JPS61234794A - Production of l-carnitine - Google Patents

Abstract

PURPOSE:L-carnitine is obtained from crotonobetaine using cell bodies with increased carnitine hydro-lyase activity which is obtained by anaerobic cultivation in the presence of a carnitine hydrolyase inducer. CONSTITUTION:A microorganism capable of converting crotonobetaine into L-carnitine such as Escerichea coli IFO 3301 or Enterobacter cloacae IFO3320 is cultured under anaerobic conditions in the presence of a carnitine hydro-lyase inducer such as DL-carnitine or crotonobetaine to effect induction of carnitine hydro-lyase. The resultant enzyme is allowed to act on a solution containing crotonobetaine to hydrate the betaine asymmetrically to give optically active L-carnitin.

Description

Detailed Description of the Invention [Object of the Invention] The present invention provides that when crotonobetaine is treated with a microorganism-derived enzyme (carnitine nitriase), L-
The present invention relates to a method for enzymatically producing Kfi from carnitine.

(Industrial application field) The novelty of the present invention is that microorganisms are treated with DL-carnitine.

To produce KL-carnitine, which is more advantageous than the inexpensively available crotonobetaine, using bacterial cells with enhanced carnitine hydrolyase activity obtained by culturing anaerobically in the presence of a carnitine hydrolyase inducer such as crotonobetaine. At the point. It is also useful to use a cell extract obtained by disrupting microbial cells, a partially purified enzyme, and a purified #substance obtained from the cell extract using a known method.

It is well known that carnitine (β-hydroxy-γ-trimethyl-7minobutyric acid) exists in two stereoisomers: 0-form and L-form. L-carnitine normally exists in living organisms and functions as a carrier that allows activated long-chain free fatty acids to pass through the mitochondrial membrane.

Although the only natural form of carnitine is left-handed L-carnitine, racemic carnitine has been used as an appetite stimulant.

However, recently it has been shown that the use of L-carnitine alone is more effective, at least for some therapeutic uses, and there is growing interest in its importance.

In fact, it is used to treat acute and chronic myocardial ischemia, angina pectoris, cardiac arrhythmia, and heart failure in the cardiovascular system.

(Prior Art) As a method for producing optically active L-carnitine, for example, the following method is known.

(1) A method of optically resolving racemic carnitine obtained by chemical synthesis. The optical resolution method is to add N-acetyl-
D-glutamic acid or N-acetyl-glutamic acid is added as a resolving agent to form a salt, which is separated using the difference in solubility, and then hydrolyzed to form L- and D-carnitine chloride ( Tokuko Sho 43-824
8).

is a typical example.

(2) A method for obtaining L-carnitine by asymmetrically reducing 3-dehydrocarnitine by the action of a microbial enzyme (carnitine dehydrogenase).

(US Pat. No. 4,221,869) (3) A method for producing L-carnitine by reacting T-butyrobetaine with a microbial enzyme (hydroxylase). (JP-A-57-39) Electronic 91) (4) A method for producing L-carnitine from crotonobetaine using an aerobic culture method or bacterial cells obtained by an aerobic culture method. (JP-A-59-183694, JP-A-59-
192095) However, in (1), the N-acetyl-D-glutamic acid used as the optical resolving agent is expensive, the operation is complicated, and the yield is poor.

(2) U, 3-dehydrocarnitine of the original H is unstable and difficult to handle, and NADH or N is expensive as a coenzyme.
Requires AD.

Regarding (3), none of the above-mentioned methods can be said to be advantageous as an industrial production method because r-butyrobetaine, which is a raw material, is expensive.

Further, according to (4), according to additional tests by the present inventors, carnitine hydrolyase activity could not be obtained in bacterial cells cultured aerobically, despite culturing in the presence of a carnitine hydrolyase inducer. There are many types of bacteria, and even among the strains that have acquired carnitine hydrolyase activity, the activity is often weak.

Therefore, the present inventors investigated the use of KI, which is cheaper than epichlorohydrin.
Focusing on crotonobetaine, which can be manufactured by J, we conducted intensive studies with the aim of developing a new method for producing L-carnitine.As a result, we found that in the presence of carnitine hydrolyase inducers such as DL-carnitine, L-carnitine, and crotonobetaine, We have discovered that when microorganisms are cultured anaerobically, strong carnitine hydrolyase activity is induced in the cells of many bacterial species, leading to the present invention.

