JPS63304991A - Production of (r)-gamma-substituted-beta-hydroxybutyric ester - Google Patents

Abstract

PURPOSE:To efficiently produce the titled compound, by treating a gamma-substituted acetoacetic ester with an NADPH-dependent reductase of reduction type derived from a fungus belonging to the genus Sporobolomyces. CONSTITUTION:Sporobolomyces salmonicolor is cultivated in a medium to produce an NADPH-dependent reductase of reduction type having 32,000 (Sephadex) mol.wt. and the following physical and chemical properties. Action: catalyzing a reaction to form an alcohol from an aldehyde or a ketone. Optimum pH and stable pH: optimum pH is approximately 7 and stable range is pH 6-10. Optimum temperature and stable temperature: optimum temperature is approximately 60 deg.C and stable range is 40-60 deg.C. Inhibitor: being inhibited with Mg<3+> or Fe<3+>. A reaction solution containing <=10% gamma-substituted acetoacetic ester is treated with the reductase in the presence of NADPH at 20-40 deg.C at pH 6-12 to produce (R)-gamma-substituted-beta-hydroxybutyric ester.

Description

[Detailed Description of the Invention] [Field of Industrial Application] In recent years, optically active oxyacid derivatives have become increasingly useful as intermediates for the synthesis of pharmaceuticals and agricultural chemicals. As a result of extensive investigation into the manufacturing method of (R)-r-substituted-β-hydroxybutyric acid ester,
The novel reductase discovered by the present inventors can be used to effectively produce γ-recombinant acetoacetate.The entire heading of the present invention? completed.

[Conventional technology]

Conventionally, it acts on γ-substituted acetoacetate,
)-r-substituted-β-... To produce hydrocyanacetic acid ester, yeast-derived L-β-hydroindicyl COA dehydrodenase (EC1, 1,
1,35] (Japanese Unexamined Patent Publication No. 118093/1983) and Thermoanaerobium derokii (Thermoanasr.
Alcohol dehydrogenase (EC1,1,1,2'+(J,A,C
, S., 1985.107.4028) is known.

[Problem that the invention seeks to solve]

However, there is room for improvement in terms of reactivity and temperature stability, and the latter requires anaerobic culture. The method for producing butyric acid ester was difficult to find practical.

〔problem? ! -β- Ninth means]

The present invention provides a method for treating γ-substituted acetoacetic esters with a reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NAr)-dependent reductase derived from the genus Sporobolomyces, thereby reducing the tIFI symptoms. (R)-γ-
This is a method for producing a substituted -β-hydroxy fluoric acid ester.

The above-mentioned reductase used in the present invention is derived from Sporobolomyces salmonicolar (SPorobolomyaeesga).
lmonicolor) It is extracted and purified from IFO1038 using an enzyme having reducing ability. This enzyme was proposed separately because it is clear that it is a new reductase that has various physical properties that are practically superior to known enzymes.

Next, the preparation of the enzyme used in the present invention and its physical properties will be specifically described.

Sporobolomyces salmonicolor τFo1038 grown in a medium consisting of 5% glucose and 51% cornstarch liquor was collected by centrifugation, washed with Linkuma buffer, and then incubated with Dynomil (manufactured by Shinmaru Enterprises). ) to crush the cells and extract the enzyme. Cell-free extract obtained after centrifugation? Fractionate ammonium sulfate and collect 60-80% saturated fractions. After overnight dialysis, DE
After adsorption onto an AE-Sephacel (manufactured by Pharmacia) column, linear gradient elution with 0 to 0.6 M sodium chloride is performed to collect active fractions. Furthermore, Dell filtration chromatography treatment (Sephadex G-10 manufactured by Pharmacia)
0) Collect the active fractions and perform Delfiltration chromatography again in the same manner as above to obtain a purified enzyme solution sample. this is,
Electrophoretically homogeneous.

Note that a crystallized product of this enzyme has not been obtained.

Next, what are the physical and chemical properties of the enzyme used in the present invention? write down

■ Action: In the presence of coenzyme NADPH, 1 mol of aldehyde or ketone as substrate, 1 mol of alcohol? generate.

(2) Substrate specificity 2 The activity for each substrate below is expressed using ethyl γ-chloroacetoacetate as a standard. The activity measurement method is as follows.

