JP2538597B2 - Novel ring enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is, for example, the production of (R) -γ-substituted-β-hydroxybutyric acid ester, which is important as a synthetic intermediate for pharmaceuticals, from γ-substituted acetoacetic acid ester. The present invention relates to a novel reductase that can be used for.

(Prior Art) In recent years, the importance of producing optically active physiologically active substances and their synthetic intermediates by an enzymatic asymmetric reduction reaction using reduced nicotinamide / adenine / dinucleotide phosphate as a coenzyme has been increasing. As an enzyme that acts on γ-substituted acetoacetic acid ester to produce (R) -γ-substituted-β-hydroxybutyric acid ester, yeast-derived L-β-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] ]
(JP-A 59-118093) and Thermoanaerobium brockii-derived alcohol dehydrogenase [EC 1.1.1.2] (J. Am. Chem. So.
c., 1985, 107 , 4028) are known.

(Problems to be Solved by the Invention) However, the former has a problem of poor reactivity and temperature stability, and the latter has a problem of poor microbial growth due to anaerobic culture and a complicated culture apparatus.

In addition, as a result of examining the activity of the following known reductases against γ-substituted-acetoacetic acid ester, no activity was observed.

Known reductases ... Glycerol dehydrogenase [EC1.1.1.6], glutamate dehydrogenase [EC1.4.1.3], alcohol dehydrogenase [EC
1.1.1.1], beta-galactose dehydrogenase [EC 1.1.1.48], lactate dehydrogenase [EC 1.1.1.27], glucose dehydrogenase [EC
1.1.1.47], formaldehyde dehydrogenase [EC 1.2.1.1], alpha glycerophosphate
Dehydrogenase [EC 1.1.1.8].

This invention acts on γ-substituted acetoacetates,
It is an object of the present invention to provide an enzyme that produces (R) -γ-substituted-β-hydroxybutyric acid ester and has the above-mentioned drawbacks improved.

(Means for Solving Problems) As a result of screening a large number of enzymes in order to achieve the above-mentioned object, the present inventors have found that a strain belonging to the genus Sporobolomyces, for example, Sporobolomyces.・ Salmoni color (Sporobolomyces salmoni
color) IFO-1038 found that γ-substituted acetoacetic acid ester has a high reducing ability, and applied for a patent (Patent application Sho
61-33935). When the present inventors extracted and purified an enzyme having a reducing ability from the above-mentioned strain, it became clear that the enzyme was a novel enzyme that fulfills the purpose of the present invention, and the present invention has been completed.

That is, the present invention is a novel reductase having the following physicochemical properties.

B) Action: In the presence of coenzyme-reduced nicotinamide / adenine / dinucleotide / phosphate (hereinafter referred to as NADPH), aldehydes and ketones are reduced to produce alcohols equimolar to them. .

(B) Substrate specificity ... Selectively reducing γ-substituted acetoacetic acid ester to produce alcohols corresponding to the (R) -form. Further, it is active against NADPH, but is inactive against NADH (reduced nicotinamide adenine dinucleotide).

C) Optimum pH and stable pH range ... The optimum pH is around 7, and the stable pH range is 6-10.

D) Optimum temperature ... The optimum temperature is around 60 ° C.

E) Deactivation condition due to temperature ... 60 ° C without deactivation at 40 ° C
30% inactivation in 10 minutes at (pH7).

F) Inhibition and activation ... Inhibited by quercetin, Fe ³⁺ ,
It is slightly inhibited by Mg ²⁺ . It is slightly activated by α, α'-dipyridyl.

G) Isoelectric point: pH 4.7.

H) Molecular weight: 32000 according to the gel filtration method of Sephadex G-100 and 36500 according to the SDS electrophoresis method.

Next, the production and purification method of the novel reductase of the present invention and various properties thereof will be specifically described. The following%
All represent% by weight. Sporoboromyces salmonicolor IFO-1038 was grown in a medium containing glucose 5% and corn stay liquor 5% and having a pH adjusted to 6.0.

The grown Sporoboromyces salmonicolor was collected by centrifugation, washed with 0.01 M phosphate buffer (pH 7.4), and then the cells were crushed with Dynomill (manufactured by Shinmaru Enterprise Co., Ltd.) to extract the enzyme. Centrifuge the mixture of crushed cells and enzyme, and add the ammonium sulfate aqueous solution (concentration of 60-80% of saturation concentration) to the obtained cell-free extract to precipitate and collect the fraction containing enzyme. It was

After dialysis against 0.01 M phosphate buffer (pH 7.4) overnight to remove ammonium sulfate, it was adsorbed on a DEAE-Sephacel (manufactured by Pharmacia) column and the concentration was 0 to 0.6 M.
Was subjected to linear gradient elution with an aqueous sodium chloride solution to collect active fractions. Furthermore, the active fractions were collected by concentrating by ultrafiltration (using an Amicon device) and performing gel filtration chromatography (using Pharmacia's Sephadex G-100) twice to obtain a purified enzyme liquid preparation. Obtained. As a result of examining the obtained preparation by electrophoresis, it was found to be a uniform substance.

