium carbonate may be added. Good. Diamino acids and / or salts thereof may be added at the beginning, or may be added at a time or in small portions from the middle of the culture. The culture period is usually 1 to 14 days, but when the amount of optically active cyclic amino acid accumulation during culture reaches the maximum, the culture is stopped, and the product is collected from the culture solution according to a general method. The culture conditions such as medium composition, medium liquidity, culture temperature, agitation speed, and aeration rate are adjusted or selected for convenience so that preferable results can be obtained according to the type of strain used and external conditions. When there is foaming in liquid culture, antifoaming agents such as silicon oil, vegetable oil and surfactant are conveniently used.

Cultivation of the microbial cells used for the microbial cell reaction of the present invention is carried out under aerobic conditions, such as stationary culture, shaking culture, and stirring culture. Usually, the culture temperature is in the range of 15 to 40 ° C, but in many cases the culture is performed at 24 to 30 ° C. The pH during the culture is preferably in the range of 3.0 to 8.0, and the pH in the culture solution may be adjusted as necessary, or a pH adjusting agent such as calcium carbonate may be added. Good. Substances that enhance the conversion activity, such as diamino acids and 6-aminohexanoic acid, may be added at the beginning, or may be added at once or in small portions from the middle of the culture. The culture period is usually 12 hours to 7 days, but the culture is stopped when a microorganism having an activity capable of converting a cyclic amino acid during the cell reaction grows, and the cell is removed from the culture solution according to a general method. to recover. The culture conditions such as medium composition, medium liquidity, culture temperature, agitation speed, and aeration rate are adjusted or selected for convenience so that preferable results can be obtained according to the type of strain used and external conditions. When there is foaming in liquid culture, antifoaming agents such as silicon oil, vegetable oil and surfactant are conveniently used.

  The bacterial cell reaction is carried out under aerobic conditions, shaking conditions, aeration stirring conditions, and the like. Usually, the reaction temperature is in the range of 15 to 40 ° C, but in many cases the reaction is carried out at 25 to 37 ° C. The pH in the mixed solution is preferably in the range of 3.0 to 8.0, and the pH in the reaction solution may be adjusted in the buffer solution or as necessary. The diamino acid may be added at the beginning, or may be added at a time or in small portions from the middle of the reaction. In addition, in order to increase the production efficiency of cyclic amino acids, substances that can increase the production efficiency, such as sugars such as glucose and xylose, organic acids, amino acids, inorganic salts, trace elements, and surfactants, are added to the medium to react. You can go. The mixing period is usually 6 hours to 7 days, but the reaction is stopped when the accumulated amount of optically active cyclic amino acid in the mixed solution reaches the maximum, and the product is collected from the reaction solution according to a general method. . The mixing reaction conditions such as the composition of the reaction solution, the reaction temperature, the stirring speed, and the aeration rate are conveniently adjusted or selected so that preferable results are obtained according to the type of strain used, the diamino acid used and the external conditions. . When there is foaming in the liquid reaction, an antifoaming agent such as silicon oil, vegetable oil and surfactant is conveniently used.

  Purification of the optically active cyclic amino acid from the culture solution or bacterial cell reaction solution is carried out by a usual method. As a specific purification method, the bacterial cells and the filtrate are separated by a centrifugal separator, a membrane separator, or a bacterial flocculant, or the solution is made alkaline and the bacterial cells are lysed. An ion exchange resin method, an electrodialysis method, a column chromatography method, a crystallization method, or the like can be used, or a combination thereof can be used for purification.

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these specific examples.
In this example, quantitative analysis and optical purity analysis of (S) -3-morpholinecarboxylic acid and (S) -pipecolic acid were carried out by the following methods.

Quantitative analysis in the culture supernatant or the mixture supernatant was calculated by internal standard method using high performance liquid chromatography. The analysis conditions are as follows.
Column: YMC Hydrosphere C18 (4.6mm ID, 25cm length)
Column temperature: 40 ° C
Detector: UV detector Measurement wavelength: 410 nm
Solvent: Liquid A; acetonitrile: water (0.04% phosphoric acid) = 35: 65
Liquid B; acetonitrile: water (0.04% phosphoric acid) = 80: 20
Flow rate: 1.0 ml / min
Gradient: A linear gradient was performed so that solution A was 100% from the start of measurement to 5 minutes, and solution A was 80% and solution B was 20% after 30 minutes, and the solvent was flowed from solution B to 100% from 30 minutes to 35 minutes.
Internal standard: 2-aminoethanol Sample preparation: 10 mg of activated carbon was added to 1 ml of the measurement solution, and the mixture was stirred well for 1 minute, and centrifuged at 15000 rpm for 10 minutes to obtain a centrifugal supernatant. The supernatant was diluted with distilled water so that the concentration of (S) -3-morpholinecarboxylic acid or (S) -pipecolic acid was 1 mg / ml or less, and 10 μl of the diluted solution was added at a concentration of 0.5 mg / ml. Aminoethanol solution (0.1 M aqueous sodium hydrogen carbonate solution) 90 μl, 186 mg / ml 2,4-dinitrofluorobenzene solution (methanol solution) 50 μl, ethanol 400 μl are mixed and stirred, then left at room temperature for 1 hour for derivatization did.
Elution time: (S) -3-morpholinecarboxylic acid 13.6 (min)
(S) -Pipecolic acid 26.7 (min)
2-Aminoethanol 9.9 (min)
The optical purity analysis method for (S) -3-morpholinecarboxylic acid and (S) -pipecolic acid was performed by high performance liquid chromatography using a chiral column. The analysis conditions are as follows.
Column: CHIRALPAK WH manufactured by Daicel (inner diameter 4.6mm, length 25cm)
Column temperature: 50 ° C
Detector: UV detector Measurement wavelength: 254 nm
Solvent: 0.25 mM copper sulfate aqueous solution Flow rate: 1.0 ml / min
Elution time: (R) -3-morpholinecarboxylic acid 36.9 (min)
(S) -3-morpholinecarboxylic acid 43.9 (min)
(R) -Pipecolic acid 13.7 (min)
(S) -Pipecolic acid 20.7 (min)
Unless otherwise indicated, ¹ H- and ¹³ C-NMR were measured in a solution of heavy water (D ₂ O) using a nuclear magnetic resonance apparatus (model JNM-LA300) manufactured by JEOL Ltd.