600 nmol of substrate and 192 nmol of NADPH? 0. - Enzyme solution (0.1M 17-phosphate buffer, p) I 7.0
), change in absorbance at 67℃ and 340nm? Measure and find.

(1) Activity against ketones Table 1 (4) Coenzyme ・Active against NADPH.

- Does not act on cotinamide adenine dinucleo ÷ -) on NADH.

(Ill) Alcohols Substantially no activity is observed against the alcohols listed in Table 2.

Table 2 ■ Optimum and stable range optimal is around 7, and Pl″16 to 1D is 55
Even when treated at ℃ for 10 minutes, more than 80% activity remains.

■ Optimal temperature and stable temperature range The optimal temperature is around 60°C. Also, FJ-17,0,
When treated for 10 minutes, an activity of 100 at 40°C and 70 at 160°C remains.

■ Effects of metal ions and various drugs ■ Reduction reaction products of optically selective r-substituted acetoacetic esters (R)
As a whole, the optical purity is 97% or more.

Optical purity of Table 4 product is (+)-α-meth=? An ester with C-α-trifluoromethylphenylacetic acid [(MTPA) is synthesized to form a diastereomeric compound, and then separated and quantified using high performance liquid chromatography (HPLC).

) (PLO condition empty A: partstst15 (
(manufactured by Watbun) (4,6φX 250sx) Mobile phase: Sansan: Tetrachlorofuran: Methanol = 600: 100:1 Speed: 2.0 d/min Detection (absorbance wavelength): 217 nm Organic solvent Stability against: Even when treated in a phosphate buffer solution of pH 7.0 containing the organic solvent shown in Table 5 at 28°C for 24 hours, it retains an activity of 80% or more.

Table 5 ■ Isoelectric point...4.7 ■ Molecular weight 52.000 (Sephadex G-100 Del filtration method) 36.500 CSD8 (Sodium dodecyl sulfate) 1 gas phoresis method] New reduction used in the present invention In order to produce the enzyme, the bacteria can be cultured according to a conventional method.

The medium used for culture contains carbon sources necessary for the growth of microorganisms,
This is a normal medium containing a nitrogen source, inorganic substances, etc. Additionally, desirable results are often obtained by adding organic trace nutrients such as vitamins and amino acids.

Cultivate! under aerobic conditions, pH 5-8, temperature 10-40'
The test is carried out for 1 to 10 days while controlling C within an arbitrary range.

In order to obtain electrophoretically uniform aldehye V reductase by culturing the bacteria, conventional ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, Delfiltration chromatography, etc. are used.

The forms of the enzyme used in the synthesis of (R)-γ-substituted-β-hydrofushibutyric acid ester from γ-fil-substituted acetoacetate include purified, semi-purified, enzyme-containing bacterial cells, processed bacterial cells, and their immobilization. Any chemical can be used. The strain used is Sboroboromyces salmonicara-U.
Since FO105B also contains an enzyme that generates (S)-γ-converted-β-hyperoxybutyric acid ester, it must be removed in advance.

The concentration of γ-g1-acetoacetate used as a substrate was 201! Although it is possible to add up to 10% by weight, since the reaction tends to be inhibited as the concentration increases, it is preferable to add less than 10% by weight, or to add in portions or continuously at low concentrations.

The γ-position substituent of the γ-substituted acetoacetic ester is preferably chloro, chloro, fluoro, acid group, etc., and the ester group is preferably an alkyl group having 1 to 10 carbon atoms.

As a coenzyme, to make NAJ)PHk essential, add NADPH' (required amount in advance or add NADP to the reaction system)
7 stems that generate Hr coexist. This/stem contains, for example, the NADP of NADP in the gluconic acid production reaction from glucose by glucose dehydrogenase PCI denase.
NADPH regeneration system, etc. that utilizes conversion to DPH
Can be used suitably.

When the reaction temperature is adjusted to 5 to 70°C, preferably 20 to 40°C, the object of the present invention can be fully achieved by adjusting the reaction temperature to 4 to 10, preferably 6 to 8.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples. In the examples, unless otherwise specified, the weight is % by weight.

Enzyme Isolation Example Glucose 5 1 wt. P) 16.0 medium consisting of 5 wt.
A seed culture was obtained by inoculating Sporobolomyces salmonicolar IF01038 and culturing at 28°C for 2 days.