To explain one example of the embodiment of the present invention, for obtaining bacterial cells, for example, KH2PO40.3%, K2HP0°0
．． 7, (NH4)2504 0. IX, MgSO
Serratia marcensus (
Inoculate one platinum loopful of IFO3736) inoculum and heat at 30°C.
The cells were cultured anaerobically for 2 days. Bacterial cells were obtained from the thus obtained culture solution by centrifugation. The obtained bacterial cells were washed with 5-saline solution and then subjected to a reaction. For the reaction, the obtained bacterial cells were treated with crotonobetaine 1.5% (105mM)
After adding Q into 1M phosphate buffer (pH 6),
This was done by shaking at 30°C for 18 hours.

After removing the bacterial cells from the resulting reaction solution by centrifugation,
Heat treatment was performed at 80°C for 5 minutes, and D and J.

The enzymatic method of Pearson et al.
Enzymo 10g5r Lt612 (1969)
) Analysis revealed that 5M of L-carnitine was produced.

Examples of microorganisms having the ability to convert crotonobetaine to L-carnitine used in the present invention include Escherichia coli IFO3301 and Enterobacter cloacani I F03320. Klebsiella #Moniae IFO3512, Aerobacter cloacani lAM1134. Erwinia Aloidea IF012380. Serratia marcensus IFO3736, Proterus vulgaris IF
03851. Salmonella typhimuricum IF0
12529. Alcaligenes faecalis AT
CC8750, 7 Labo Bactericum estero aromaticum IFO3751, Achromobacterium palplus IFO13181, Bacillus su7aericum IF
O3526, Agropactericum radiobaccu I
FO12664, Azotobacter vinelandii IF01
2018. Micrococcus roseras IF037
68゜Corynepactericum rapus IFO12161
, Staphylococcus aurelus IFO3060,
Pseudomonas marginalis IFO3925, Arthrobacter simplex, Russ IFO12069, Plevipactericum linens IFO12141, Cellulomonas 7 Lapigena IF0374B, Hafnia alpei IFO3731, Acetobacter orleanens IF03259. Chromobacterium iodinum IFO3558, Xanthomonas campestris IFO13303, Vibrio parahaemolyticus IFO12711, Krepsiae 2 pneumoniae IFO3319, Aeromonas hydrophylla IF0129) Electronic 8. Protaminobacter alboflavas IFO 3707, Acinetobacter calcoaceticus IFO 13006, Citrobacter intermedellus IFO 13544, B.
5sil IFO3231゜Clostridium butyricum IF0385 & Rhizobium japonicum I
F013338. Gluconobacter Serinus IF
O3264 Streptococcus 7 Aecalis IFO
312 & Pediococcus bentosacillus IF
03893. Leuconostoc mesenteroides IFO3426, Lactobacillus acidophilus IF
O3205, Probionipactericum technicum I
F 012428. Aerococcus viridans
IFOl 2317, Mycopactericum avicum IF03082゜7 Chaldea asteroides
IFO3423 Streptomyces alps IF0
There are 13014 etc.

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

7 Lactose, Shakerose, maltose, carbohydrates such as glycerol, ammonium sulfate as a nitrogen source,
Ammonicum chloride, potassium nitrate.

Mediums containing inorganic salts such as urea, amino acids, peptones, cabumino acids, cornstarch liquor, bran, rice bran, and yeast extract are used, but are limited to these. Furthermore, a carnitine hydrolyase inducer such as 0.01 to 2XODL-carnitine, L-carnitine, crotonobetaine, etc. is usually added to this medium, but only at a degree that allows carnitine hydrolyase activity to be obtained. If so, it is not limited to this.

The bacterial strain is inoculated into this medium and cultured anaerobically.

The temperature suitable for culturing is usually 15 to 60°C, but
More preferably, the temperature is 25 to 40°C. The initial pH of the medium is usually 3-9. Preferably it is in the range of 5-8.

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

It is possible to produce L-carnitine by bringing the bacterial cells removed by a conventional method from the anaerobic culture solution thus obtained into contact with a solution containing crotonobetaine. A reaction pH of usually 2 to 10, preferably 5 to 7 gives good results. The reaction temperature is usually 1
0-60°C, preferably 25-40°C. The amount of crotonobetaine initially added is usually 0.1 to 10%, preferably 1 to 5%. Adding crotonobetaine in portions may give good results. In addition, during the reaction [glucose, shakerose, maltose,
Adding glycerol or yeast extract may give good results. The reaction system contains zinc, calicum, calcicum, chromium, cobalt, stronticum, iron, copper, sodium, nickel, varicum, magnesium, manganese,
Addition of molybdenum and lyticum compounds often improves the reaction yield.

