JPS61293392A - Production of crotonobetaine - Google Patents

Abstract

PURPOSE:To produce the titled substance readily and at a low cost, by reacting D-carnitine or L-carnitine or gamma-butyrobetaine with a microorganism belonging to the genus Escherichia, Bacillus, Nocardia, etc. CONSTITUTION:D-Carnitine or L-carnitine or gamma-butyrobetaine is reacted with a microorganism belonging to the genus Escherichia, Enterobacter, Klebsiella, Aerobacter, Erwinia, etc., e.g. Escherichia coli or Enterobacter cloacae. The microorganism is cultivated in a culture medium suitable for each strain and the resultant microbial cells are brought into contact with a solution containing a substrate to afford the aimed crotonobetaine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a method for producing crotonobetaine enzymatically by allowing microorganisms to act on at least one maple compound.

(Industrial Application Field) The target compound, crotonobetaine, is the di & K form of L-carnitine. It is well known that carnitine (β-hydroxy-r-trimethyl-aminoacetic acid) exists in 2s stereoisomers, D-form and L-form. L-carnitine normally exists in living organisms and has an export function as a carrier that allows activated long-chain m-r1h-carnitine to pass through the mitochondrial membrane.

Racemic carnitine has been used as an appetite stimulant, although only left-handed L-carnitine is available in the form of a natural product.

However, recently, it has been revealed that it is more effective to use L-carnitine alone as C for at least some therapeutic uses, and interest in its importance is increasing.

In fact, it is used in the treatment of saturation and chronic myocardial ischemia in the cardiovascular system, angina pectoris, cardiac arrhythmia, or heart failure.

For example, the following method is known as a method for producing optically active carnitene.

(1) A method of optically resolving racemic carnitine nitrile obtained by chemical synthesis. The optical resolution method involves adding C to DL-carnitine nitrile as a precursor using N-acetyl-D-glutamic acid or N-acetyl-L-glutamic acid as a resolving agent to generate a salt.
A common method is to split C using the difference in solubility and then hydrolyze it to form L- and D-carnitine chloride (Japanese Patent Publication No. 8248/1982).

(2) A method of optically resolving racemic carnitine obtained at °C by a chemical synthesis method. D- and L-shi1
Hoppe Sailer's Zeitschrift Physiologem
8eylers Z, Physiol, chern
) Bd, 851°8, 618-622 (197
2) This method is not commonly used.

(8) A method for producing L-carnitine from C-crotonobetaine using bacterial cells obtained by anaerobic culture (Japanese Patent Application No. 187878-1982).

However, in (1) and (2), the optical resolution agent is expensive and the operation is complicated, and in C, it is necessary to recycle the by-product υ-carnitine.

At present, it is considered that the most effective method is to dehydrate D-carnitine, convert it to C-crotonobetaine, and convert it to L-carnitine using the method (8) above. For dehydration, DL-carnitine is treated with acetic anhydride.

Biochimi
caat Biophysica Acta),
187.98 (1967) can be applied. However, this method requires the use of a large excess of acetic anhydride relative to carnitine.

The reaction temperature is also low (a tar-like substance is produced, which causes a decline in the quality and coloration of the product.Furthermore, in order to obtain crotonobetaine from the reaction solution, it is necessary to remove unreacted acetic anhydride and acetic acid produced). For this purpose, an organic solvent such as propatool is required.Furthermore, since this reaction is a non-aqueous reaction, the carnitine used as a raw material is obtained by dividing crystallized carnitine nitrile or carnitine using an ion exchange resin. After separation from the agent, carnitine nitrile must be hydrolyzed to form an aqueous C carnitine solution, and then water must be removed by concentration to dryness.Since carnitine is a hygroscopic substance, drying is the first step.

In the present invention, C is, for example, D-carnitine containing L-carnitine obtained by crystallizing L-carnitine with L-sulfuric acid, and is used as a raw material to obtain crotonobetaine. Furthermore, an aqueous DL-carnitine solution obtained by chemical synthesis can also be used as a raw material, making it possible to provide an inexpensive method for producing L-carnitine. Furthermore, since it is a microbial reaction, the reaction conditions are mild and there is no need to worry about coloring.

