JPH01222796A - Production of l-carnitine - Google Patents

Abstract

PURPOSE:To efficiently obtain L-carnitine of high optical purity by making a culture of specific microorganisms in the presence of L-carnitine to specifically derive L-carnitine-amide-hydrolase and by acting this hydrolase on DL- carnitineamide. CONSTITUTION:Firstly, microorganisms capable of producing either L-carnitine or both L-carnitine and D-carnitine from racemic carnitrineamide [e.g., classified as Pseudomonas sp. CA27B1 (FERM No.8912)] is put to culture in the presence of L-carnitine to obtain such microorganisms as to be specifically enhanced in L-carnitine-amide-hydrolase activity or an enzyme preparation therefrom. Thence, said microorganisms or enzyme preparation therefrom is acted on a mixture of L-carnitineamide and D-carnitineamide normally at 5-60 deg.C and pH of 4-10 to produce the objective L-carnitine.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing L-carnitine by enzymatically asymmetrically hydrolyzing racemic carnitinamide. L-carnitine produced by the present invention was once called vitamin BT as a substance related to fatty acid metabolism. Conventionally, the racemic form has been used in stomach-healthy medicines, etc., but recently the L-carnitine has attracted particular attention, and It is useful as a therapeutic agent for diseases and lipidemia, and is also used as a high-calorie infusion. Since it has been found that D-carnitine antagonizes the effect of L-carnitine on fatty acid metabolism, there is currently a high demand for L-carnitine that does not contain D-form.

Conventional methods for producing carnitine include a method of optically resolving racemic carnitine (for example, Japanese Patent Publication No. 43-8248), 5
- Enzymatic asymmetric reduction of dehydrocarnitine (Appl
, Environ, Microbiol, 39
Vol. 529, 1980), a method via 4-chloro-3-hydroquine butyrate (Japanese Patent Application Laid-Open No. 59-118
093), a method using crotonobetaine as a substrate (JP-A-59-'183694).

JP-A-59-11' 8095, JP-A-59-192
095. JP-A-6O-137295), method for dihydroxylating γ-butyrobetaine using microbial enzymes (JP-A-57-39791), asymmetric hydrolysis of DL-o-A VIL/carnitine by esterase (Biotechn
ol, Bioeng, ) vol. 26, p. 911, 198
4 years) are known. These methods are industrially unsatisfactory because the raw materials are expensive, the enzymes are unstable, and the supplementation of expensive coenzymes is required. The present inventors have developed a new L-
After repeated research to establish a method for producing carnitine, he invented a method for biochemically hydrolyzing carnitinamide and filed a separate application 7'C (Patent Application No. 61-19a173). Problems and problems that the invention aims to solve Means for Solving the Problems The above-mentioned separate invention of the present inventors is a method for enzymatically hydrolyzing carnitinamide, which is an intermediate of a very efficient carnitine synthesis method starting from epichlorohydrin, to obtain L-carnitine. This is an excellent method in which synthesis steps and resolution are performed in one step and L-carnitine with high optical purity can be obtained, but the present inventors have further improved this method.

In other words, we continued improvement research with the goal of increasing the purity of L-carnitine in the product and making the process more efficient by increasing the activity of the enzyme preparation used. We researched the cultivation method of microorganisms used to act on mixtures of amides, such as racemic carnitinamide, and found that the microbial culture ~enzyme preparation specifically decomposed only L-carnitinamide and improved the optical purity. The present inventors have completed the method of the present invention for obtaining a high amount of L-carnitine.

Effect The method of the present invention is similar to the method of Japanese Patent Application No. 198173/1983, in which microorganisms having the activity of hydrolyzing carnitinamide to produce nithine are made to act on carnitinamide to generate a force of 2,000 μm. However, especially L
- specifically enhances L-carnitinamide hydrolase (patent application 1981-091938), an enzyme that specifically hydrolyzes ditinamide to produce L-carnitine but does not act on D-carnitinamide. In order to produce a high concentration of L-carnitine, microorganisms are cultured in a medium in which L-carnitine is present as a specific inducer.

The microorganisms used can be selectively isolated from nature based on their ability to produce carnitine from carnitinamide. Specifically, the bacterial strains CA32-C, CA-10-1-5, and CA271 that were isolated by the present inventors
31, CA30-11. B%CA2B-50A%CA
I can give you 30-55. These strains were isolated by the present inventors and deposited at the National Institute of Microbial Technology as representative strains that can be used in the present invention, and their taxonomic properties are as follows.