As an enzyme source for the reaction, a carnitine hydrolyase active fraction obtained from #1 crab cells by a known treatment method can also be used. For example, fixed bacterial cells or immobilized enzymes obtained by known methods are also effective. Carriers used for immobilization include carrageenan, alginic acid, agar, collagen, and gelatin.

Natural compounds such as pectin, or synthetic polymers such as polyacrylamide, polyacrylic acid, ethylene acrylic acid copolymers, and photocrosslinkable resins can be used. Also,
In some cases, good results may be obtained by using bacterial cells treated with acetone powder or dry cells, or by adding a surfactant. Even bacterial cells subjected to mutation treatment are included in the present invention as long as they have carnitine hydrolyase activity.

The present invention will be specifically explained by giving examples below.0 Example 1 KH2PO40.3%, K2HPO40.7%,
(NH4)25040.1%, MgSO4・7H20
0.01, peptone 0.5%.

Yeast extract 0.5%, carnitine hydrolyase inducing substance (DL-carnitine is crotonobetaine) 0.
One platinum loopful of the strains shown in Table 1 was inoculated from the slant medium into 20 m of a 3% liquid medium, and cultured anaerobically at 30°C for 2 days. Bacterial cells were obtained from the culture solution thus obtained by centrifugation. After washing the desired bacterial cells with 5-saline and adding them to 0.1M phosphate buffer (pH 6) containing 1.5% (105mM) of crotonobetaine,
The reaction was carried out by shaking at 30°C for 18 hours.

The resulting reaction solution was centrifuged to remove bacterial cells, then heated at 80° C. for 5 minutes and analyzed. The amount of L-carnitine produced is shown in Table 1.

As a control, Table 1 also shows the results obtained when the reaction was carried out using bacterial cells obtained by aerobic culture. The conditions were: 2% glycerol, 0.3 ammonium sulfate, KH2PO40, IN, K2HPO40, 3%,
MgSO4・7H200,05X, FeSO4φ7
H201ml/di, MnSO4-4H201W/d
jL, yeast extract 1%;

The strains shown in Table 1 were added to a 5- medium consisting of peptone ic After inoculation, the cells were cultured with shaking at 30°C for 16 hours.

Bacterial cells were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and collected. The bacterial cells were treated with crotonobetaine hydrochloride 1.5% (83.6mM
) containing 0-IM phosphate buffer (pH 6,0) 5d
K was added thereto, and the reaction was maintained at 30°C for 16 hours.

The amount of L-carnitine in this reaction solution was analyzed in the same manner as described above, and the results shown in the control in Table 1 were obtained.

Example 2 The reaction was carried out in the same manner as in Example 1, except that crotonobetaine added to the reaction was 3% (210 mM) and the reaction time was 48 hours, and the results shown in Table 2 were obtained. The numerical value indicates the amount of L-carnitine produced.

Table 2 Example 3 According to the method of Example 1, Escherichia coli (IFO3301) was cultured using crotonobetaine as an inducer, and the resulting bacterial cells were washed with physiological saline.

(Intact cells) After the bacterial cells were disrupted by ultrasonication, a precipitated fraction was collected by centrifugation, and the precipitated fraction was treated with surfactant Tritonx-100.
After treatment, a cell-free extract was prepared by centrifugation.

Enzyme reaction was carried out using 50mM phosphate buffer CpH7.0),
The test was carried out at a temperature of 37° C. for 3 hours using the above-mentioned starch preparation which is compatible with 50 mM croto/betaine and 2 cape/− protein.

For reactions using intact cells as the enzyme, 15mM L
- In the reaction using carnitine and cell-free extract as enzyme, 21 mM of L-carnitine was produced.

Example 4 The same procedure as in Example 2 was carried out except that the culture scale and reaction scale were both 100 times larger and Serratia marcensus (IFO3736) was used. At this reaction scale, the initial amount of crotonobetaine added is 7.5 g (52
, 4 m-mol). The supernatant obtained by removing the bacterial cells from the reaction solution by centrifugation was passed through a Dowex column with a length of 80 cm and an inner diameter of 5 cttt K to obtain a carnitine fraction.