When L-carnitine is obtained from crotonobetaine using microorganisms, r-butyrobetaine is produced as a by-product depending on one condition. According to the present invention, by-product r-
It is possible to convert butyrobetaine to crotonobetaine, a monofilamentary compound.

When hydrogen is added to crotonobetaine, it is also useful as a raw material for capronium chloride, which is used to treat various medical conditions such as r-butylene, hypochlorhydria, erectile stools, and decreased parasensory nerve tone. be.

In view of the above circumstances, the present inventors focused on DL-carnitine, which can be produced at a lower cost than epichlorohydrin, and conducted intensive studies with the aim of developing a new method for producing crotonobetaine. It was discovered that crotonobetaine is produced from D-carnitine, DL-carnitine, and r-butyrobetaine, leading to the present invention.

The reaction of the present invention is a novel reaction that has not yet been reported.

(Prior Art) As a method for producing crotonobetaine, for example, the method described below is known.

(1) Method of acting on DL-carnitine with acetic anhydride ■) Method of acting on L-carnitine with Escherichia coli, Proteus vulgaris, or Salmonella typhimurium.

Full 7, j Clobiol (Aroh Microbio)
l) IL2゜91-95 (1982) Fuems,
Microbiology Letters (FEM8 Microbi
ology LetterS) 18.201-20
5 (1982) sZeitschrift, Hühr, Allgeaine Microbio-/l/ (Z, fiir,
Al1g, A11krobiol) IJ, 758
-758 (1979) However, (1) is DL-
It is necessary to use acetic anhydride with a large excess of C relative to carnitine, and the reaction temperature is also high.

Furthermore, in order to obtain crotonobetaine from the reaction solution, it is necessary to remove unreacted acetic anhydride and produced acetic acid.

For this purpose, an organic bath medium such as ethanol is required in the wreck.

Only eight genera of Escherichia, Proteus, and Sarunella are known for C, and the substrate C is L-
Only carnitine was present.

Therefore, the moss of the present invention is cheaper than epichlorohydrin.
1) Focusing on L-carnitine, we conducted a CfQ study with the aim of developing a new production method for Ritonobetaine. It was found that a-tonobetaine was produced. Furthermore, it was discovered that r-butyrobetaine is oxidized to produce crotonobetaine, leading to the present invention. The production of crotonobetaine from D-carnitine and r-butyrobetaine is a novel reaction. To explain one example of the embodiment of the present invention, the following steps are taken: 1. For example Eva, KIl12PO40,8%, K2
HPO40°7%, (NH4)28040.1%,
Mg804・7HzOo, ot%.

One platinum loop of Serratia marcensus (IF03786) inoculum was inoculated from a slant medium into 20 ml of a limb culture medium consisting of 0.5% peptone and 0.5% yeast extract.

The cells were incubated for 2 days at 80°C for 9 seconds. Bacterial cells were obtained from the thus obtained culture solution by centrifugation. The obtained bacterial cells were washed with 5 ml of physiological saline and then subjected to reaction.

For the reaction, the obtained bacterial cells were treated with L-carnitine 1% (62mM).
containing 0.1 M phosphoric acid dlli & (pH 6) 511t
The test was carried out by shaking at 80° C. for 2 days. The resulting reaction solution was centrifuged to remove bacterial cells.
After the remainder was heated at 80'C for 5 minutes, a portion of the treated solution was analyzed by high performance liquid chromatography (L(3)), and gQmM of crotonobetaine was produced.