[Example 1]
Production of (S) -3-morpholinecarboxylic acid by culture conversion using Paracoccus sp. AB10292 (Paracoccus sp. AB10292) strain L-4-oxalidine monohydrochloride 3 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate 100 ml of 80 μg / L, pH 8.0 medium was placed in a 500 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a strain of Paracoccus sp. AB10292 (Paracoccus sp. AB10292) was inoculated from the slant into the medium. A total of 10 Erlenmeyer flasks were subjected to aerobic culture at 27 ° C. for 3 days on a rotary shaker at 180 rpm. After completion of the culture, the (S) -3-morpholinecarboxylic acid concentration in the culture medium was measured and found to be 375 mg / L.

About 1 L of the culture solution collected from these 10 Erlenmeyer flasks was separated into cells and supernatant by centrifugation. After adding 5 g of activated carbon to the supernatant and stirring for 1 hour, the activated carbon was removed using filter paper. The filtrate was passed through a column packed with 500 ml of strongly acidic cation exchange resin Duolite C-20 (H ⁺ type) to adsorb (S) -3-morpholinecarboxylic acid, and 2 L of deionized water was added. The column was washed by passage and then eluted with 2.5 L of 2% aqueous ammonia. The eluate containing (S) -3-morpholinecarboxylic acid was concentrated under reduced pressure to dryness, and then 250 ml of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 250 ml of Duolite C-20 (NH ₄ ⁺ type), and the column was washed with 2 L of deionized water to recover (S) -3-morpholinecarboxylic acid. The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and then 80 ml of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 80 ml of weakly basic anion exchange resin Duolite A-368S, and then the column was washed with 800 ml of deionized water to recover (S) -3-morpholinecarboxylic acid. .

The passing liquid and the recovered liquid obtained as described above were concentrated under reduced pressure, and methanol was added for crystallization. The crystals were collected by filtration and dried to obtain 152 mg of white crystals.
The chemical shift values of ¹ H-NMR and ¹³ C-NMR in the NMR analysis of this crystal coincided with those of (S) -3-morpholinecarboxylic acid described in Non-Patent Document 2.
¹ H-NMR
d (ppm): 3.98 (1H, dd), 3.77 (1H, dt), 3.51-3.68 (3H
, M), 3.17 (1H, dt), 3.01 (1H, ddd)
¹³ C-NMR
d (ppm): 171.3, 66.8, 64.0, 57.7, 42.9
Moreover, although HPLC analysis was performed in order to measure optical purity, the peak of (R) -3-morpholine carboxylic acid was not detected, and the obtained crystal was only (S) -3-morpholine carboxylic acid.

[Example 2]
Preparation of high-concentration L-lysine hydrochloride resistant mutant Paracoccus sp. AB10302 (Paracoccus sp. AB10302) Succeeded inoculated with one platinum ear Paracoccus sp. The cells were cultured for 18 hours on a reciprocating shaker at 27 ° C. 1 ml of the culture broth was diluted 10-fold with 0.85% saline and dispensed into a sterile petri dish with a diameter of 9 cm, and then this cell suspension was irradiated with ultraviolet rays for 90 seconds using a Toshiba GL-15 germicidal lamp. Irradiated.

L-lysine monohydrochloride 55 g / L, yeast extract 0.1 g / L, sodium chloride 1 g / L
L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate One flask was prepared by putting 100 ml of 80 μg / L, pH 8.0 medium in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask. Further, 10 ml of the bacterial cell suspension irradiated with ultraviolet rays was inoculated into the flask, and a rotary shaker at 27 ° C. and 180 rpm. And aerobic culture for 7 days.

  A soybean casein digest agar medium (manufactured by Nippon Pharmaceutical Co., Ltd.) adjusted so that the concentration of L-lysine monohydrochloride is 50 g / L was dispensed into a 9 cm diameter sterilized petri dish to prepare an agar medium and cultured on a rotary shaker The culture solution was smeared and cultured at 27 ° C. Colonies that grew on the agar were isolated and selected to prepare a Paracoccus sp. AB10302 strain.

[Example 3]
Production of (S) -3-morpholinecarboxylic acid by culture conversion using Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain In the same manner as in Example 1, except that Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain is used. Carried out. The production amount of (S) -3-morpholinecarboxylic acid in the culture solution was 501 mg / L.

