JPH01222797A - Production of d-carnitine and l-carnitineamide - Google Patents

Abstract

PURPOSE:To produce D-carnitine and L-carnitineamide in high efficiency by acting on DL-carnitineamide etc. microorganisms which specifically derived D-carnitineamide-hydrolase or an enzyme preparation therefrom. CONSTITUTION:Firstly, microorganisms capable of producing either D-carnitine or both D-carnitine and L-carnitine from racemic carnitineamide [e.g., classified as Pseudomonas sp. CA27B1 (FERM No.8912)] is put to culture in the presence of D-carnitineamide to obtain such microorganisms as to be specifically enhanced in D-carnitine-amide-hydrolase activity or an enzyme preparation therefrom. Thence, said microorganisms or enzyme preparation therefrom is acted on D-carnitineamide or a mixture of D-carnitineamide and L-carnitineamide to produce the objective D-carnitine, the other objective L-carnitine-amide unreacted being left.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to D-carnitinamide or a mixture of D-carnitinamide and L-carnitinamide, such as DL-carnitinamide.
The present invention relates to a method for producing D-carnitine or D-carnitine and L-carnitinamide from carnitinamide by enzymatic hydrolysis reaction. D-carnitine produced according to the present invention is a substance that antagonizes the effect of L-carnitine on fatty acid metabolism, and is therefore a compound that has the potential to be used as a medicine and as an intermediate for pharmaceutical synthesis. It also serves as an intermediate for the synthesis of other useful quaternary ammonium compounds (for example, surfactants). Also, since it has a carboxyl group and a hydroxyl group, it can also be used as an intermediate in the synthesis of various optically active compounds. L-carnitinamide becomes L-carnitine through hydrolysis, and L-carnitine is a compound necessary for the metabolism of higher fatty acids, which is essential for the transport of higher fatty acids to the mitochondria, and is associated with ischemic heart disease and lipemia. It is used as a therapeutic agent and a component of high-calorie infusions.

Conventional technology - Carnitine is produced by optically resolving racemic carnitine (for example, Japanese Patent Publication No. 43-8248).
-The only known method is to obtain it as a by-product of carnitine production.

Problems to be Solved by the Invention and Means for Solving the Problems The present inventors discovered that although D-carnitine is a useful compound as described above, the only production method is to optically produce racemic carnitine. Since this is a complicated method of dividing, we researched a more efficient production method and found that when it is applied to DL-carnitinamide, which is an intermediate in the racemic carnitine synthesis method, it acts on monocarnitinamide and converts D-carnitine into D-carnitine. An enzyme that produces but does not interact with L-carnitinamide, D
- As a result of discovering and isolating microorganisms that produce carnitine and hydrolase, and researching ways to increase the production of this enzyme, we found that by culturing the enzyme-producing bacteria in a medium containing D-carnitinamide, D-carnitinamide was produced. - We have completed the present invention by discovering that hydrolase activity is specifically induced and produced with high activity.

Effect The method of the present invention is similar to the method of the invention (
D-
What is the method for efficiently producing carnitine and L-carnitinamide? /G3's. The method disclosed in Japanese Patent Application No. 1983 hydrolyzes carnitinamide to produce carnitine, and the process of the present invention involves hydrolyzing carnitinamide to produce carnitine, and allowing active microorganisms to act on carnitinamide to produce carnitine. D-carnitinamide hydrolase, an enzyme that specifically hydrolyzes to produce D-carnitine but does not act on L-carnitinamide, is produced with high activity, so D is used as a specific enzyme inducer. - Cultivating the microorganism in a medium in the presence of carnitinamide. Thus, when a microorganism that specifically produces D-carnitinamide hydrolase with high activity or a microorganism derived from the microorganism iD-carnitinamide is reacted with a mixture of lunitinamide, for example, DL-carnitinamide, D-carnitinamide is produced. is produced and L-carnitinamide remains, so both compounds can be separated and recovered from the reaction solution.

The microorganisms used can be selectively isolated based on their ability to produce carnitine from carnitinamide in nature. Specifically, the bacterial strain CA32-C was isolated by the present inventors.

0A-10-1-5, CA27E1, (!A3゜-11
B, 0A2B-50A, and 0A30-35. These strains were isolated by the present inventors, and the present invention (
It has been deposited with the National Institute of Microbial Technology as a representative method that can be used, and its taxonomic properties are as follows.