The microorganism used in the present invention is 2°C, for example Escherichia coli IF03801. Enterobacter cloacanie IF0882Q, Klebsiella pneumoniae IFOa512. Nyalba)y 9-Croacani IAM1134. Erwinia aloideae IFO12880, Serratia marcensus IF08
786, Propus vulgaris LF08851
゜- Narmonella tephimurium IF012529.
Alcaligenes faecalis AT (IC8750,
Flavobacterium Nistyloaromatoicum IF08
751゜Achromobacter Palplus IF01818
1゜Baculas sphaericum XF08526. Agrobacterium radiobacter IFO12664,
Azotobacter violandii IF01201g,
Micrococcus roseus IFOa768. Corynebacterium Rasayu IFO12161, Staphylococcus aureus IF08060. Pseudomonas marginalis IFO8925, Arthrobacter simplex LFO12069, Brevibacterium linens 11?012141. Cellulomonas flavigena IFOlA8. Hafnia alvei IrO8781, Acetobacter orleanens
IrO2259, Chromobacterium iodinum I
F08558. Xanthomonas campestris IFO18i8. Vibrio parahaemorichii”
Kasu XFO12711, Aeromonas hydrophila ll101297g, Protaminobacter alboflavus IFOi3707. Acinetobacter Calcoaceticus IF018006. Citrobacter
Naka7a indie “i 39 knowledge=#≠
≠;IFO1354=4 Bacterium Gracil
IF08281. Clostridium butyricum
IFOf1m85g, Rhizobium japonicum I
FO19888, Gluconobacter selinus IFO82
64, Streptococcus faeris IFO812
8. Hediocophcus bentosasius IF08898
, Leufstock Mescentroides IF034
26. Lactobacillus acidophilus IFO82
05, Propionibac zolium ticunicum IF0
12428. Aerococcus viridans If”0
12817. Mycobacterium avium IF0
8082. Nocardia Asteroides IFO8
423, Streptomyces alps IFO130
14, Mucor racemosus Ii'04581゜ Rhizolapus oryzae 1FO4705, Absidia ferrulea 1FO4011, Phycomyces nitens
IF05695. Eremothecium asibii IF
OO557, Aspergillus niger IrO2416
, Penicillium oxalicum IFOr+74g.

Monascus Anca IFO5965, Neurospora
Cracosa IFO6660, Ospora viscosa 1
FO4604, Pluralia Bernecchi 11'064
Q7. Fusarium solani IFO5282,
Gibberella fujikuroi IFO6604, Ustoiragozeae IFO6907, Keratinominus ajielloi IF07865. Sporothrix dienchii IF05988. Trichoderma viride IFO4
847゜Ferticillium alboatrum Ik
'05922, Isaria Kogane IFO5299
, Bryofradium deliquescens IFO6617
, Trichophyton mentagrophytes IF0546
6. Phytophthora infestans IFO4872
, cylindrocarvone destraftans I Fo
6796゜f-y Calomyces cerevisiae Ii'
00259゜Elmaskus fertilis IFOO
691, Endomyces cashier IFO1112, Endomycobsis capsularis IFOO672, Schizosaccharomyces bombe IFOO846, Vitia
Polymorph, IFOO195, Hansenula Anomala I
FOO149, Sibaniomyces occidentalis
IFOO371, Debario Nyises Hansenyi IF
OOO21, Satsukalo Mycodes Ludbigii IP01
04B, Hanseniaspora barbiensis IF
OO115, Nadsonia Elongator IFOO665
, Nematosbo, La colili If"00658゜Ribomyces lipopher 1FOO6723, Sporobolomyces phorsatiicus Ii'01034゜Cributococcus albidus IFOO878, Torulopsis candida IJ'00768. Pityrosborum obare IFOosss, Brettanomyces anno Malas IFOO642, Candy Dalgosa IFOO591, Chloe Lakera Javani Power I
FO1094, Trigonopsis palabilis IFO
O671, Uikahamia Fluorescence IFO1
116, Kluyperomyces polisplus IFOO
996, Brera alba IFO1192゜Rhodotorula minuta IFOO187, Trichosporon capitatum If00748. Gleophyllum trapeum IFO6429, Schizophyllum commune IFO4
928, Tramenus Sanguinea IF06490, etc.

Bacterial culture conditions vary somewhat depending on the strain used, but generally speaking, glucose is used as the carbon source.

Carbohydrates such as fructose, sucrose, and maltose, glycerol, and nitrogen sources include ammonium sulfate, ammonium chloride, and potassium nitrate.

Urea, amino acids, peptones, casamino acids, cornstew liquor, JS suma, rice bran, yeast extract, etc. Inorganic carriers include magnesium sulfate, sodium chloride, calcium carbonate, phosphate-hydrogen kawatam. A medium containing other nutritional sources such as potassium dihydrogen phosphate, malt extract, meat extract, Pharmamedia, etc. may be used, but is not limited to these.

The m strain is inoculated into this medium and cultured aerobically or anaerobically. The temperature for culturing is usually 15 to 60°C, more preferably 25 to 40°C. Initial P of medium
H is 9 usually 8-9. Preferably, it is a concavity of 5 to 8.

