JPS62134093A - Production of carnitine - Google Patents

Abstract

PURPOSE:To efficiently convert an alkyl ester derivative of dehydrocarnitine into the aimed substance by using a microorganism of the genus Bacillus, etc., in an aqueous medium in a method for converting the alkyl ester derivative of the dehydrocarnitine into the carnitine by action of a microorganism. CONSTITUTION:A microorganism, belonging to any of the genuses Bacillus, Ashbya and Penicillium and having the ability to convert a (3-alkoxycarbonyl-2- oxopropyl)trimethylammonium halide into carnitine, etc., is cultivated in a culture medium containing the former substrate to give microbial cells, extract solution thereof or purified enzymic specimen. The above-mentioned microbial cells, etc., are reacted with the substrate (3-alkoxycarbonyl-2-oxopropyl) trimethylammonium halide and the reaction solution is subjected to an ion exchange resin column, eluted and concentrated to afford the aimed carnitine.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing carnitine 1x by allowing microorganisms to act on ro-alkoxycarbonyl-2-oxopropyl)trimethylammonium halide. Carnitine produced according to the present invention has been called vitamin BT as a substance related to fatty acid metabolism, and is a useful substance used as a medicine for heart diseases and the like. Conventionally, the DL isomer has been used as a medicine, but recently the L isomer has been attracting particular attention (for example, History of Medicine, 1).
25, p. 660, 1982).

BACKGROUND OF THE INVENTION Various methods have been known for producing carnitine, but each method has its own problems and is difficult to achieve industrially. A method closely related to the present invention is a method for producing carnitine by enzymatic reduction of 3-dehydrocarnitine (App'1. Environ, Microb
io:L, vol. 39, p. 327, 1980). The disadvantages of this method are that it is necessary to combine a method of recycling the auxiliary element, that the synthesis of the raw material 3-dehydrocarnitine is complicated and expensive, and that 3-dehydrocarnitine is unstable. It is.

Problems to be Solved by the Invention and Means for Solving the Problems As described above, the present inventors have solved the conventional carnitine production method,
In particular, the production method for L-carnitine has some drawbacks as an industrial production method, so we conducted research to establish a more efficient production method for L-carnitine.In particular, 3-dehydrocarnitine is unstable, and its synthesis is difficult. To overcome the complexity,
Its alkyl ester, (3-alkoxycarbonyl-2-oxopropyl)trimethylammonium halide, is easy to synthesize (for example, published in Japanese Patent Publication No. 51-6312
In 5), we focused on its stability and researched a method to obtain L-carnitine by enzymatic reduction. As a result, they discovered that various microorganisms convert (3-alkoxycaribonyl-2-oxopropyl)trimethylammonium halide into carnitine ester, and filed a separate patent application. Furthermore, it was discovered that a microorganism that has an enzyme that decomposes carnitine ester and converts it into carnitine can produce L-carnitine from (3-alkoxycarbonyl-2-oxopropyl) trimethylammonium halide all at once, thereby completing the present invention. It's arrived.

Effects and Operations of the Invention According to the present invention, (5-alkoxycarbonyl-2-oxopropyl)trimethylammonium halide, which can be easily obtained by trimethylamination of industrially available γ-haloacetate, can be converted into carnitine at once. When microbial cells (including immobilized microbial cells) are used, the coenzyme regeneration system of the microorganism itself can be utilized, providing an extremely advantageous method for producing carnitine.

The microorganism used in the present invention can be used regardless of its classification position as long as it has an enzyme system capable of converting (3-alkoxycarbonyl-2-oxopropyl)trimethylammonium halide into L-carnitine. That is, as shown in the Examples, Pseudomonas (Pθ
θ-udomonas) genus, Bacillus
Examples include microorganisms such as the genus Ashbya, genus Penicillium, and the genus Penicillium.

Cells obtained by culturing microorganisms with such abilities in a medium or their processed products are (3-alkoxycarbonyl-
When catalytically reacted with 2-oxopropyl) trimethylammonium halide, carnitine is converted. 1. The cultivation method for obtaining the microorganisms used in the present invention may be any conventional cultivation method, and does not require any particular explanation, but (5-alkoxycarbonyl-2-okineprobyl)trimethyl used as a substrate may be used. When microorganisms are grown in a medium containing ammonium halide, cells with high conversion activity can be obtained. Either a solid medium or a liquid medium can be used.

The bacterial cells obtained in this way may be allowed to act on the substrate, or a bacterial cell extract, a purified enzyme preparation, or an immobilized preparation thereof may be conjugated with a coenzyme regeneration system and allowed to act on the substrate. . Alternatively, a substrate may be added to the culture solution for growth culture and reacted without separating the microbial cells from the culture solution.