The enzyme activity was determined by measuring substrate 600nmol and NADPH192nmol 0.6
It was determined by adding to ml of enzyme solution (0.1 M phosphate buffer, pH 7.0) and measuring the absorbance at a wavelength of 340 nm at a temperature of 37 ° C. Then, the ability to produce 1 μmol of γ-chloro-β-hydroxybutyric acid ethyl ester per minute from γ-chloroacetoacetic acid ethyl ester was defined as 1 unit.

The physicochemical properties of the reductase of the present invention are as follows.

Action: In the presence of coenzyme NADPH, aldehydes and ketones are used as substrates and alcohols equimolar to them are produced.

Substrate specificity (i) Activity against ketones and aldehydes ... As described in Tables 1 and 2. These activity values show 100% activity against ethyl γ-chloroacetoacetate.
Is expressed as.

The ketones with zero activity are as follows. Two
-Ketoglutaric acid, 2-ketobutyric acid, 2-ketovaleric acid,
2-ketocaproic acid, 2-ketoadipic acid, 2-ketoisovaleric acid, 2-ketoisocaproic acid, 2-ketomalonic acid, 2-keto-3-methylvaleric acid, 2-keto-D-gluconic acid, 3-indole Glyoxylic acid, pyruvic acid,
Phenylpyruvic acid, benzoylpyruvic acid, glyoxylate, oxalic acid, oxypyruvic acid, p-oxyphenylpyruvic acid, indole-3-pyruvic acid, calcium ketopantothenate, ketopantothenamide, ketopantothenic acid, ketopanto Lactone, acetoacetic acid, levulinic acid, dihydro-4,4-diethyl-2,3-furandione, dihydro-4-methyl-4-ethyl-2,3-furandione, dihydro-4-methyl-4-propyl- 2,3-
Frangion, ethyl 2-keto-3-valerate, methyl 2-keto-3-caproate, 2-keto-3-phenylbutyric acid, dihydro-5-isopropyl-4,4-dimethyl-2,3
-Flangion, dihydro-5- (3-pentyl) -4,
4-dimethyl-2,3-furandione, dihydro-5- (3
- pentyl) -4,4-diethyl-2,3-furandione, de hydro carnitine ethyl ester, S- acetoacetyl Ko Enzyme A, acetoin, benzoin, acetol, vitamin K _3, vitamin K _5, isatin, alpha- Naphthoquinone, parabanic acid, 2,5-toluquinone, chloranil, 3-methyl-1,2-cyclohexadione, acenaphthenequinone. Aldehydes having zero activity are as follows.

Formaldehyde, acetaldehyde, crotonaldehyde, D-glyceraldehyde-3-phosphate
Diacetylacetal, glycolaldehyde, propionaldehyde, salicylaldehyde, betaine aldehyde, p-dimethylaminobenzaldehyde, p-hydrobenzaldehyde, glutaric anhydride, n-butyraldehyde, glutaraldehyde, D-gluconic acid sodium salt, (ii) coenzyme・ Active against NADPH.

・ Does not act on NADH (nicotinamide adenine dinucleoside).

(Iii) Alcohols Substantially no activity is observed against the alcohols shown in Table 3.

Optimal pH and stable pH range The optimal pH is around 7. Moreover, 80% or more of the activity remains even when treated at pH 6 to 10 at 55 ° C for 10 minutes.

Optimal temperature and stable temperature range The optimal temperature is around 60 ° C. Moreover, 100% activity at 40 ° C and 70% activity at 60 ° C remain when treated at pH 7.0 for 10 minutes.

Effects of additives (metal ions and compounds) ... As shown in Table 4. Note that the activity (%) is a relative value with 100 when the addition of these metal ions or compounds is not performed.

Optical Selectivity The reduction reaction product of γ-substituted acetoacetic ester is the fifth
As shown in the table, all of them are (R) -form and optical purity is 97% ee
That is all.

The optical purity of the product was determined by high-performance liquid chromatography (HPLC) after synthesizing an ester with (+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) to obtain a diastereomeric compound. Separate and quantify.