The bacteria are usually grown by culturing for 8 hours to 10 days.

Bacterial cells were extracted from the culture solution obtained in this manner by a conventional method, and the cells were collected from D-forcenitine, L-carnitine,
contact with a solution containing at least one compound selected from the group consisting of r-butyrobetaine (thus, it is possible to produce crotonobetaine. p[ of the reaction is usually 2 to 10, preferably 5 to 7 gives good results.

The reaction temperature is 9 usually 10 to 60°C, preferably 25
~40°C. The initial substrate concentration is usually 0.1
-10%, preferably 1-5%. Adding the viscous material in portions may give good results. Also, adding glucose, sucrose, maltose, glycerol, yeast extract, etc. during the 9 reaction may give good results.

Zinc, potassium, calcium, and chromium in the reaction system.

Cobalt, strontium, iron, copper, sodium.

Nifkel, barium, magnesium, manganese.

Addition of molybdenum or lithium compounds often improves the reaction yield.

Can be used. For example, immobilized heptadons or immobilized enzymes obtained by known methods are also effective. Carriers used for immobilization include carrageenan and alginic acid.

Natural compounds such as agar, collagen, gelatin, and pectin, or synthetic polymers such as polyacrylamide, polyacrylic acid, ethylene acrylic acid copolymer, and photocrosslinkable +11 fat can be used. Also acetone po
In some cases, good results may be obtained by using cells treated with waer or dry cells, or by adding a surfactant. Bacterial cells subjected to mutation treatment are also included in the present invention as long as they have carnitine hydrolyase activity.

The present invention will be specifically explained below with reference to Examples.

Example 1 U (2PO40.8%, K2HPO40.7 type, (
NHa)28040.1%, Mg8U4・11420
0.01%, peptone 0.5%, yeast extract 0.5
20d of liquid medium consisting of
The bacterial strains shown in the table were inoculated with IB Kaimin, and the cells were cultured anaerobically at 30°C for 2 days.

Bacterial cells were obtained by centrifugation from the culture solution thus obtained. After washing the obtained bacterial cells with 5 yxl of physiological saline, D-carnitine 15@(62mM), L-carnitine 1'7y (62mM), or γ-
After adding it to 5 ml of 0.1M phosphoric acid weak I solution (PM6) containing 1% (69mM) of butyrobetaine, 8
Reactions were carried out by shaking r#J for 2 days at 0°C.

The resulting reaction solution was centrifuged to remove bacterial cells.

Heat treatment was performed at 80°C for 6 minutes, and analysis was performed by LO.

The main constituents of crotonobetaine are shown in Table 1.

Table 1 Nijieri Seventh Acoli lPO880126″ 22″
51″ Enterobacter cloacani lPO8820
252661 Klebsiella pneumoniae lPO8
512212842 Fn Bacter ri o7 Kae I person M1184 16 19 12 Erwinia aloideae lPO1288018292
7 Seladea marcennas lPO878681806
8 Proteus BullLrtf Squirrel lPO385126
2861 Salmonella tefimurium lPO1252
9298044 Alcaligenes F1 Echarism TC
C8750171229 Flavo Cutylium esteraromateicum UFO8751281747 Achromobacter parvulus lPO18181141549 Bacillus suf 1 Ericum lPO8526181987 Agropacterium radiobacter lPO126642
22159 Azotobacter vinelandii lPO120
18111628 Miku a Kotsuka x a-tttsu x
IFO876g 24 88
FJ2 Cori Honbacterium Rasayu lPO1216
1198245 Staphylococcus aureus IF
Oa060 29 26 20 Syedomonas marginalis lPO8925242818
Fluthrobacter cissyplefuchs lPO12069
278548 Brevibecterium Linens lPO1
2141211788*ka%fsu 75t'r'su
IFOB748 10 18 4
4 hafnia amibai troutt
28 28 51 Acetobacter orleanens lPO3259282658 Chromobacterium iodinum lPO3558111185 Xanthomonas campestris IFO1880J1
16 25 8B Vibrio parahaemoriticus IFO12?11 18 18
80 F0 Monas Hydrophylla lPO12
978111645 Brotaaminopacter alboflavus lPO8707142651 Acinetopacter carloacenaicus IP018QO6192048 Sit
-asyp-31Fo18ss* 29
82 B.