The substrate concentration varies depending on whether the method is a batch method or a continuous method, but in a batch method it is generally about 0.1 to 30%, preferably about 0.5 to 10%, and in a continuous method it is less than this. It is better to lower the

The reaction is generally carried out at a temperature of 10 to 60°C, preferably around 25 to 45°C, and at a pH of around 4 to 1o. The reaction time varies depending on the method of standing, stirring, flowing down, etc., and the form and activity of the enzyme preparation, so it is not uniform, but in a batch method, it is usually 50
The time is approximately 72 minutes to 72 hours. The reaction can be tracked, for example, by thin layer chromatography using a solvent system of n-propanol: 38% aqueous ammonia: water (85:5:10) and the concentration of Rfo, 13 spots generated by spraying with Dragendorff's reagent. In addition, the produced carnitine can be analyzed by high-performance liquid chromatography or by the method of Pehan et al. (MethocL).
of EnzymaticAnalysis Part 2
It can also be traced by analysis using L-carnitine acetyltransferase according to the method described in 1974).

After the reaction is completed, υcarnitine is recovered by a known method such as applying the reaction solution to an ion exchange resin column and eluating and concentrating.

Next, examples of the present invention will be shown.

Example 1 A large test tube containing 5 d of the medium shown in Table 1 was inoculated with the microorganisms shown in Table 2, and cultured at 26°C for 48 hours for bacteria and 7 days for mold.

After washing the bacteria obtained from culture solution 5 by centrifugation or filtration twice with water, add phosphate buffer Q, pH 7.0, containing (3-ethoxy-2-oxopropyl)trimethylammonium chloride at a concentration of 1 ozg/rd, 5 ．． - was added, and the reaction was carried out by shaking culture at 60°C for 18 hours. As a result, L-carnitine was produced as shown in Table 2.

KH2PO4' 2.5 //
8. Q ItMgSO4・7H200,1tt
Q, 1ttFeSO4・7H400,01/I
O,01//NH4CL
1.0 // 1. Q nNaCA
5. Q tt 5. O
tt glucose 10. Ott 10. O
tt meat extract 5. Q // 5. Q
//Polypeptone 5. □ p 5. OW
Yeast extract -30〃 Substrate cage IG, O// 10.0 /
1 Methylammonium chloride 2nd sign Organism L-lunitin producing Pseudomonas cru a, 3w+g/r
dSipie (Pseudomonas cruciviae) Engineering FO12047 Paterseuscereus Q, 3 /1 (Ba
cilliuBcereus) ATCo 9139 Ashveer Gossippy 1.0〃(Ash
bya gossypi soil) engineering FO0560 Penicillium trzebinskii 1.0
ATC! 010507 Example 2 In Example 1, (3
-Methoxycarbonyl-2-oxopropyl)trimethylammonium chloride was used in the same manner as in Example 1, and L-carnitine was produced in any of the microorganisms shown in Table 1.

Example 3 Bacillus cereus ATO grown by the method of Example 1
Add 10ff17 of 4% sodium alginate solution to IL0d suspended in physiological saline to a concentration of C9139 cells 'i 200 Q/mt! After adding and mixing, 1
This mixed solution was gradually added dropwise to 5 g of calcium chloride solution to completely cover the granular immobilized bacterial cells. This total amount of immobilized bacterial cells is
(5-ethoxycarbonyl-2-oxopropyl)trimethylammonium chloride 1%, glucose 1 part,
When the mixture was added to 20 ml of phosphate buffer at pH 7.0 containing 0.1% magnesium sulfate and reacted at 30°C for 18 hours, L-carnitine was produced at a concentration of 13 vg/L.

Example 4 Using Bacillus cereus ATCO9139i, Example 1
The supernatant liquid (300 mA) obtained by removing the bacterial cells from the reaction liquid carried out under the same conditions as above was subjected to ion cross-fertilization (J fat (Dowex 50
) column, eluted with dilute hydrochloric acid, concentrated, and added ethyl alcohol to obtain 45m9 crystals of L-carnitine chloride.1 Patent distributor: Representative of Piall Co., Ltd. Presentation: Representative of Sei Chuo Kasei Co., Ltd. Yoshi Mizutori Three parts

Claims (1)

[Scope of Claims] 1, (3-Alkoxycarbonyl-2-oxopropyl)trimethylammonium halide in an aqueous medium by the action of a microorganism or an enzyme system derived therefrom that has the ability to convert trimethylammonium halide into carnitine. 1. A method for producing carnitine, which comprises converting alkoxycarbonyl-2-oxopropyl) trimethylammonium halide into carnitine. 2. The microorganism used is Bacillus (￥Bacillus￥
2. The production method according to claim 1, wherein the microorganism is a microorganism belonging to any one of the genus ), Ashbya genus, and Pen-icillium genus.