[Example 4]
Production of (S) -3-morpholinecarboxylic acid by culture conversion using Paracoccus reference strain The same procedure as in Example 1 was carried out except that various reference strains belonging to the genus Paracoccus shown in Table 2 were used. The production amount of (S) -3-morpholine carboxylic acid in the culture solution of each strain was as shown in Table 2.

［実施例５］
パラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株を利用した、菌体反応による（Ｓ）−３−モルホリンカルボン酸の製造
６−アミノヘキサン酸７．２ｇ／Ｌ、酵母エキス２ｇ／Ｌ、塩化ナトリウム１ｇ／Ｌ、リン酸２水素カリウム２ｇ／Ｌ、リン酸水素２カリウム１ｇ／Ｌ、塩化マンガン４水和物
８０μｇ／Ｌ、ｐＨ８．０の培地１００ｍｌをバッフル付き５００ｍｌ容三角フラスコ
に入れ、１２１℃で２０分間オートクレーブ滅菌した。植菌直前そのフラスコに別滅菌したグルコース溶液（２５０ｇ/Ｌ）を８ｍｌ添加し、その培地中にパラコッカス・エスピ
ー ＡＢ１０３０２（Paracoccus sp.AB10302）株をスラントから一白金耳接種し、２７℃で回転数１８０ｒｐｍのロータリーシェーカーで２４時間好気培養して前培養液を作成した。

[Example 5]
Production of (S) -3-morpholinecarboxylic acid by bacterial cell reaction using Paracoccus sp. AB10302 strain (Paracoccus sp. AB10302) 6-aminohexanoic acid 7.2 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate
100 ml of 80 μg / L, pH 8.0 medium was placed in a 500 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a platinum loop of Paracoccus sp. AB10302 strain was inoculated into the medium from the slant. A preculture was prepared by aerobic culture for 24 hours on a rotary shaker at 180 rpm.

Next, a 65-liter Jafermenter was charged with 7.2 g / L of 6-aminohexanoic acid, 8 g / L of yeast extract, 1 g / L of sodium chloride, 2 g / L of potassium dihydrogen phosphate, 1 g / L of dipotassium hydrogen phosphate, and chloride. Manganese tetrahydrate 30 L of a medium with 80 μg / L and pH 8.0 was added and steam sterilized at 121 ° C. for 25 minutes. Separately sterilized glucose solution immediately before inoculation (300 g / L
2) was added to the jaffer mentor, and 300 ml of the preculture solution was inoculated, and cultured for 38 hours at 27 ° C., a stirring rotational speed of 180 rpm, and an aeration rate of 1 vvm.

Bacterial cells were collected from the culture broth with a centrifugal separator and sterilized by filtration (L-4-oxalidine monohydrochloride 30 g / L, glucose 22.5 g / L, 300 mM, pH 6.98, 300 mM).
3 L was dissolved in the collected bacterial cells and suspended, and the bacterial cell suspension was added to a 5 L minijar fermenter. The cell reaction was carried out at a reaction temperature of 27 ° C., a stirring speed of 450 rpm, and an aeration rate of 0.5 vvm for 25 hours. It was 13.8 g / L when the (S) -3-morpholine carboxylic acid density | concentration in the reaction liquid after completion | finish of reaction was measured.

Bacteria were removed from the reaction solution by centrifugation, 24 g of activated carbon was added to the supernatant and stirred for 1 hour, and then the activated carbon was removed using filter paper. The filtrate was passed through a column packed with 2 L of strongly acidic cation exchange resin Duolite C-20 (H ⁺ type) to adsorb (S) -3-morpholinecarboxylic acid, and further with 5 L of deionized water. The column was washed and eluted with 2% aqueous ammonia. The eluate containing (S) -3-morpholinecarboxylic acid was concentrated under reduced pressure to dryness, and then 1 L of deionized exchange water was added to dissolve the solid matter. The solution was passed through a column packed with 1 L of Duolite C-20 (NH ₄ ⁺ type), and the column was washed with 2 L of deionized water to recover (S) -3-morpholinecarboxylic acid.

The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and 900 ml of deionized water was added to dissolve the solid matter. Strongly basic anion exchange resin Duolite A-113 (CH ₃ COO ^- form) was passed through a column packed with 200 ml, and then washing the column with deionized water (S)-3-morpholine carboxylic acid It was collected.

  The passing liquid and the collected liquid obtained above were concentrated under reduced pressure, decolorized with activated carbon, and filtered with a 0.45 μm filter, and then the filtrate was concentrated and crystallized with methanol. The crystals were collected by filtration and dried to obtain 34.5 g of white crystals of (S) -3-morpholinecarboxylic acid.

  In order to measure the optical purity, HPLC analysis was performed, but the peak of (R) -3-morpholinecarboxylic acid was not detected, and the obtained crystal was only (S) -3-morpholinecarboxylic acid.