rCterium Gracil lPO3281181715
Clostridium butyricum 11i'08858
26 27 55') Zobium Di+
Poeca A IFO188B8 21
80 57 Gluco/Bacillus IF'08
264 10 15 40 Streptococcus f1 Industrial squirrel lPO312818145
G-pedioftucus bentosasius IFO1e8
12 17 810 Iconostoc Mesenteroides IFOFJ426 22 2
6 26 lac)/<Cirrus acidophilus lP
O8205152045 Propionivecnarium technicum lPO12428111429 Aerocoffus
Viridans lPO12317111784 Phycobacterium avium lPO8082151846 Noca tl Via Asteroides lPO84281810
58 Streptomyces al″fx IFOIJ1
014 16 18 59 Example 2 The same procedure as in Example 1 was carried out except that the liquid medium 5xl shown in Example 1 was used and the strains shown in Table 2 were cultured aerobically, and the results shown in Table 2 were obtained. I got it.

Table 2 Enterobacter cloacani lPO88202t=
27″ 45″ Serratia marcensus lPO
3786272954 Hafnia A I4 lPO878
1222685 Acinetobacter Calcoaceticus
IF'013006 80 26 89 Nocaldia Asteroides lPO84281017
59 Example 3 PH consisting of 5% malt extract, yeast extract o, s S
Using the liquid culture medium No. 6, the same procedure as in Example 2 was carried out except that bacteria were collected by filtration if necessary, and the results shown in Table 3 were obtained.

Table 3 Mucor Racemosus lPO458116” 1
8”M 44′ Rhizotpus oryzae Il=’04?
05 16 24 18Absidia ferrea lPO4011161929Phycomyces nitens lPO569521218g
Eremothecium asibii I)'00557
12 11 88 Aspergillus niger lPO4416171848 Penicillium oxasicum lPO5748151685 Monascus 7inca lPO5965162480 Neurospora clavosa lPO6660151686 La viscosa lPO4604202825 Pluralia bel Nefuki IP064G7 12 19
15 Fusarium solani lPO528218242
2 Gibberella Fujikuroi lPO6604111514
Usteilagozeae Ili'06907
IFO598a 16
20 42 Trichoderma viride lPO484
7171520 Fuerteicillium Atrum
lPO592214141G Isaria Kogane lPO
5299141621 Gliocladium deliquescens lPO6617182147 Trichophyton mentagrophytes lPO5466121611 Phytophthora infestans lPO4872151g
48 Cylindrical Rocker I Shigin Dex Tractance I
F'06796 17 15 46 Safcalomyces cerevileae IFOO259101?
20 Elmmascus Fe M Tiris IFOO691
161944 Endomyces cash register lPO11121
61487 Endomycopsis gabusularis IFO
O67216152B Schizosatucharomyces cylinder
lPO0846121542ζshu apolymorphy I
FOO195152251/) Ncessura Anomara I
FOO149201622 Shiba-omyces occidentalis IFOO87111202G Debaryomyces hansenii IFOO028111620 Satsukalomycodes rudopicii lPO1048161622 Hanseniaspora barbiensis IFOO11514
1519 Nadsonia Elongator IFOO6652
82085 Nematosbora coryli IFOO6681
41842 Sl Myces Lipoffy-IFOO6782
21821 Sporobolomyces torsateicus lP
O1084111288 Krybutokofcus albidus IFOO878151724 Torulopsis candida IFOO76816164g Pityrosgilum
Obare IFOO666192120 Brettanomyces anomalus IFOO642152248*Yandi';f Rugosa IFOO591121581 Kroetschella javanica lPO1094171889
Trigonopsis palabilis IFOO671121
120 Wicker Tamemia Fluorescence IFO111
6151587 Kluyveromyces polysporus 1
poosss 10 12 19 Brera alpa IFO11921211250 Dotorula minuta IFOO887121480 Trichosporon capitatum IFO0748181946 Gleophyllum trabeum IFO6429151819 Schizophyllum commune IFO4928192086 Tiger Me→S Sanguinea IF06490 10
18 17 Example 4 The culture scale and reaction scale were both 100 times larger, and Serratia marcensus IF03786 was used.