A culture medium having the same composition as above was placed in a 50011jt-2J volume flask, seed culture 5cjQ was added, and lesion culture was performed at 28°C for 4 days. Next, the 10 flasks mentioned above? In total, 5
Centrifugation (2800G) from the culture solution of 1.

After washing the cultured bacteria collected after 20 minutes with 0.01 MIJ acid buffer (pH 7.4), Dynomil (beads with 0.
25 to 0.5 myxφ) for 20 minutes, 2
Centrifuge at 8000G for 20 minutes to remove suspended matter.
A crude enzyme solution was obtained. Add ammonium sulfate to this and add 60 to 80
The saturated fraction was collected by molecular centrifugation (28,000 G
) for 20 hours. Next, it was adsorbed on DEAR-Sephacel column chromatography (1.6φ x 60mm), and after washing with the above buffer, sodium chloride 0 to 0.6
Linear gradient elution was performed using the same buffer contained in M'. Both fractions that showed activity were collected and concentrated using an ultrafilter (Amicon, YM 10'), then supplied to a Dell filtration column (Sephadex, G-100, 2,0φ x 90cvL) containing 0.1M NaCjk. Perform chromatography using the above buffer, collect the two fractions that showed activity, and perform chromatography using Delfiltration in the same manner as above. A purified enzyme solution was prepared. This is electrophoretically a single pyH
ef showed.

Example 1 Using the purified enzyme obtained in Enzyme Isolation Example, a reduction reaction was carried out under the conditions shown in Table 6. The t0 reaction completed solution was extracted twice with 200 d of ethyl acetate, and after dehydration with Na8S04, the ethyl acetate was removed under reduced pressure. Removed. The remaining colorless and transparent liquid 7 was analyzed by NMR and the following results showed that it was ethyl γ-chloro-β-hydroxybutyrate. Please confirm.

NMR (CDCj3) δ (pp-): 1-25 (3
H, tr, CH3CH2-)0)T03-6 (2Hs d, C1-C'H2・CH-)CH3,22(IE(,br, -OH)2.62 (2
E(,a,-cH-cHL-coaR)CH Also, the yield was 0.474 with an Eri purity of 98.2% for GQ analysis.
g (yield: 92 g). This product had an optical purity of 974ee according to HPLC analysis of MTPA ester.

The enzyme activity U in Table 6 is as follows.

1μ per minute from γ-chloroacetoacetic acid ethyl ester
The ability to produce moles of γ-chloroβ-hydrobutyric acid ethyl ester is defined as t1 units (U).

Table 6 Example 2 Conditions including the NADPH regeneration system shown in Table 7.
Start the reaction with the r-fl converted acetoacetate r substrate shown in Table 8, pJ4 k with Q, 5 N-NaOH 6.
The reaction was carried out for 20 hours while maintaining the temperature at 5. Example 1 after completion of reaction
and the product as well? Analyze the hole. NMR analysis results,
Product? confirmed. The yield (rate), optical purity, etc. are shown in Table 8.

Table 7 γ-chloro-β-hydrobutyrate methyl NMR (CD
Cl2) δ(pptn): 6-6 (3H1s,
CH3-) 4.20 (IH, -CHzCH-CHz)OH OH 5-22 (IH, br, -OH) OH γ-chloro-β-hyP ethyl chloride butyrate Same as Example 1.

γ-chloro-octyl β-hydroxybutyrate NMR (C
r) C13) δ (ppm): o-ss (3H% t
rs CH3-(CHz)n-)1.28 (10H,
s, -(CH2)5-)OH 3-22(IH, br, -0H) OHO γ-Zolomoβ-hydrobutyric acid ethyl γ-fluoro-β-Il Both are almost identical to chloride ethyl ester 1γ- Horse mackerel?
-β-hyP ethyl butyrate NMR (cDCj3) δ
(ppm): 1-25 (3H%"r% CH3
CHz-)OH0 3,22 (IH, br, -OH) [Effects of the invention]

Claims (1)

[Claims] It is characterized by the action of a reduced nicotinamide adenine dinucleotide phosphate-dependent reductase derived from the genus Sporobolomyces on γ-substituted acetoacetate (
R) Method for producing -γ-substituted-β-hydroxybutyric acid ester.