JPS63123387A - Production of gamma-halo-beta-hydroxybutyric ester - Google Patents

Abstract

PURPOSE:To efficiently produce the titled compound useful as a raw material for synthesizing drugs such as L-carnitine, etc., by treating a gamma-haloacetoacetic ester with a culture solution, cell or a treated substance of the cell of bacteria. CONSTITUTION:A bacterium [e.g. Cellulomonas sq. AKU672 strain (FERM P-9026), etc.] capable of converting a gamma-haloacetoacetic ester shown by formula I (R1 is halogen; R2 is arbitrary organic residue such as alkyl, phenyl, aryl, etc.) into a corresponding gamma-beta-hydroxybutyric acid ester is cultivated in a nutri tive medium containing a carbon source, nitrogen source, inorganic substance, etc., at pH3-8 at 10-40 deg.C for 1-10 days to give a culture solution and culture cells. 0.001-50wt% gamma-haloacetoacetic ester is contacted with the culture solution, the cell, a treated substance of the cell or an immobilized material thereof etc.) in an aqueous medium such as water, a buffer solution, a hydrous organic solvent, etc., at pH3-8 at 10-60 deg.C for 48hr at the longest to give the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing γ-halo-β-hydroxybutyric acid ester by reacting γ-haloacetoacetate with bacteria. It is useful as a raw material for pharmaceutical synthesis such as γ-halo-β-hydroxybutyric ester HLIJ lunitin.

[Problems to be solved by conventional technology and invention] γ-
... Aceto's previous enemy - γ corresponding to dal by reducing it chemically
- a lifting platform for producing halo-β-hydroxybutyric acid ester;
The disadvantage is that side reactions are likely to occur and the yield of the target product is low. In order to solve the problem, a method using the fermentation enzyme action of microorganisms that produce L-β-hydroxyacyl COA dehydrogenase (% 1987-1) 80
Publication No. 93) was proposed. However, the microorganisms that have been reported are yeasts and molds, and in addition to the history of bacteria that have been improved through mutations and other steps, it is difficult to understand what bacteria is. I- There is a need to establish a method for utilizing it.

〔problem? Predetermined means to solve the problem]

The present invention provides γ-haloacetoacetic esters and corresponding γ-
Converting to halo-β-hydroxybutyric acid ester
This is an im method for producing γ-halo-β-hydroxyface acid ester, which is characterized in that it acts on γ-haloacetoacetate and collects the product.

The r-haloacetoacetate used in the present invention has the general formula: R, -CH2Co -CE(2COOR2 (in the formula R
1 is a halogen, and R2 is an optional M residue of an alkyl group, phenyl group, or aryl group.

The γ-haloacetoacetic acid ester used in the present invention can be obtained, for example, by reacting a halogen with a diketene in a bath medium, but the heart wall can also be produced from γ-haloacetoacetic acid ester by a conventional Grignard reaction.