Example 1 except that eDL-carnitine was used.
I did the same thing. On this reaction scale, the added +1
) L-carnitine was 5 f (84 mmol). The supernatant obtained by removing the bacterial cells from the reaction solution by centrifugation was loaded onto a Dowex column, length 801. Inner diameter!
A crotonobetaine fraction was obtained through MIIC. The amount of crotonobetaine obtained was 2.81 (16 mmol)
Met.

〔Effect of the invention〕

1) It can be produced cheaper than epichlorohydrin, and L
- It becomes possible to easily and inexpensively produce crotonobetaine, a precursor of L-carnitine, by acting microbial cells on carnitine at °C.

2) After separating the γ-butyrobetaine produced as a by-product during the above reaction, it is possible to return it to the raw material crotonobetaine again.

8) When L-carnitine is produced from crotonobetaine by the anaerobic culture method in the above-mentioned Japanese Patent Application No. 59-187878, the resulting microbial cells (waste 4) can be used in the present invention. be.

Applicant: Steel Chemical Industry Co., Ltd. Representative Hiroshi Sasaki

Claims (2)

[Claims] (1) D-carnitine or L-carnitine or γ
- converting at least one compound selected from the group consisting of D-carnitine, L-carnitine, and γ-butyrobetaine into crotonobetaine by the action of a microorganism capable of converting butyrobetaine into crotonobetaine; A method for producing crotonobetaine. (2) The microorganism is Escherichia.
) genus, Enterobacter
Genus Klebsiella, Aerobacter genus, Erwinia (E
genus rwinia, genus Serratia,
Proteus genus, Salmonella
monella), Alcaligenes (Alcalig)
enes), Flavobacterium (Flavobacterium
terium), Achromobacter (Achromobacter)
bacter) genus, Bacillus genus,
Agrobacterium genus, Azotobacter genus, Micrococcus genus, Corynebacterium genus, Staphylococcus genus,
Genus Pseudomonas, Genus Arthrobacter, Genus Brevibacterium, Genus Cellulomonas, Genus Hafnia (
Genus Hafnia, Acetobacter
ter) genus, Chromobacterium (Chromobacterium
terium genus, Xanthomonas
nas), Vibrio, Aeromonas, Protaminobacter (P
rotaminobacter), Acinetobacter (Acinetobacter), Citrobacter (
Citrobacter genus, Bacterium
erium genus, Clostridium
ium) genus, Rhizobium genus, Gluconobacter genus, Streptococcus genus,
Pediococcus spp., Leuconostoc spp., Lactobacillus spp., Propionibacterium spp.
Genus, Aerococcus, Mycobacterium, Nocardia, Streptomyces, Mucor
or) genus, Rhizopus genus, Absidia genus, Phycomyces (Phycomyces)
genus Eremothesium
ecium genus, Aspergillus
us), Penicillium, Monascus, Neurospora (
Neurospora genus, Oospora
a) Genus, Pullularia, Fusarium, Gibbe
genus Rella, genus Ustilago, genus Keratinomyces, genus Sporothrix, genus Trichoderma, genus Verticillium, spp.
aria), Gliocladium (Gliocladium)
um) genus, Trichophyton
) genus, Phytophthora genus,
Cylind-rocarpon
), genus Saccharomyces
) genus, Eremascus genus, Endomyces genus, Endomycopsis genus, Schizosaccharomyces genus,
Pichia genus, Hansenula
ula), Schiwaniomyces
myces), Debaryomyces
yces), Saccharomycodes
mycodes), Hanseniaspora (Hansen)
iaspora) genus, Nadsonia
Genus, Nematospora, Lipomyces, Sporobolomyces, Cryptococcus, Torulopsis (
Torulopsis genus, Pityrosporum (Pit
yrosporum), Brettanomyces (Bret
genus Candid
a) Genus Kloeckera, Genus Trigonopsis, Genus Wickerhamia, Genus Kluyveromyces, Genus Bul.
lera), Rhodoto-rula
), the genus Trichosporon, the genus Gleophyllum, the genus Schizophyllum, and the genus Trametes. Claim (1) Method described. However, when L-carnitine is used as a substrate, the genus Escherichia, Proteus, and Salmonella are excluded.