[Example 6]
Production of (S) -pipecolic acid by culture conversion using Paracoccus sp. AB10292 (Paracoccus sp. AB10292) strain L-lysine monohydrochloride 10 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, phosphoric acid Potassium dihydrogen 2g / L, Dipotassium hydrogen phosphate 1g / L, Manganese chloride tetrahydrate 8
100 ml of 0 μg / L, pH 8.0 medium was placed in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a strain of Paracoccus sp. AB10292 was inoculated from the slant into the medium. A total of 10 Erlenmeyer flasks were subjected to aerobic culture at 27 ° C. for 3 days on a rotary shaker at 180 rpm. The (S) -pipecolic acid concentration in the culture was measured and found to be 5.8 g / L.

About 1 L of the culture solution collected from these 10 Erlenmeyer flasks was separated into cells and supernatant by centrifugation. After adding 5 g of activated carbon to the culture supernatant and stirring for 1 hour, the activated carbon was removed using filter paper. The filtrate is passed through a column packed with 1 L of strongly acidic cation exchange resin Duolite C-20 (H ⁺ type) to adsorb (S) -pipecolic acid, and the column is further washed with 4 L of deionized water. And then eluted with 5 L of 2% aqueous ammonia. The eluate containing (S) -pipecolic acid was concentrated under reduced pressure to dryness, and then 500 ml of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 500 ml of Duolite C-20 (NH ₄ ⁺ type), and the column was washed with 2 L of deionized water to recover (S) -pipecolic acid. The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and 200 ml of deionized water was added to dissolve the solid matter. The solution was used as a weakly basic anion exchange resin Duolite A-368S 1
(S) -pipecolic acid was recovered by passing 1 L of deionized water through this column.

The passing liquid and the recovered liquid obtained as described above were concentrated under reduced pressure, and methanol was added for crystallization. The crystals were collected by filtration and dried to obtain 3.2 g of white crystals.
Elution time of this crystal by HPLC analysis and ¹ H-NMR and ¹³ C-NM in NMR analysis
The chemical shift value of R was consistent with that of (S) -pipecolic acid, which is a standard product.
¹ HNMR data:
d (ppm): 1.37-1.59 (3H, m), 1.70-1.77 (2H, m), 2.08 (1H, m), 2.86 (1H, m), 3 .27 (1H, m), 3.43 (1H, m)
¹³ C NMR data:
d (ppm): 175.4, 59.8, 44.5, 27.3, 22.6, 22.4
Moreover, although HPLC analysis was performed in order to measure optical purity, the peak of (R) -pipecolic acid was not detected, and the obtained crystal was only (S) -pipecolic acid.

[Example 7]
Production of (S) -pipecolic acid by culture conversion from DL-lysine monohydrochloride using Paracoccus sp. AB10292 (Paracoccus sp. AB10292) strain DL-lysine monohydrochloride 3 g / L, yeast extract 2 g / L, chloride Sodium 1g / L, Potassium dihydrogen phosphate 2g / L, Dipotassium hydrogen phosphate 1g / L, Manganese chloride tetrahydrate 100 ml of 80 μg / L, pH 8.0 medium was placed in a 500 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a strain of Paracoccus sp. AB10292 (Paracoccus sp. AB10292) was inoculated from the slant into the medium. The Erlenmeyer flask was subjected to aerobic culture at 27 ° C. for 3 days on a rotary shaker at 180 rpm. When the (S) -pipecolic acid concentration in the culture was measured, it was 0.76 g / L.

[Example 8]
Production of (S) -pipecolic acid by culture conversion using Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain L-lysine monohydrochloride 5 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, phosphoric acid Potassium dihydrogen 2g / L, Dipotassium hydrogen phosphate 1g / L, Manganese chloride tetrahydrate 80
100 ml of a medium of μg / L, pH 8.0 was placed in a 500 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a Paracoccus sp. AB10302 strain was inoculated from the slant into the medium. 27 ° C
And aerobic culture on a rotary shaker at 180 rpm for 24 hours to prepare a preculture solution.

L-lysine monohydrochloride 65 g / L, yeast extract 5.5 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate 80 μg / L 100 ml of L, calcium carbonate 2 g / L, pH 8.0 medium was placed in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Just before inoculation, 16 ml of a separately sterilized glucose solution (375 g / L) was added to the flask, and 5 ml of the preculture was inoculated into the medium. The aerobic culture was carried out at 27 ° C. for 12 days on a rotary shaker with a rotation speed of 180 rpm, and the concentration of (S) -pipecolic acid in the culture medium was measured.
It was 8.4 g / L.

[Example 9]
Production of (S) -pipecolic acid by culture conversion using Paracoccus standard strain L-lysine monohydrochloride 3 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, 100 ml of a medium of dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate 80 μg / L, pH 8.0 was placed in a 500 ml Erlenmeyer flask equipped with a baffle, and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and various strains belonging to the genus Paracoccus shown in Table 3 were inoculated from the slant into the platinum medium. Thereafter, the cells were aerobically cultured on a rotary shaker at 180 rpm for 3 days at 27 ° C. Table 3 shows the amount of (S) -pipecolic acid produced in each strain culture solution.

