JPS63233785A - Novel 3alpha-hydroxysteroid dehydrogenase and production thereof - Google Patents

Abstract

PURPOSE:To produce 3alpha-hydroxysteroid dehydrogenase having high stability, by cultivating a microorganism, belonging to the genus Cellulomonas and having the ability to produce the 3alpha-hydroxysteroid dehydrogenase having specific properties. CONSTITUTION:A microorganism, belonging to the genus Cellulomonas and having the ability to produce 3alpha-hydroxysteroid dehydrogenase having the following characteristics is cultivated. With reductive ability for steroid compounds having oxo group at the 3-position and carbonyl group-containing compounds, 98,000 molecular weight measured by a native polyacrylamide electrophoresis, 24,000 molecular weight measured by sodium dodecyl sulfate (SDS) electrophoresis and stable when allowed to stand at pH6-8 (1/15M phosphoric acid buffer solution) and 30 deg.C for 24hr and pH7 and 60 deg.C for 2hr. The aimed 3alpha- hydroxysteroid dehydrogenase is then collected from the culture thereof.

Description

[Detailed Description of the Invention] [Field of Industrial Application] In recent years, various attempts have been made to utilize enzymes industrially. One is as a clinical diagnostic enzyme that utilizes the enzyme's substrate specificity to selectively measure the concentration of a specific compound from among a mixture of miscellaneous compounds, and the other is as a conventional energy-intensive enzyme. It is used in bioreactors to establish energy-saving processes by using enzymes in chemical reaction processes. Since the enzyme of the present invention specifically acts on 3α-hydroxysteroid compounds such as androsterone, deoxycholic acid, chenodeoxycholic acid, and cholic acid among steroid compounds, it is useful in clinical trials for measuring bile acids in blood. It is expected to be used as a diagnostic enzyme. In addition to 3α-hydroxysteroid compounds, the enzyme acts on various compounds with carbonyl groups, such as cyclohexanone and methyl isobutyl ketone.
Termentone can be reduced to the corresponding alcohol or asymmetrically reduced to menthol, which is useful as a fragrance, so it can be used to produce useful substances using a bioreactor.

[Prior Art] As an enzyme having a similar function to the enzyme in the present invention, 3α-hydroxysteroid dehydrogenase (hereinafter referred to as 3α
-H8DH) is known (for example, r P, 1
．． Marcus, p. Talay et al., J. Biol.
Chem, vol. 190, pp. 661-674.

1955”). , 3α-H5DH has great activity against so-called 3α-hydroxysteroid compounds such as androsterone, sodium cholate, cholic acid and de14° xycholic acid.

[Problems to be Solved by the Invention When coenzymes are used industrially, they are required to have high stability. Conventional 3α-HSDH loses all its activity when heated at 60°C for 10 minutes.If this problem of low thermostability could be solved, that is, a more stable enzyme could be developed. It is expected that its practical value will further increase.

The present invention provides such a novel 3α-hydroxysteroid dehydrogenase with high stability and a method for producing the same.

[Means for solving the problem] In order to obtain an enzyme with higher stability than the conventional 3α-H8DH, microorganisms were screened from soil and the like. As a result, it was discovered that the Ichishu strain, which is recognized as a member of the genus Cellulomonas, has properties suitable for the purpose. That is, a microorganism belonging to the genus Cellulomonas and having a novel ability to produce 3α-H3DH. The present invention relates to a novel method for producing 3α-HSDH, which is characterized by culturing and collecting the novel 3α-H3DH from the culture.

The production of the novel 3α-I (tDH) according to the present invention is carried out by culturing the enzyme-producing bacteria in a medium. Examples of the enzyme-producing bacteria include Cellulomonas, which the present inventors isolated from soil. The mycological properties of Honkan are shown in Tables 1 and 2.

Table 1 Physical properties of Cellulomonas tourbata KE31 Morphological properties On broth agar medium (so”c) Shr Length # (1-2μ1) 48h
r Short 4 (0,5-Lμm) motility
Decaspore formation - Durham stain Decacultural properties Gelatin liquefaction forming circular yellow colonies 1 mm in diameter + (weak) Physiological properties Catalase + Bovine citase + (weak) Casein decomposition Decacellulose decomposition Decomposition of dexanthin - Salt Resistance 7% or less Growth 10% or more Use of citric acid that does not grow - Extracellular production of pigment - Cell wall analysis Temperature range for growth including lysine 20-45°C (33°C is good)
Growth pH range 6.5-8.5 (7.2 is good)
Attitude towards oxygen Facultative anaerobic Table 2 Assimilation of carbon sources °−′″ Glycerin + Acetic acid Decarabinose + Glufonic acid Deca ribose
＋ Lactic acid Decoglucose ＋
Propionic acid Decagalactose + Citric acid -Fructose + Cellobiose
Based on more than 10 mycological properties, this strain is taxonomically classified as “E, 5
tackebrandt et al., Zbl, Bakt,
HYg,, 1. Abt, Or4gC3,401
A novel method for producing 3α-H3DH using the Shichimoto bacterium described in 1982, p. 409 will be described.