These isolates are apparently Durham-negative, aerobic bacilli, and have been tested in the Eer-gey'a Manu Systematic Bacteriology.
al Of Elystematic BaOt
According to eriOIOg7) Volume 1 (1984), from the properties described below, it is classified as Pseudomonadaceae in Classification Section 4 of the same book.
e) Pseudomonas (Paeuaomon)
as) belongs to the genus.

The taxonomic properties of the isolates will be described below based on the common characteristics of the isolates, divided into several groups.

First, to describe the common characteristics of all the isolates, as mentioned above, they are all Durham-negative, aerobic bacilli, and the size ranges from relatively small α5-0.8 x α7-50 microns to 1. There are some that are relatively thick, measuring .2 to 1.5 x 2.4 to 4.2 microns (see Table 1).No polymorphism is observed under normal conditions, and there is no acid resistance. It does not produce spores, is motile, and has extremely flagella. When cultured on broth agar, the colony is small, the periphery is full, the ridge is semi-lenticular to convex, the surface is smooth, the gloss is translucent, milky white to grayish white (C!A30-35
(As noted separately, the bacterial cells are yellow), by liquid culture in broth,
There is no surface growth, and the growth is moderately cloudy. Some bacteria ('OA-32Of: group V, which is a representative strain) produce leaf-shaped deposits, but other strains do not produce deposits.

Gelatin was not liquefied, and Ml't test, vP test, indole production, and starch hydrolysis were all negative. Utilization of inorganic nitrogen sources (nitrates and ammonium salts) were both positive in succinate medium. Both oxidase and catalase are positive, and the O-F test is oxidative.

The utilization of citric acid was positive in all cases in the 51 mOH medium. In lidmus milk, one group, the representative strain 0A27B11, makes the reaction alkaline and reduces lidmus, but other strains do not change (of course, there is no coagulation or liquefaction).

For acid production from sugar, in addition to those shown in Table 1, CA30-3
All except 5i, L-arabinose, D-mannose, D-fructose, sucrose, maltose, ])
-) Negative for L/haO-s, D-rubit, D-mannose, D-7 lactose, glycerin, and starch.

Hereinafter, based on the properties of the isolates, those with common properties are further classified into groups of ■, ■, m, tv, v, and v+, and their taxonomic properties are listed in Table 1.

Table 1 "Bacterial bodies: yellow Table 1 (Continued)" In the 5tani13r method, bacteria having the above-mentioned properties are isolated one by one for each group. These bacteria were identified in the Baseise Manual of Systematic Bacteriology, Volume 1.
(1984), all were recognized to belong to the genus Pseudomonas.

Group 1 is Pseudomonas putida (P, putida).
), and Pseudomonas brachiilii (P.

delafieldii), but differs from the former in the accumulation of poly-β-hydroxybutyric acid, and from the latter in the production of water-soluble pigments and growth at 4°C, and there are no other corresponding bacterial species. Pseudomonas sp.
domonas sp, ) and representative method C.
A27E1i was deposited with the Microbial Technology Research Institute C (hereinafter abbreviated as FAI). ■The group shares many properties with Pseudomonas blaphyllii, including the use of arginine,
It varies depending on the growth.

It is positive for hydrogen sulfide production and grows well at pH 5.

Since there is no corresponding bacterial species, Pseudomonas species (Pse
udomonas sp, ) and representative method 0A50-11Ej (deposited at the National Institute of Fine Technology).
It differs depending on the production of water-soluble pigment and growth at 4°C. There is no corresponding bacterial species, and Pseudomonas species
sp, ) to identify ~ representative method 0A28-50At
Deposited at the National Institute of Fine Technology.

■The group differs from Pseudomonas blaphyllii in the use of arginine, inositol, and growth at 4°C.
omonas sp, ) and one representative strain CA
10-1-5i deposited with the Institute of Fine Technology.

Group V is Pseudomonas alcaligenes (Pseu-d
omonas alcaligenes), but they differ in the utilization of glucose and arginine, and as there is no corresponding bacterial species, Pseudomonas species (Pse.
udomonas sp, ) and representative method 0
A32-C was deposited with the Microtech Institute. ■The group requires growth factors for growth, and the fact that the bacterial cells are yellow is that Xanthomonas (
It appears to be Xanthomonas) ii Ic liJ, but since the absorption of the dye does not identify it as xanthomonasin, it was thought to belong to the genus Pseudomonas.