［実施例１０］
パラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株を利用した、菌体反応による（Ｓ）−ピペコリン酸の製造
６−アミノヘキサン酸７．２ｇ／Ｌ、酵母エキス２ｇ／Ｌ、塩化ナトリウム １ｇ／Ｌ
、リン酸２水素カリウム２ｇ／Ｌ、リン酸水素２カリウム１ｇ／Ｌ、塩化マンガン４水和物 ８０μｇ／Ｌ、ｐＨ８．０の培地１００ｍｌをバッフル付き５００ｍｌ容三角フラス
コに入れたフラスコを１０本作成し、１２１℃で２０分間オートクレーブ滅菌した。植菌直前そのフラスコに別滅菌したグルコース溶液（２５０ｇ/Ｌ）を８ｍｌ添加し、その培
地中にパラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株をスラントから一白金耳接種して、２７℃で２日間、回転数１８０ｒｐｍのロータリーシェーカーで好気培養した。

[Example 10]
Production of (S) -pipecolic acid by bacterial cell reaction using Paracoccus sp. AB10302 strain (Paracoccus sp. AB10302) 6-aminohexanoic acid 7.2 g / L, yeast extract 2 g / L, sodium chloride 1 g / L
, Potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate Ten flasks were prepared by putting 100 ml of 80 μg / L, pH 8.0 medium in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a platinum loop of Paracoccus sp. AB10302 strain was inoculated from the slant into the medium. The aerobic culture was performed on a rotary shaker with a rotation speed of 180 rpm for one day.

  Next, the culture solution was collected to about 1 L, and centrifuged at 8000 rpm for 20 minutes to remove the supernatant. To the precipitated cells, 200 ml of 50 mM phosphate buffer (pH 6.98) was added and stirred well, and centrifuged again at 8000 rpm for 20 minutes to remove the supernatant to obtain cells for cell reaction.

  100 ml of a substrate solution (L-lysine monohydrochloride 30 g / L, dissolved in 300 mM phosphate buffer having a pH of 6.98 so that glucose is 22.5 g / L) was added to the cells and suspended. The bacterial cell suspension was added to an Erlenmeyer flask. After the bacterial cell reaction was carried out for 27 hours with a rotary shaker at 27 ° C. and 180 rpm, the (S) -pipecolic acid concentration in the reaction solution was measured and found to be 16.2 g / L.

[Example 11]
Production of trans-5-hydroxy- (S) -pipecolic acid and cis-5-hydroxy- (S) -pipecolic acid by culture conversion using Paracoccus sp. Strain AB10292 (Paracoccus sp. AB10292) 5-hydroxy-DL -Lysine hydrochloride 3.3 g / L (manufactured by Wako Pure Chemical Industries), yeast extract 2 g / L
Sodium chloride 1 g / L, potassium dihydrogen phosphate 0.29 g / L, disodium hydrogen phosphate 12 hydrate 6.86 g / L, manganese chloride tetrahydrate 100 ml of 80 μg / L, pH 8.0 medium was placed in a 500 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, the flask was separately sterilized with a glucose solution (250 g / L
8 ml) was added, and a platinum ear was inoculated from the slant with a strain of Paracoccus sp. AB10292 (Paracoccus sp. AB10292). The three Erlenmeyer flasks were aerobically cultured on a rotary shaker at 180 rpm for 3 days at 27 ° C.

When the culture supernatant was analyzed by TLC, two blue-violet ninhydrin coloring substances showing Rf values clearly different from 5-hydroxy-DL-lysine hydrochloride were observed (developing solvent acetonitrile: methanol: water = 3: 1: 1, Rf value = 0.308,0.356, Merck TLC plates: silica gel 60F _254).

About 300 mL of the culture solution collected from the Erlenmeyer flask was separated into cells and supernatant by centrifugation. 100 ml of the centrifugal supernatant was strongly acidic cation exchange resin Duolite C-20 (H ⁺ type)
The ninhydrin color-developing substance was adsorbed by passing through a column filled with, and the column was further washed with 500 mL of deionized water, and then eluted with 500 mL of 2% aqueous ammonia. The eluate was concentrated under reduced pressure to dryness, and then 50 ml of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 50 ml of strongly acidic cation exchange resin Duolite C-20 (NH ₄ ⁺ type), and the column was further washed with 250 mL of deionized water to recover the ninhydrin coloring material. The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and 50 ml of deionized water was added to dissolve the solid matter. Its solution strongly basic anion exchange resin Duolite A-113 (CH ₃ COO ^- type) 10 ml was passed through a column packed with further washing the column with deionized water 50ml recovered ninhydrin coloring material did.

  The passing liquid and the collected liquid obtained above were concentrated under reduced pressure to dryness, and then 1 ml of deionized water was added to dissolve the solid matter. Column chromatography of the dissolved solution was performed using a column packed with 70 ml of activated carbon (granular, manufactured by Wako Pure Chemical Industries, Ltd.) to recover two substances that develop ninhydrin color.

The recovered liquid obtained above was concentrated to dryness under reduced pressure, and 0.5 ml of deionized water was added to dissolve the solid matter. Column chromatography of the lysate was performed using a column packed with 200 ml of strongly acidic cation exchange resin Diaion UBK530 (Ca ²⁺ type), and 44 mg of substance showing Rf value = 0.356, Rf value = 0.308. 84 mg of the substance showing was isolated.