All of these isolates are Durham-negative, aerobic bacilli, and are described by Bergey's Manual Systematic Bacteriology.
l of Systematic Bacteriol
ogy), Volume 1 (1984), it belongs to the genus Pseudomonas in the family Pseudomonadaceae in classification section 4 of the same book, based on the properties described below.

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, all of the above are Durham-negative, aerobic bacilli, and the size is relatively small at 0.5-0.8 x 0.7-50 microns. They range from small ones to relatively large ones of 1.2 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 golden, the ridge is semi-lens-convex, the surface is smooth, the gloss is translucent, milky white to grayish white (CA30
Only in case of -35, the bacterial cells are yellow (as noted separately), and the growth is moderately cloudy with no growth on the surface when cultured in broth liquid. Some bacteria (group V, of which CA-520 is a representative strain) produce leaflet-like sediments, but other strains do not produce sediments.

It does not liquefy gelatin, and the MR test, VP test, indole production, and starch hydrolysis are all negative. Utilization of inorganic nitrogen sources (nitrates and ammonium salts) were both positive in succinate medium. Oxidase and catalase are both positive, and O-F tenuto is oxidative.

When using citric acid, Simon's medium results in positive reduction, but there is no change in other strains (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 5 are L-arabinose, D-mannose, D-fructose, sucrose, my)-s, D
-Trehalose, D-sonovit, D-mannose,
Negative for D-fructose, glycerin, and starch.

In the following, strains with common characteristics are further classified into groups based on the characteristics of the isolates: i, n, m, ■, v, and vx.

The classification properties of the groups are listed in Table 1.

Table 1: Bacterial bodies: yellow Table 1 (Continued) In the Tatami 5tanier method, bacteria with the above properties have been isolated in numbers for each group.・Bacteriology, Part 1
(1984), all were recognized to belong to the genus Pseudomonas.

Group 1 is Pseudomonas putida (P, putidaλ)
and Pseudomonas brachiilgii (P.

delafieldii), but the former differs from the latter in its accumulation of poly-β-hydroxybutyric acid, and 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 the representative strain C
A27B1 was designated by the Microbial Technology Research Institute (hereinafter referred to as the Institute of Microbial Technology).

(abbreviated as Shimo-Koken). ■The group shares many properties with Pseudomonas blaphyllii, but differs in the use of arginine and growth at 4°C.

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 the representative strain CA30-11B was deposited at the Microtech Institute. The ■ group also shares many properties with Pseudomonas blaphyllii, but differs in the production of water-soluble pigments and growth at 4°C. There is no corresponding bacterial species, and Pseudomonas species
sp, ), and the representative strain CA28-5OA was deposited at the Microtech Institute. The group ■ differs from Pseudomonas blaphyllii in the use of arginine, the use of inositol, and growth at 4°C, and since there is no corresponding bacterial species, it was identified as Pseudomonas sp., and the representative strain CA10-1- 5 was deposited with the National Institute of Fine Technology. 7
The group is Pseudomonas alcaligenes (Pseu-do
monas alcaligenes), but differs in the utilization of glucose and arginine, and there is no corresponding bacterial species, so Pseudomonas species (Pseudomonas species)
Domonas sp, ) was identified as a representative strain CA.
32-C was deposited with the National Institute of Fine Technology. Xanthomona 7. (
Although it appears to belong to the genus Xanthomonas, since the absorption of the dye did not identify it as xanthomonadin, it was assumed that it belonged to the genus Pseudomonas. The strains of this group contain many sugars besides diabetes listed in Table 1 (L-abbinose, D-mannose, D-fructose, D-galactose, maltose, sucrose, trehalose, D-mannitol, inosyto-/I). /) to produce acid. No bacterial species corresponding to the genus Pseudomonas was found, so it was identified as a species of the genus Pseudomonas (Pseudomonas sp-), and the representative strain CA30-35 was deposited at the Microtech Institute.

The correspondence between the name of the strain deposited at the Institute of Fine Technology and the deposit number of the Institute of Fine Arts is as follows.