The bacterium used in the present invention is a bacterium that has the ability to convert γ-haloacetoacetate to the corresponding γ-halo-β-hydroxybutyrate, such as Aureobacterium.
) fi alkaline rness (Alcaligenes)
) Genus Agrobacterium (Agrobacterium)
m) Jf4 ant, xo bacter (Arthroba
cter ) genus Amorphosboranium (Amor
pho e po rangenium) Ampullariella family Brevibacterium
) Genus Bacillus h Genus Corynebacterium ((: orynebacterium
m) P! 4 Cellulomonas
a) Genus Escherichia
Genus Enterobacter
Genus 7 M Bacterium (FlaVOk) aiQ'lri
um ) FA Hafinia ) A Genus Kurtia Genus Lactobacillus Micrococcue 754 Methanomonas ) 94 Methylobacillus () 4 athylobacil Mlcrobispora (Mlcrobispora) Mlcrobispora (Micromonospora) Nocardia (Nocaraia) Genus Proteua Genus Pseudomona, X (P8eu (LOmOnaa)
h4 Pediococcua Q
4 Planomonospora (Planomonospora)
) Genus protomonaa Genus Rhodoccus Genus Serratia (5err
atia) belonging to the genus Streptomyces - Moactinomyce;
omycea), genus Xanthomonas (CXanthOmOnaa), 1 is a bacterium belonging to the genus Yersinia. To give a more specific example, Aureobacterium teledensae FO12961 (
Aureobacterium terragens
) Alcaligenes faecalis E F'012669
(AlcaligeHes faacalis) Agrobacterium radiobacter air AM 152
6 (Agrobacterium radiobacterium
er) Arylobacter simplex eFO
12069 (Arthrobacter aimpl
ec ) Amorphosporangium aranticolor JCM 3 [] 38 (Amorphosporangium
gium auranticolor) Ampurariella kiriniblica JCM 3329 (Ampul
lariella cylindrica) Brevibacterium ammoniagenes FO12071 (
Brevibacterium ammonium
ee) Bacillus subtilis E F0 3037 (
Bacillus 5ubtilia) Corynebacterium daltamicum 4534 ATCC13
032 (Corynebacterium gluta
micum) Cellulomonas sp. AKU 67
2 (Cellulomonas sp, ) Escherichia coli K12 Eng F'03208 (Ksher
ichia coli) Enterobacter aerodenes JCM 1235 (1nterobacter
aerogenes) Flavobacterium Nisteroaromaticum EFO37b1 (1avobac
teriu+n eat, eroaromaticum
) Hafin: La alvei IFO3731 (Hafin:La alvei) Kurtia zopfi F0 12083 (Kurthia zopfi
Lactobacillus Amylophilus JCM 1) 24
(Lactobacillus: Llus amylophil
us ) Micrococcus luteus eFO1270
8 (Micrococcus 1uteua) Methanomonas Methylobora JCM 2848 (Meth
anomOnas methylovora) Meterobacillus glycogenes JCM 2850 (M8t
h71ObaC1llu8 g17cOgene8)
Microbispora aerata JCM 3076 (Mi
crobispora aerata) Micromonospora grisea JCM 3182 (Micro
onos・pora grisea) Nocardia coralinae AM 12121 (Nocardia
a corallina) Proteus mirabillus E1'0 3849 (Protθus mirab
illg) Pseudomonas crucibiae ayo
12047 (Pseuaomonaa cruciv
iae ) Pedeococcus bentosaceus JCM
2023 (peaiococcus pentos
aceus) Planomonospora venezueleschensis JCM 3167 (PlanomOnoapora
venezuelensis) Protomonas
Extroquence JCM 281) (Protom
onas extroquens) Rhodococcus
Coralina JCM3199 (Rhodococua
corallina ) Serratia marcescens AM 1) 05 (Eleratia marcesc
ens ) Streptomyces arabicus JcM4
161 (streptomyces arab
icus) Thermoactinomyces Sarakari :r
c>i 3137 (Thermoactinomyc
es aacchari) Xanthomonas maltomilia, TCM 1975 (Xanthomona
s maltophilia ) Yersinia rukeri JCM 2429 ( Yersinia ruk
eri) etc. These strains were collected by Hakko Research Foundation (Engineering FO) and Institute of Applied Microbiology (IAM), the University of Tokyo.
, or the RIKEN Microbial System Conservation Yi1) establishment (JC
M) It is a strain that is maintained by ATCC etc. under its respective number and can be easily obtained as needed ◎Among these, Cellulomonasnis t-AxU672i is a strain discovered by the present inventors, and 1. It has been deposited with the Institute of Microbial Technology, Agency of Technology under deposit number 9026. Mycological 5
! i' is shown below.

1. Morphology (1) Cell shape and thickness: Qld culture: Coccus, 1). 5-0.6
μff1Fresh culture: Amorphous, rod, diameter 0.5-0.7 μm, length > 2.0 μm (21 Polymorphic publication M (3) Presence or absence of motility, page 2 (4) Presence or absence of flagella: page (5) Presence or absence of spores: None (6) Durham staining: Positive 2, Growth status on each medium (1) Color of broth cultured on broth agar plate Colony: Yellow (cultured for 2 days) Colony shape: dicircular, smooth colony Elevation: Periphery of central convex colony: Full line (2) Meat juice liquid culture concave ring, slightly precipitated ■ (3) Meat juice gelatin puncture culture: Liquefies (4)
Lidomus milk: produces acid 6, physiological properties (1) Reduction of nitrate: ■ (2+MR test: Negative (31VP test: Negative (4) Production of indole: Negative (5); Production of hydrogen sulfide: Negative (6) Starch Hydrolysis site: Positive (7) Utilization of citric acid: Negative (8) Pigment formation: None (9) Urease: Negative a (2) Oxidase: Negative αD Catalase: Positive α21 Attitude towards acid systems: Aerobic αj Growth range temperature 37S42℃ PH6.0-7.5 Q41OF test: Fermentation (151 Effect on cellulose: Negative (16) ■ Sugar-free of acid and gas production from sugars
Acid gas■ L-arabinose -■ Arb
utln + -■ Cellulomonas ten -■ Dextrin
+ -■ D-fructose +
−■ D-galactose 10 −■ D
−Glucose 10 −■ Glycogen 10 −■ Maltose
＋ −〇 Starch
−■ Sucrose 10
-@) L/ha0-su(treha108li + -O xylose 10 - ■ Glycerol - -〇 Inulin -- @lLa --O Manitol
- -O Mannose
− −◎ α-Methylglucoside −
-(α-meth)rlglucosiae)O raffinose - - [phase] Rhamnose --〇 Lubitol
−− 〇 Nrubosu − −Rin71
DNA degradability: Positive b Caseinolytic aminopeptidase activity: Negative σ Salt tolerance: NIL (Grows at 15 Qi 1 ■; Cell wall amino acid: Ornithine c!1) Cell division: Bend + 221 DNA (D GCt*: 74.7
%(c) Heat treatment on skim milk instep: 66℃, 60
Due to the mycological properties of the bacteria, which were more than viable during the separation process, this bacterium was classified as a coryneform bacterium, and the method of Yamada et al.