The substance having Rf value = 0.356 was analyzed by NMR, and its ¹ H-NMR and ¹³ C-NM were analyzed.
The chemical shift value of R is trans-5-hydroxy- described in Non-Patent Document 7.
Consistent with that of (S) -pipecolic acid. The specific rotation of this substance was [α] _D = -20.2 ° (c; 1.0, H ₂ O, 23 ° C.).
¹ HNMR data:
d (ppm): 1.54-1.64 (1H, m), 1.68-1.81 (1H, m), 2.01-2.10 (1H, m), 2.24-2. 33 (1H, m), 2.78-2.86 (1H, m), 3.43 (1H, dd), 3.59 (1H, dd), 3.94 (1H, m)
¹³ C NMR data:
d (ppm): 173.8, 63.2, 58.0, 47.2, 29.8, 23.8

Ｒｆ値＝０．３０８を示す物質は、¹Ｈ-ＮＭＲと¹³Ｃ-ＮＭＲの分析結果及び生成に使
用した基質から考えてciｓ−５−ヒドロキシ-（Ｓ）-ピペコリン酸と考えられる。また、この物質の比旋光度は［α］_D＝−３０．６°（ｃ；２．０、Ｈ₂Ｏ、２３℃）であった。¹ＨＮＭＲデータ：
d(ｐｐｍ)：１．７７−２．１４(４Ｈ，ｍ)、３．１９(１Ｈ，ｄｄ)、３．３２(１Ｈ，ｄｔ)、３．６３(１Ｈ，ｄｄ)、４．１５−４．１９(１Ｈ，ｍ)
¹³ＣＮＭＲデータ：
d(ｐｐｍ)：１７３．４、６０．８、２、５８．０、４７．５、２７．６、２０．５

A substance exhibiting an Rf value = 0.308 is considered to be cis-5-hydroxy- (S) -pipecolic acid in view of the analysis results of ¹ H-NMR and ¹³ C-NMR and the substrate used for production. The specific rotation of this substance was [α] _D = −30.6 ° (c; 2.0, H ₂ O, 23 ° C.). ¹ HNMR data:
d (ppm): 1.77-2.14 (4H, m), 3.19 (1H, dd), 3.32 (1H, dt), 3.63 (1H, dd), 4.15-4 19 (1H, m)
¹³ C NMR data:
d (ppm): 173.4, 60.8, 2, 58.0, 47.5, 27.6, 20.5

［実施例１２］
パラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株を利用した、菌体反応によるｔｒａｎｓ−５−ヒドロキシ-（Ｓ）-ピペコリン酸及びｃｉｓ−５−ヒドロキシ-（Ｓ）-ピペコリン酸の製造
６−アミノヘキサン酸 ７．２ｇ／Ｌ、酵母エキス２ｇ／Ｌ、塩化ナトリウム １ｇ／Ｌ、リン酸２水素カリウム２ｇ／Ｌ、リン酸水素２カリウム １ｇ／Ｌ、 塩化マンガン４水和物 ８０μｇ／Ｌ、ｐＨ８．０の培地１００ｍｌをバッフル付き５００ｍｌ容三角フラ
スコに入れたフラスコを２本作成し、１２１℃で２０分間オートクレーブ滅菌した。植菌直前そのフラスコに別滅菌したグルコース溶液（２５０ｇ/Ｌ）を８ｍｌ添加し、その培
地中にパラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株をスラントから一白金耳接種し、２７℃で４０時間、回転数１８０ｒｐｍのロータリーシェーカーで好気培養した。

[Example 12]
Production of trans-5-hydroxy- (S) -pipecolic acid and cis-5-hydroxy- (S) -pipecolic acid by bacterial cell reaction using Paracoccus sp. AB10302 strain 6-aminohexane 7.2 g / L of acid, 2 g / L of yeast extract, 1 g / L of sodium chloride, 2 g / L of potassium dihydrogen phosphate, 1 g / L of dipotassium hydrogen phosphate, manganese chloride tetrahydrate Two flasks were prepared by putting 100 ml of 80 μg / L, pH 8.0 medium in a baffled 500 ml Erlenmeyer flask and sterilized by autoclave at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a Paracoccus sp. AB10302 strain was inoculated into the medium from a slant at 40 ° C. for 40 hours. The aerobic culture was performed on a rotary shaker with a rotation speed of 180 rpm.

Next, the bacterial cells were collected from the culture broth with a centrifuge and filtered to sterilize a substrate solution (5-hydroxy-DL-lysine hydrochloride 20 g / L, glucose 3 g of pH 6.98 to 15 g / L).
(Dissolved in 00 mM phosphate buffer) 20 mL was added to the collected cells and suspended, and the cell suspension was added to a 100 ml Erlenmeyer flask. Thereafter, the cell reaction was carried out for 32 hours using a rotary shaker with a reaction temperature of 27 ° C. and a rotation speed of 160 rpm.

When the reaction supernatant was analyzed by TLC to confirm production, trans-5-hydroxy- (S) -pipecolic acid and cis-5-hydroxy- (Rf value = 0.308 and 0.356, respectively. S) -pipecolic acid ninhydrin color was observed (developing solvent acetonitrile: methanol: water = 3: 1: 1, TLC plate manufactured by Merck & Co., Ltd .; silica gel 60F ₂₅₄
).