CA27B1 ... Hui Koken Bacteria No. 8912 CA
30-11B --- Microtechnical Research Institute No. 8910 CA 28
-5 OA --- Microtechnical Research Institute No. 8909 CA10-1
-5-----Feikoken Bakuyori No. 89o8 CA! +2-C・1
Both wild strains and mutant strains of these microorganisms can be used, and enzymes extracted from microorganisms or their culture fluids are also applicable to the present invention. Can be used for 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 required preparations with high L-carnitinamide hydrolase activity, L-hydrochloride is added to a medium containing carbon sources, nitrogen sources, and inorganic salts necessary for the growth of the microorganisms used. Microorganisms are cultured in a medium to which 1,000 liters of microorganisms are added. The concentration of L-carnitine used in the medium is usually 0.
1 to 2% is desirable. It can be used at concentrations below or above this concentration range, but below this concentration range, the enzyme activity of the resulting brave organism culture will be low. Further, above this concentration range, the effect of added L-carnitine on the conut becomes large.

To generate carnitine from carnitinamide,
If microorganisms are cultured in a medium containing carnitinamide or carnitine, a high degree of conversion activity can be observed, but for example, when racemic carnitine or carnitinamide is used, it acts on L-carnitinamide and converts it into D-carnitinamide. In addition to L-carnitinamide hydrolase, which does not act on D-carnitinamide, D
- Since the enzyme that produces carnitine is produced by induction of D-carnitinamide hydrolase, when a microorganism or an enzyme derived from it is applied to DL-carnitinamide or a mixture of L-carnitinamide and D-carnitinamide, L- In addition to carnitine, D-carnitine is also produced, which lowers the optical purity of L-carnitine as a product. This was not clear in studies using commonly found DL-carnitinamide or DL-lunitine, but was first clarified by the present inventors in studies using optically active carnitinamide or carnitine. be.

By culturing the bacteria in the presence of L-carnitine in the culture medium of the bacteria used, the production of L-carnitinamide hydrolase is specifically increased, and at the same time, the percentage of L-carnitine in the product carnitine (optical purity) is increased. ) is the intention of the present invention. Therefore, even if other carnitine-related substances such as D-carnitine are present in addition to L-carnitine, the addition of L-carnitine is effective, but D-carnitine has a low inducing effect on L-carnitinamide hydrolase production. Therefore, if the amount of carnitine is the same, the effect is inferior to that when using pure L-carnitine. Thus, it is desirable to use substantially pure L-renitin.

A method in which a microorganism with high L-carnitinamide hydrolase activity prepared as described above or an enzyme preparation prepared therefrom is allowed to act on a mixture of L-carnitinamide and D-carnitinamide is a method in which the enzyme is added to a solution containing the substrate. It is sufficient to add the standard sample and incubate until the reaction progresses, but
For culturing microorganisms as enzyme preparations, a substrate may be added to the microorganism culture solution and reacted.Also, enzyme preparations isolated from the microorganism culture solution, bacterial cells, washed bacteria, freeze-dried bacterial cells, and acetone-dried It is also possible to contact the substrate in the form of physicochemically or biochemically treated bacterial cells, extracts, purified products, immobilized preparations, etc.

The substrate concentration differs depending on whether it is a patch method or a continuous method, but in a patch method, it is generally 0.1 to 40% in the medium.
The concentration is preferably about 0.5 to 20%, and in a continuous system, it is better to lower the concentration by one level.

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. The reaction time varies depending on the means of standing, stirring, and flowing down, or the form and potency of the enzyme preparation, but it is usually about 1 to 100 hours in the patch method.

The progress of the reaction is monitored by tracking the production of L-renitin by thin layer chromatography, or by analyzing L-callenitin by the enzymatic method of Pearson et al. The ratio of L-form and D-form in total carnitine or the optical purity can be determined by separating carnitine from the reaction solution by chromatography on silica gel and converting it into carnitinamide of L-phenylalanine. TeL-
It can be determined by the area ratio of both L-carnitinamide of phenylated p-alanine and D-carnitinamide of L-phenylalanine. After the reaction is completed, the reaction solution is applied to an ion exchange resin column, eluted with dilute aqueous ammonia, and concentrated using a known carnitine purification method.
) Recovered as L-carnitine chloride.

Next, examples of the present invention will be shown.

Example 1 Glucose 2%, corn steeple force - 2%.

NaCA o, 5%, K2HPO4G, 75
%, KH2PO40,25%.