ApploMlcroblol, 18. 41
7 (1972)) ■ Cellulose decomposition activity is lacking ■ Cell division or bending type ■ Amino acid in the cell wall is ornithine ■ () C content is narrow in the range of 71 to 76 and the amount of cylindrical ■ A wide range of sugars From the point of making pancakes through fermentation,
The fourth group was identified as Cellulomonas sp.

The above-mentioned bacteria are generally mutated naturally or artificially, but all of the bacteria that reduce γ-haloacetoacetate and convert it to γ-halo-β-hydroxybutyrate are the ones of the present invention. Can be used in manufacturing methods.

The bacteria used in the present invention can be cultured according to conventional methods. The medium used for culturing is a conventional medium containing carbon sources, nitrogen sources, inorganic P4IJ substances, etc. necessary for the growth of bacteria. Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids.

The culture is carried out under aerobic conditions for 1 to 10 days while controlling the pH to 3 to 8 and the temperature to an appropriate range of 10 to 40°C. For the reaction, either a bacterial culture solution or a culture isolated from the culture solution can be used. Im bacterial cell processing products include one dried cell obtained by methods such as freeze-drying or acetone drying, Kokutai crushed liquid obtained by methods such as integral sonic grinding, autolysis, and ultrasonic treatment, as well as γ-710 acetoacetic acid. An enzyme protein class having the enzymatic activity of converting an ester into the corresponding γ-halo-β-hydroxy-enzymatic ester, and any identified product of the bacterial cells 17c or 7 can be used.

γ-haloacetoacetate corresponding to γ-halo-β-
The method for converting to hydroxybutyrate ester involves culturing γ-haloacetoacetate and the above-mentioned bacteria in an aqueous medium, immobilizing them individually, treating them together, or immobilizing them by a known method and bringing them into contact with a friend. stomach.

As the aqueous medium for such a reaction, water, a buffer solution, and a water-containing bath medium can be used.

In order to act on the bacterial tγ-haloacetoacetate, the pH is usually 3 to 8 and the reaction temperature is 10 to 6.
The temperature is controlled to be within the range of 0°C.

The r-haloacetoacetic acid ester is added to the reaction system as it is, in a solvent, or after being dispersed.

The reaction system ester@tomo is preferably in the range of 60.001~50''M.The addition of such γ-haloacetoacetic ester is possible at any stage of the reaction; It can also be carried out in the middle stage.

The reaction can also be carried out in the presence of sugars such as glucose, nutrients for microorganisms, surfactants, and the like. The reaction time can be adjusted depending on conditions such as # degree, or at most 48
After a period of time, the r-haloacetoacetic ester is converted to the corresponding γ-halo-β-hydroxybutyric ester.

To collect the thus obtained γ-halo-β-hydroxybutyric acid ester from the culture solution or reaction solution, remove one body or a box using conventional methods such as centrifugation or ultrafiltration. Conventional methods such as extraction using a medium such as ether, carbon tetrachloride, benzene, or ethyl acetate can be employed.

〔Example〕

Next, the invention-〇 method @ will be further described with reference to Examples.

Example 1 A medium (p[(6,5)) consisting of 5 parts glucose and 5 parts by weight of Company's L Corn Steep Liquor was placed in a test tube, the microorganisms shown in the table were inoculated, and the mixture was heated at 28°C. A statement of shaking culture for 48 hours.