In addition to the sugars listed in Table 1, the strains of this group contain a large number of sugars (L
-arabinose, D-mannose, D-7 lactose,
Acid is produced from D-galactose, maltose, sucrose, trehalose, D-mannitol, inositol). No bacterial species corresponding to the genus Pseudomonas was found, and the species of the genus Pseudomonas (pseudomona8sp-) was found.
Identified with representative method C! A30-35 was deposited with the Microtech Institute.

What is the name of the strain deposited with the Microtech Institute and the deposit number of the Microtech Institute? The corresponding expression is as follows.

0A27B1 ... Microtechnical Research Institute No. 8912 CA
30-11B...Microtechnology Research Institute No. 8910C! A
2 B -5OA --- Microtechnical Research Institute No. 89o9 OA
10-1-5 ... Microtechnical Research Institute No. 8908 ch32-
c ... Microtechnical Research Institute No. 8911 C! A30-3
5...Feikoken Kyoiku No. 8907 Both wild strains and mutant strains of these microorganisms can be used, and enzymes extracted from the microorganisms or their culture solutions can also be used in the present invention. Furthermore, immobilized enzymes and immobilized microorganisms having this enzymatic activity can of course also be used.

In order to culture these microorganisms and obtain the necessary preparations with high monocarnitinamide hydrolase activity, D-carnitinamide is added to the basal medium containing the carbon source, nitrogen source, and inorganic salts necessary for the growth of the microorganisms used. Microorganisms are cultured in the added medium. The concentration of D-carnitinamide used in the medium is usually preferably 0.1 to 2%. Concentrations below or above this concentration range can be used, but below this range, the enzyme activity of the microbial culture observed will be low. Moreover, above this concentration range, the influence of added D-carnitine on the cost increases.

To generate carnitine from carnitinamide,
If microorganisms are cultured in a medium containing carnitinamide or carnitine, a culture with high conversion activity will be observed, but for example, if racemic carnitine or carnitinamide is used, it will act on D-carnitinamide and convert it into L-carnitinamide. Not only D-carnitinamide hydrolase, which does not act on L-carnitinamide, but also L-carnitinamide hydrolase, which acts on L-carnitinamide.
- Since the enzyme that produces carnitine, L-carnitinamide hydrolase, is also induced, a mixture of D-carnitinamide and L-carnitinamide, such as DL
- When microorganisms or enzymes derived from microorganisms are allowed to act on carnitinamide, L-carnitine is also produced in addition to D-carnitine, lowering the optical purity of D-carnitine as a product. This was not clear in studies using commonly obtained 1:1 L-carnitinamide or DL-carnitine, but was clarified for the first time by the present inventors in studies using optically active carnitinamide or carnitine. be.

By culturing the bacteria in the presence of D-carnitinamide in the culture medium of the bacteria used, the production of D-carnitinamide hydrolase is specifically increased, and at the same time, the percentage of D-carnitine in the product carnitine (optical It is the intention of the present invention to increase the purity. Therefore, even if other carnitine-related substances N, t, D-carnitine and L-carnitine are present in addition to D-carnitine, D
-There is an effect of adding carnitine, but D-carnitine and L
- It is disadvantageous that carnitine has little inducing effect on the production of D-carnitinamide hydrolase. Furthermore, when L-carnitinamide is present at the same time, L-carnitinamide hydrolase is also induced and produced, which is disadvantageous for obtaining D-carnitine with high optical purity. Therefore, it is desirable to use a substantially pure carnitinamide.

The microorganism with high D-carnitinamide hydrolase activity prepared as described above or the enzyme preparation prepared thereby i. may be added and cultured until the reaction progresses, but
For culturing microorganisms as enzyme preparations, a substrate may be added to the culture solution of the microorganisms for reaction.Also, enzyme preparations, cells, washed bacteria, freeze-dried cells, and acetone isolated from the culture solution of microorganisms can be used. It is also possible to contact the substrate in the form of dried bacterial cells, physicochemically or biochemically treated bacterial cells, extracts, purified products, immobilized preparations, etc.

The substrate concentration varies depending on whether it is a batch method or a continuous method, but in a batch method, it is generally 0.1 to 40% in the medium.
Preferably, it is about 15 to 20%, and in a continuous system, it is better to lower the concentration more than this.