HPLC conditions Column: Partisil5 (Watman) (4.6
φ × 250 mm) Mobile phase: Hexane: Tetrachlorofuran: Methanol = 600: 100: 1 Speed: 2.0 ml / min Detection (Absorbance wavelength): 217 nm Stability to organic solvents pH 7.0 including organic solvents in Table 6 28 in phosphate buffer
It retains 80% or more of activity even when it is treated at ℃ for 24 hours.

Isoelectric point pH 4.7 Molecular weight 32,000 (Sephadex G-100 gel filtration method) 36,500 [SDS (sodium dodecyl sulphate) electrophoresis method] To produce the novel reductase used in the present invention, a conventional method is used. Then, the bacterium can be cultured. The medium used for culturing is an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of microorganisms. In addition, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results.

The medium is aerated under aerobic conditions for 1 to 10 days while controlling the pH to 3 to 8 and the temperature to 10 to 40 ° C.

To obtain the electrophoretically uniform reductase by culturing the bacterium, ordinary ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like are used.

〔Example〕

Next, the method of the present invention will be described in more detail with reference to Examples.

Glucose 5% by weight, corn staple liquor 5
Take 5 ml of a medium (pH 6) containing 10% by weight in a test tube and use Sporobolomyces salmonicolor.
nicolor) IFO1038 was inoculated and shake-cultured at 28 ° C. for 48 hours to obtain a seed culture solution.

Next, 10 2-volume flasks were prepared, 500 ml of the medium having the same composition as described above and 5 ml of the seed culture were added to each flask, and shake culture was performed at a temperature of 28 ° C. for 3 to 4 days.

Then, the cultured cells recovered from the culture solution of 5 by centrifugation (28000 G, 20 minutes) were mixed with 0.01 M phosphorus buffer solution (pH 7.
After washing with 4), Dyno Mill (Bead diameter 0.25 ~ 0.5
mm) for 20 minutes and centrifuged at 28000G for 20 minutes to remove suspended substances to obtain a crude enzyme solution. By adding ammonium sulfate aqueous solution (concentration of 60-80% of saturation concentration) to this crude enzyme solution, the fractions containing the enzyme were precipitated and collected, and the fraction containing the enzyme was centrifuged (28000G, 30 minutes). It was recovered and dialyzed against 0.01 M phosphate buffer (pH 7.0) for 20 hours to remove ammonium sulfate.

The obtained enzyme solution was adsorbed on a DEAE-Sephacel column (column diameter 1.6 cm, length 30 cm). After the column was washed with the above buffer, a linear gradient elution was carried out with the same buffer containing 0 to 0.6 M sodium chloride to collect fractions showing activity.

The active fraction was concentrated by ultrafiltration (Amicon, YM10),
It was supplied to a gel filtration column (Sephadex G-100, column diameter 2.0 cm, length 90 cm). Chromatography was performed with the above buffer containing 0.1 M sodium chloride, the active fractions were collected, and gel filtration chromatography was performed in the same manner as above to prepare a purified enzyme solution. When this purified enzyme solution was subjected to electrophoresis, it showed a single band. The purification results are shown in Table 7.

(Effect of the invention) The novel reductase obtained by the present invention has a high reducing ability of γ-substituted acetoacetic acid ester, and thus is useful as a reduction catalyst in an organic synthesis reaction, particularly an asymmetric synthesis reaction,
Great industrial value.

Claims (1)

(57) [Claims] 1. A novel reductase having the following physicochemical properties. B) Action: In the presence of coenzyme-reduced nicotinamide / adenine / dinucleotide / phosphate (hereinafter referred to as NADPH), aldehydes and ketones are reduced to produce alcohols equimolar to them. . (B) Substrate specificity ... Selectively reducing γ-substituted acetoacetic acid ester to produce alcohols corresponding to the (R) -form. Further, it is active against NADPH, but is inactive against NADH (reduced nicotinamide adenine dinucleotide). C) Optimum pH and stable pH range ... The optimum pH is around 7, and the stable pH range is 6-10. D) Optimum temperature ... The optimum temperature is around 60 ° C. E) Deactivation condition due to temperature ... 60 ° C without deactivation at 40 ° C
30% inactivation in 10 minutes at (pH7). F) Inhibition and activation ... Inhibited by quercetin, Fe ³⁺ ,
It is slightly inhibited by Mg ²⁺ . It is slightly activated by α, α'-dipyridyl. G) Isoelectric point: pH 4.7. H) Molecular weight: 32000 according to the gel filtration method of Sephadex G-100 and 36500 according to the SDS electrophoresis method.