In this system, γ-chloroacetyl)I! 25 μl of methyl rl-acid
was applied, and the reaction was continued for 24 hours at r=4.

The resulting reaction solution was subjected to deweighting M treatment by centrifugation, and then the reaction h
Extracted with 2rnit acetate ether 4 ut and subjected to gas chromatography (Gc-1, I APF, P
Concave G20MX1rn, 150℃, N230m17 m1
n) to analyze it. Shown in Table 7.

Example 2 A reaction was carried out and analyzed in the same manner as in Example 1 using ethyl γ-chloroacetoacetate as the base bond. The results are shown in the table.

Below is the margin Example 6 Glucose 5fKit%, corn staple liquor 5
Medium (pH 6,5) consisting of 51) Lt't- tester and Cellulomonas sp. AKU 672 (
Seed culture at was obtained by inoculating the seedlings and culturing them with shaking at 28° C. for 24 hours.

Next, take 1001) Lt of culture medium with the same composition as above into a 500-liter flask, and add 500ml of food culture solution. Males were added and cultured with shaking at 28°C.

The resulting culture solution was centrifuged, washed with 0.9% NaCl water, and then diluted with 1 (W/V) '36 of glucose.
O,IM phosphate buffer (pi(6,0) 100WLt
Suspended in ethyl γ-chloroacetoacetate 1-0. ! i'
To? After adding Fi, the mixture became unreactive for 18 hours while being aerated and shaken.

The obtained anti-core liquid r? After sterilization treatment by separation, vinegar%
Extract with x chill 300mA (1 (10rILt x 3 times)?
I did it. Anhydrous magnesium sulfate is added to this ethyl acetate layer.
After adding water and dehydrating, it was vacuum-dried to obtain an oily product with a weight of 0.989. This product is distilled under reduced pressure.
435), NmR (JEOL PMX608 Engineering), gas chromatography (Shima*GC-9APFS PE020
MX1m, 150℃, N23 Qrnl/min
When the sample was checked with a sample, it was confirmed that it was ethyl γ-chloroβ-hydroxybutyrate.

NMR δ (in CDC13): δ(1)Pm) 1.25 (3H
, t), 2.60 (2H, a), 3.35 (I H,
s, exchangeable, OH)3
-60 (2Ht d), 4-2 (2a, q
) CR-TC min) 4.6 Example 4 Micrococcus luteus e FO12708' was cultured and reacted in the same manner as in Example 6, and the product r was separated, yielding 0.85 g of an oily raw material.

Furthermore, γ-
kr4 was confirmed to be chloroβ-hydroxy@ethyl acid.

Example 5 Using γ-chloroacetoacetate octyl 'kM'Th,
Seven reactions were carried out in the same manner as in Example 3, and gas chromatography was carried out. 4tGC-9APF, oV-IXI style, 1
25°Cs N230'/min, IR (Shimadzu R-
455), NMR (JKOLGX-270) T confirmed.

As a result, it was confirmed that it was octyl γ-chloroβ-hydroxy@acid. 1-Reaction yield was 50%.

The basic ingredient is 10% Tween 80 (KA
(O-ATLAS) and added to the reaction system.

5ji! Th Example 6 10 g of fc wet bacterial cells obtained in the same manner as in Example 6
．． Suspended in 1M phosphate buffer (pH 6.5), cooled with ice water, sonicated for 4 times for 5 minutes, and then centrifuged to remove any residue. 6? ! By removing lk, a crude fermentation compound was obtained.

'NADPH (Sigma) 20 to this crude #accumulated liquid 101d
0■? In addition, 20 ethyl γ-chloroacetoacetate was immediately added in portions over a period of 4 hours, and after further reaction for 4 hours, the reaction solution was added in the same manner as in Example 6. Analysis revealed that γ-chloroβ-
The yield of ethyl hydroxybutyrate was 90%.

Example 7 Cultivation was carried out in the same manner as in Example 6, 10 gkti of sucrose was added to the resulting culture solution, and after aeration culture, 1 g of ethyl γ-chloroacetoacetate was dispensed every 8 hours. When the reaction noise was analyzed in the same manner as in Example 1, the yield of γ-chloroβ-ethyl butyrate was 40%. [Effects of the invention]

Claims (1)

[Claims] (1) Produced by reacting γ-haloacetoacetate with a culture solution, bacterial cells, or treated bacterial cells that have the ability to convert γ-haloacetoacetate into the corresponding γ-halo-β-hydroxybutyrate. 1. A method for producing γ-halo-β-hydroxybutyric acid ester, which comprises collecting a substance.