From this carnitine fraction, crotonobetaine was removed using a method of treating with sodium bisulfate (Japanese Patent Application No. 187377/1987), and 3.6 g (214 mmol) of L
-, Karunichi, /'tm. The specific optical rotation of this L-carnitine is [α]. = -30.5° ((, ++1.Q aqueous solution).

Example 5 Medium lj with the composition shown in Example 1! was inoculated with Escherichia coli (IF03301) and cultured anaerobically at 30°C for 2 days.

Bacterial cells were obtained by centrifugation from the culture solution thus obtained. After washing the bacterial cells with 100% physiological saline, 1 g of the cells was immobilized on 2-gianan to obtain 10 g of immobilized bacterial cells.

Crotonobetaine 200 m genus, sodium citrate 1
0 m genus, KNO30,5X, MgSO4・7H2
05mM.

CaCl20.5m, phosphate buffer 100mM
5 g of immobilized element 14 was added to a reaction solution containing (pHa, o) sotnt and reacted at a temperature of 30° C. for 24 hours. After completion of the reaction, L-carnitine in the reaction solution was quantified.

Next, the immobilized bacterial cells were taken out, washed, and added again to the above reaction solution, and the reaction was repeated. The results are shown in Table 3.

The immobilized bacterial cells were able to stably produce L-carnitine for 15 days or more.

Table 3 Example 6 The same procedure as in Example 1 was carried out, except that the compounds shown in Table 4 were used as carnitine hydrolyase inducers at a concentration of 0.1X, and the results shown in Table 4 were obtained.

The strain used was Citrobacter intermedellus (IF
Table 4: O13539) The 0 value indicates the concentration (mM) of L-carnitine produced.

Claims (1)

Scope of Claims: (1) By culturing a microorganism capable of asymmetrically hydrating crotonobetaine to produce L-carnitine under anaerobic conditions in the presence of a carnitine hydrolyase inducer, carnitine L-carnitine is characterized by inducing hydrolyase and asymmetrically hydrating crotonobetaine to produce optically active L-carnitine through the action of the enzyme.
Method for producing carnitine. (2) The method according to claim (1), wherein the carnitine hydrolyase inducer is DL-carnitine. However, the genus Escherichia,
Salmonella spp., Proteus (
It does not contain microorganisms of the genus Proteus. (3) The carnitine hydrolyase inducer is at least one selected from the group consisting of D-carnitine, L-carnitine, crotonobetaine, DL-carnitine nitrile, crotonic acid, allyl alcohol, acrolein, acrylonitrile, acrylamide, and acrylic acid. The method according to claim (1), which is a compound. However, this does not apply when D-carnitine and L-carnitine are added at the same time. (4) The microorganism is Escherichia.
) genus, Enterobacter
Genus Klebsiella, Aerobacter genus, Erwinia (E
genus rwinia, genus Serratia,
Proteus genus, Salmonella
mo-nella), Alcaligenes
gene), Flavobacterium (Flavobacterium
cterium), Achromobacter (Achrom)
(obacter) genus, Bacillus (Bacillus)
Genus, Agrobacterium
) genus. Azotobacter genus, Micrococcus genus, Corynebacterium genus, Staphylococcus genus, Pseudomonas genus, Arthrobacter genus genus Brevibacterium Brevibacterium genus, Cellulo-monas genus, Hafnia genus, Acetobacter genus
Chromobacterium (Chromo-acter), Chromobacterium (Chromo-
bacterium), Xanthomonas (Xanth
omonas genus, Vibrio genus, Aeromonas genus, Protaminobacter genus, Acinetobacter genus, Citrobacter genus, Bacterium (B
genus acterium, Clostridium
Rhizobium
Genus, Gluconobacter
) genus, Streptococcus
s) genus, Pediococcus
Genus, Leuconostoc,
Lactobacillus genus,
Propionibacterium (Propioni-bacterium)
erium), Aerococcus
s) genus, Mycobacterium
The method according to claim (1), wherein the microorganism is at least one type of microorganism belonging to a genus selected from the group consisting of the genus Um, Nocardia, and Streptomyces. (7) The method according to claim (1), in which a microbial cell or a processed product thereof is used as carnitine hydrolyase. (8) The method according to claim (1), wherein a compound capable of accepting electrons produced by microorganisms is added during culturing under anaerobic conditions. (9) The compound that can accept electrons is KNO_3 or N
The method according to claim (8), which is aNO_3.