The reaction is normally carried out at a temperature of 5 to 60°C, preferably around 25 to 40°C, and at a pH of around 4 to 10. Reaction time: standing still;
The batch method usually takes about 1 to 100 hours, although it varies depending on the means of stirring and flowing down, and the form and potency of the enzyme preparation.

Progress of the reaction is monitored by thin layer chromatography to monitor the production of D-carnitine. The ratio of the 5- and 9-isomers in the total carnitine or the optical purity can be determined by separating carnitine from the reaction solution by silica gel atomography.
- After conversion of phenylalanine to carnitinamide,
It can be determined by high performance liquid chromatography based on the area ratio of both L-lunitinamide of L-phenylalanine and D-carnitinamide of L-phenylalanine. After the reaction is completed, carnitine is recovered by known carnitine purification methods. For example, by applying the reaction solution to an ion exchange resin column,
D-carnitine chloride is recovered by elution with dilute ammonia water and concentration.

Next, examples of the present invention will be shown.

Example 1 2 parts glucose, 7' corn stew! 7 car 2%
, Na(,t α5%, K, HPO, 0,75%, KH
2PO40, 25%, MgSO4-7H,O0,01%
, FeSO4-7H20L-forcernitine hydrochloride or D
- Medium containing carnitine hydrochloride at a concentration of 30 -
Place each in a 300-capacity Erlenmeyer flask and sterilize it.
Pseudomonas sp. 1140A50-11B and O
Al 0-1-5f: Inoculated and cultured at 26° C. with shaking at a speed of 220 revolutions per minute. Bacterial cells were collected from the culture solution by centrifugation and diluted with phosphoric acid 94.4%, D-body = L-body ratio, 9
a4: 1.6) The amount of D-carnitine produced when suspended in a phosphate buffer containing a concentration of t-5 (same volume as the culture solution volume for raw stomach culture) and allowed to react at 26° is It was as shown in Table 1. That is, for the production of D-carnitinamide hydrolase, an enzyme that produces D-carnitine from D-carnitinamide, L-carnitine and D
- There is virtually no effect of carnitine; on the other hand, bacterial cells cultured in a medium containing carnitinamide have a high activity of D.
-Carnitine amide hydrolase was produced, and its action on D-carnitine produced D-carnitine at a high concentration.

Table 1 Example 2 In a medium containing 9-island nitinamide at the concentrations shown in Table 2, strain 0A28-% as in Example 1, pH 7,
The production rate of total carnitine and the rate of D-carnitine in total carnitine when reacted for 12 hours in a phosphate buffer of 0.0 was as shown in Table 2. Although the strain used originally had a high ability to produce L-carnitinamide hydrolase and a low ability to produce D-carnitinamide hydrolase, the effect of D-carnitine on D-carnitine production was demonstrated.

Table 2 °C Example 3 Strain 0A50-11B at a concentration of 0.5% plus the following medium:
The total carnitine production rate and the proportion of D-carnitine in total carnitine when inoculated with OAI 0-1-5 and reacted were as shown in Table 3. D
-In addition to the produced carnitine, L-carnitinamide, which remained unreacted, was found in the reaction solution obtained by reacting live-stomached bacterial cells with carnitine aclidemide m at the residual rate shown in the table. It was done. The bacterial cells were removed from the reaction solution, and D-carnitine and L-carnitine amide were recovered by adsorption onto a cation exchange resin and elution.

Table 3 ■ Effects of the present invention on DL-force nitinamide base As mentioned above, the present invention has various uses for D-
It can be efficiently produced from carnitine, L-carnitinamide 1r, DL-arnitinamide, which is an intermediate for DL-carnitine synthesis, and the like.

Patent applicant: Pyall Co., Ltd. Representative Kiyoshi Nakayama Chuo Kaseihin Co., Ltd. Representative Kisanbe Mizushima

Claims (1)

[Claims] 1. D-carnithiamide hydrolase is produced by culturing a microorganism capable of producing D-carnitine or D-carnitine and L-carnitine from racemic carnitinamide in the presence of D-carnitinamide. The specifically induced culture or the enzyme derived from the culture is
D-carnitine, or D-carnitine and L-carnitine, which is produced by reacting carnitinamide or a mixture of D-carnitinamide and L-carnitinamide to produce D-carnitine, leaving unreacted L-carnitinamide. Method for producing carnitinamide. 2. The microorganism used is Pseudomonas (￥Pseu￥-
The method according to claim 1, wherein the microorganism is of the genus Domonas.