[Example 13]
Production of (R) -3-thiomorpholinecarboxylic acid by culture conversion using Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain L-alanine 20 mg / L, L-sodium hydrogen glutamate monohydrate 20 mg / L, L-valine 20 mg / L, L-isoleucine 20 mg / L, L-leucine 20 mg / L, thiamine hydrochloride 5 mg / L, (+) biotin 0.1 mg / L, cyanocobalamin 2.5 m
g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, iron sulfate heptahydrate 250 μg / L, copper sulfate pentahydrate 250 μg / L, calcium chloride 2.5 mg / L, zinc sulfate heptahydrate 500 μg / L, manganese chloride tetrahydrate 500 μg / L, nickel chloride hexahydrate 250 μg / L, sodium molybdate dihydrate 250 μg / L, potassium iodide Ten flasks were prepared by putting 100 ml of 5 μg / L, boric acid 25 μg / L, pH 8.0 medium in a 500 ml Erlenmeyer flask with baffle, and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 10 ml of an L-4-thialysin monohydrochloride aqueous solution (20 g / L, pH 7.0) was added to each flask after filtration sterilization. Similarly, 8 ml of a separately sterilized glucose solution (250 g / L) was added to each flask, and a platinum loop of Paracoccus sp. AB10302 strain was inoculated from the slant into the medium. Aerobic culture was performed on a rotary shaker at 180 rpm.

Next, the cells are removed from the culture broth with a centrifugal separator, and the supernatant is passed through a column packed with 500 ml of strongly acidic cation exchange resin Duolite C-20 (H ⁺ type) (R) -3. -Thiomorpholine carboxylic acid was adsorbed, further washed with 2 L of deionized water, and eluted with 5 L of 2% aqueous ammonia. The eluate was concentrated under reduced pressure to dryness, and 250 ml of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 250 ml of strongly acidic cation exchange resin Duolite C-20 (NH ₄ ⁺ type), and the column was further washed with 2 L of deionized water. The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and then 80 ml of deionized water was added to dissolve the solid matter. The solution strongly basic anion exchange resin Duolite A-116 a ^- was passed through a column packed with (CH ₃ COO type) 80 ml, the column was washed further with deionized water 800 ml.

The passing liquid and the recovered liquid obtained above were concentrated under reduced pressure to dryness, and then 20 ml of deionized water was added to dissolve the solid matter. When the solution was analyzed by TLC, a ninhydrin coloring substance was observed at an Rf value = 0.494 (developing solvent acetonitrile: methanol: water = 3: 1: 1, TLC plate manufactured by Merck; silica gel 60F ₂₅₄ ).

The solution was concentrated to dryness under reduced pressure, and 4 ml of deionized exchange water was added to dissolve the solid matter. The column chromatography of the solution was performed using a column packed with 250 ml of weakly acidic cation exchange resin Amberlite CG-50 (NH ₄ ⁺ type), and a fraction containing a substance exhibiting an Rf value = 0.494 was collected. . The solution was concentrated to dryness under reduced pressure, 20 ml of deionized exchange water was added to dissolve the solid matter, decolorized with activated carbon, filtered through a 0.45 μm filter, and then methanol was added to the solution while concentrating the crystals. Made it. The crystals were collected by filtration and dried to obtain 84 mg of white crystals.

The ninhydrin coloring substance showing this Rf value = 0.494 is considered to be (R) -3-thiomorpholine carboxylic acid in view of the analysis results of ¹ H-NMR and ¹³ C-NMR and the substrate used for production. The specific rotation of this substance was [α] _D = −53.2 ° (c; 2.0, H ₂ O, 23 ° C.).
¹ HNMR data:
d (ppm): 2.74-2.82 (1H, m), 2.89-3.13 (3H, m), 3.29 (1H, ddd), 3.69 (1H, dt), 3 .84 (1H, dd)
¹³ C NMR data:
d (ppm): 172.3, 59.3, 45.3, 27.5, 23.8

［実施例１４］
パラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株を利用した、菌体反応による（Ｒ）−３−チオモルホリンカルボン酸の製造
６−アミノヘキサン酸７．２ｇ／Ｌ、酵母エキス２ｇ／Ｌ、塩化ナトリウム１ｇ／Ｌ、リン酸２水素カリウム２ｇ／Ｌ、リン酸水素２カリウム１ｇ／Ｌ、塩化マンガン４水和物
８０μｇ／Ｌ、ｐＨ８．０の培地１００ｍｌをバッフル付き５００ｍｌ容三角フラスコ
に入れたフラスコを４本作成し、１２１℃で２０分間オートクレーブ滅菌した。植菌直前そのフラスコに別滅菌したグルコース溶液（２５０ｇ/Ｌ）を８ｍｌ添加し、その培地中
にパラコッカス・エスピー ＡＢ１０３０２（Paracoccus sp.AB10302）株をスラントから一白金耳接種し、２７℃で４４時間、回転数１８０ｒｐｍのロータリーシェーカーで好気培養した。

[Example 14]
Production of (R) -3-thiomorpholinecarboxylic acid by cell reaction using Paracoccus sp. AB10302 (Paracoccus sp. AB10302) strain 6-aminohexanoic acid 7.2 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate
Four flasks were prepared by putting 100 ml of 80 μg / L, pH 8.0 medium in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Immediately before inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a Paracoccus sp. AB10302 strain was inoculated into the medium from a slant at 44 ° C for 44 hours. The aerobic culture was performed on a rotary shaker with a rotation speed of 180 rpm.