MPSO4-7H, O0,01%, Fe3O3-7H
L-carnitine, D-carnitinamide or D-carnitine was added to the basal medium with a composition of 200,001% at the concentrations shown in Table 1.
Medium containing either carnitine (both hydrochloride) (p
Pseudomonas bacterium CA28-5 was added to the sterilized sterilized 30-mL Erlenmeyer flask.
Inoculate 0A and incubate at 26°C and 72°C at a speed of 220 revolutions per minute.
The cells were cultured for a period of time. From the culture solution - Bacterial cells were collected by centrifugation, washed twice with phosphate buffer (pH 7,0), suspended in the same amount of culture solution (the same volume as the culture solution), and incubated at 26°C for 72 hours. As a result, L-carnitine was produced at the production rate shown in Table 1. The production rate of L-carnitine by bacterial cells from a culture without these compounds added to the growth culture was 2.9, which was not completely pure and contained a trace amount of L, but L-carnitine/(hydrochloride) ) of L-
It is clear that the effect on carnitine production is significantly higher than that of D-carnitinamide (&)'J or D-carnitine (hydrochloride) used. What is even more noteworthy is that
It is the percentage of L-μnitine in the total carnitine produced.

That is, when L-carnitine (hydrochloride) was used as an inducer of the enzyme, the ratio of L-carnitine was 99X. D-force) v When Nisenine (hydrochloride) is used, the production rate of L-carnitine is low, and L-carnitine in the total carnitine is
-The carnitine rate was 95-97%, which was a rather low value.

Table "'+ l@fJi9 a, 4X (DB: LKa
=9 a4: 1.6, purity 99.48% (
D-form: L-form ratio = 99.48: (152) Example 2 F solution from which bacterial cells were removed from the reaction solution carried out in Example 1 using a medium containing L-carnitine hydrochloride (1.5%) p
After adjusting to H1,0 with hydrochloric acid, it was passed through a cation exchange resin column, the adsorbed L-carnitine was eluted with 2% aqueous ammonia, and the eluted fraction of L-kat V-nithine was concentrated under reduced pressure to dryness. was dissolved in warm ethanol and cooled to obtain L-carnitine crystals. The optical purity of this crystal, expressed as optical isomer excess (ee), was 98%.

Example 3 Glucose 1%, Peptone CL 5%, Meat Extract Q, 5%
, NaC2Q, 5%, NH4Ct0.1%, KH2
O4Q, 75%, KH2PO4Q, 25%, Mr
SO4-7E (20Q, 01%, Fe3O3”7H
Pseudomonas strains shown in Table 2 were planted in a medium (pH 7.2) with a composition of 1% 200,00 and 15% L-carnitine hydrochloride, and cultured with shaking at 26°C at a speed of 295 revolutions per minute. After that, the bacterial cells were collected by centrifugation, washed twice with phosphate buffer (pH 7,0), and then suspended in DL-carnitinus 0 phosphate buffer (same volume as the culture solution volume for the growth culture). The production rate of L-2,000 ton when the reaction was allowed to stand at 26° C. was as shown in Table 2. In addition, when using bacterial cells cultured in a medium from which L-carnitine hydrochloride was removed from the above medium, the lunitin production rate was 2.0 to 2.9% after 144 hours of reaction.

Outer Table Reference Examples Carnitine hydrochloride or D-carnitine hydrochloride 1.0
Pseudomonas bacterium CA3 in culture medium added to a concentration of %
0-11B and CAi O-1 body ratio 9a4:
1.6) in a phosphate buffer solution containing 5% concentration (same volume as the culture solution volume for growth culture) and allowed to stand at 26°C for a static reaction. Table 3 shows the amount of nitine produced. It was shown in . That is, p-force μ = L-carnitine and D-carnitine have no effect on the production of D-carnitinamide hydrolase, an enzyme that generates D-carnitine from tinamide; It has been shown to be effective.

Table Power / Upa'.

Effects of the Invention As described above, the present invention produces L-carnitinamide with high optical purity by allowing enzymes derived from microorganisms to act on DL-carnitinamide, which is an intermediate for carnitine synthesis. The present invention provides an industrial method for producing L-runitin, which efficiently produces renitin and is in demand as a raw material for pharmaceuticals and other uses.

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

Claims (1)

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