Next, the cells are collected from the culture broth with a centrifuge and dissolved in a substrate solution (L-4-thalidine monohydrochloride 10 g / L, glucose 7.5 g / L in 300 mM phosphate buffer having a pH of 6.98). 40 mL was added to the collected cells and suspended, and the cell suspension was added to a 200 ml Erlenmeyer flask. Thereafter, the cell reaction was carried out for 27 hours on a rotary shaker with a reaction temperature of 27 ° C. and a rotation speed of 160 rpm.

When the supernatant of the reaction solution was analyzed by TLC, L-4-thalidine monohydrochloride almost disappeared, and ninhydrin coloration of (R) -3-thiomorpholinecarboxylic acid was observed at an Rf value = 0.494 (development). Solvent acetonitrile: methanol: water = 3: 1: 1, TLC plate manufactured by Merck & Co .; silica gel 60F ₂₅₄ ).

The cells are removed from the reaction solution by centrifugation, and the filtrate is passed through a column packed with 40 ml of strongly acidic cation exchange resin Duolite C-20 (H ⁺ type) to give (R) -3-thiomorpholine. Carboxylic acid was adsorbed, and the column was further washed with 200 ml of deionized exchange water, and then eluted with 2% aqueous ammonia. The eluate containing the ninhydrin coloring substance was concentrated under reduced pressure to dryness, and then 40 L of deionized water was added to dissolve the solid matter. The solution was passed through a column packed with 20 ml of strongly acidic cation exchange resin Duolite C-20 (NH ₄ ⁺ type), and the column was further washed with 100 ml of deionized exchange water (R) -3-thio. The morpholine carboxylic acid was recovered.

The passing liquid and the collected liquid were concentrated under reduced pressure to dryness, and 10 ml of deionized water was added to dissolve the solid matter. The solution was added to the strongly basic anion exchange resin Duolite A-113 (CH ₃
The (R) -3-thiomorpholine carboxylic acid was recovered by passing through a column packed with 3 ml of (COO ⁻ type) and further washing the column with deionized water.

The passing liquid and the collected liquid obtained above were concentrated under reduced pressure, decolorized with activated carbon, filtered through a 0.45 μm filter, and then crystallized by adding methanol while concentrating the solution. The crystals were collected by filtration and dried to obtain 70.5 mg of white crystals of (R) -3-thiomorpholinecarboxylic acid. The specific rotation of this crystal was [α] _D = −53.7 ° (c; 2.0, H ₂ O, 23 ° C.).

[Comparative Example 1]
D-lysine monohydrochloride 3 g / L, yeast extract 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, manganese chloride tetrahydrate 80 μg / L, 100 ml of pH 8.0 medium was placed in a baffled 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. Immediately before the inoculation, 8 ml of a separately sterilized glucose solution (250 g / L) was added to the flask, and a strain of Paracoccus sp. AB10292 (Paracoccus sp. AB10292) was inoculated from the slant into the medium. The Erlenmeyer flask was subjected to aerobic culture at 27 ° C. for 3 days on a rotary shaker at 180 rpm. Neither (S) -pipecolic acid nor (R) -pipecolic acid was detected in the culture broth after completion of the culture.

Claims (3)

Paracoccus sp. AB10292 (Paracoccus sp. AB10292) deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depository Center under the deposit number of FERM P-21605 or FERM at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center Paracoccus sp. AB10302 deposited under the deposit number of P-21606 is selected from L-4-oxalysine, L-lysine, 5-hydroxy-L-lysine, or L-4-thialysine From a medium containing one L-diamino acid and / or a salt thereof, or from the L-diamino acid and / or a salt thereof and a D-diamino acid and / or a salt thereof which is an enantiomer of the L-diamino acid In a medium containing a mixture of enantiomers corresponding to the L-diamino acid One cyclic amino acid selected from (S) -3-morpholine carboxylic acid, (S) -pipecolic acid, 5-hydroxy- (S) -pipecolic acid, or (R) -3-thiomorpholine carboxylic acid and / or Alternatively, a method for producing an optically active cyclic amino acid, comprising producing a salt thereof. Paracoccus sp. AB10292 (Paracoccus sp. AB10292) deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depository Center under the deposit number of FERM P-21605 or FERM at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center The cells of Paracoccus sp. AB10302 deposited under the deposit number of P-21606 are treated with L-4-oxalysine, L-lysine, 5-hydroxy-L-lysine, or L-4-thialysine. A solution containing one kind of L-diamino acid and / or a salt thereof selected from the above, or a D-diamino acid which is an enantiomer of the L-diamino acid and / or a salt thereof and the L-diamino acid and / or its Suspended in a solution containing an enantiomeric mixture consisting of salts, and the L-diamino acid One cyclic amino acid selected from (S) -3-morpholine carboxylic acid, (S) -pipecolic acid, 5-hydroxy- (S) -pipecolic acid, or (R) -3-thiomorpholine carboxylic acid And / or a salt thereof. A method for producing an optically active cyclic amino acid, comprising:   Paracoccus sp. AB10292 (Paracoccus sp. AB10292) deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number of FERM P-21605, or a mutant strain thereof and the National Institute of Advanced Industrial Science and Technology Paracoccus sp. AB10302 (Paracoccus sp. AB10302) deposited at the Patent Biological Depositary Center under the deposit number of FERM P-21606.