The medium contains assimilable carbon, nitrogen and other inorganic substances, vitamins,
Media commonly used for culturing microorganisms, including Ami, °jm, yeast extract, etc., are widely used.

Examples of the carbon source include glucose, galactose, arabinose, sucrose, fructose, sorbose, nrubitol, glycerin, and ethanol. Nitrogen sources such as peptone, meat extract, yeast extract, cornstarch liquor.

Malt extract, ammonium sulfate, ammonium phosphate,
Examples include ammonium chloride. Examples of inorganic salts include phosphates, magnesium salts, calcium salts, and the like.

Using a medium containing these components, the temperature is 20-45°C, preferably 30-35°C, and the p■ of the medium is 6.5-8.5, preferably pl+7-7.5 for 10-100 hr, under microaerobic conditions. Cultivate under. Various known treatment methods can be used to extract the enzyme from the culture obtained by culturing the bacteria. For example, after separating the bacterial cells using a conventional method such as centrifugation or filtration, the cells can be separated by autolysis, French press, Dyno Mill, ultrasonic treatment, etc., using live bacterial cells, acetone-dried bacterial cells, or freeze-dried bacterial cells. After crushing, a bacterial cell extract is obtained. To separate and purify the enzyme from the bacterial cell extract, the precipitate obtained by salting out ammonium sulfate (20-8oz saturated fraction) was mixed with 1/30M Tris buffer (pH
After desalting, the extract is passed through a column packed with an anion exchanger such as DEAE-Sepharose, which has been equilibrated with the same buffer in advance, to adsorb the enzyme. Next, when chromatography is performed in the same buffer using an elution method in which the potassium chloride concentration is increased stepwise, the enzyme is eluted at a potassium chloride concentration of 0.1M. The active fraction containing the enzyme is further purified by various known purification methods, such as gel filtration, affinity chromatography, and electrophoresis.

The physicochemical properties of the novel 3α-H3D produced by the present invention are shown below.

39 action The enzyme is nicotinamide adenine dinucleotide (N
AD) (alkaline conditions are preferred) converts 1 mole of a substrate such as 5α-antrostane-3α-oleur into 1 mole of 5α-antrostane-3,17-dione and menthone, respectively.

On the other hand, reduced nicotinamide adenine dinucleotide (
NADH) (acidic conditions are preferred), 1 mol of 5α-antrostan-17β-ol-3-one (
Substrates such as 4,5-dihydrotestosterone) or 1-menthone are mixed with 1 mole of 5α-antrostane-3,
Convert to 17-diol and menthol. It converts various other compounds with carbonyl groups into the corresponding alcohols, and also performs the reverse reaction.

b. Substrate specificity As shown in Table 3, steroid compounds having a ketone at the 3-position, menthone, ethyl 4-chloroacetoacetate, etc. are reduced in the presence of NADH. However, it has no activity against 4-androstene-3,17-dione and prednisolone, which have unsaturated ketones.

Table 3 A-reactive ability for various substrates 5α-an)-
oXtan-17β-ol-3-one 0.24
1005α-and n-stan-3,17-sion 0.24 435β-7and 0-stan-3,17-sion 0.17
7 block a cabinet stage 0
0.16 0.054
-1nd 0sten-3,17-v"on
0.24 0 Firednisolone
0.14
0toku0oethyl acetoacetate
6.1° 251-centen-3-
On 53°°4.8
3-hen-2-on
5 1.44-to Asen-3-
on 45
6.22-Sodium okifbentanoate
14.5° 1.6o, Menthong
2.4 0.3 Substrate is dissolved in ethanol (final concentration of ethanol is 10%) and 1/15
The reaction was carried out in M acetate buffer (pH 4, 6) at 30°C.

° Completely dissolved in the buffer solution and reacted.

The reaction occurred in the state of the °° pendant.

Further, as shown in Table 41, steroid compounds having a hydroxyl group at the 3α position and various hydroxy compounds were oxidized in the presence of NAD. 5α-Anthrostane-
It has the highest activity against 3α-ol-17-one, but
It had no effect on steroids with a 3β-ol structure such as the similar 5α-androscun-3β-ol-17-one.

Table 4 Oxidizing capacity for various substrates 5α-and0 stan-3α-ol-17-one
0.24 1005α-T-drostane-3α
, 1-fluor β-thiol 0.24 815
α-77)”o stane-3β-ol-17-one0.
240 Teocholic acid
0.17 3.1
Chenoteochoncholic acid
0.17 2.1 Cholic acid
0.17
0.5 with menthol
2.5 0.14 I1 B 08° No & 8
00 0.254-methyl-2-h°
ntano k 150”
No. 1 nol to 4.9 cyclo
Self; gift” 4,
6(+,-)6-methyl-5-hebuten-2-ol
38° 10.4 Nerol
3.3
2.6 keraniol
3.3 0.18
The substrate was dissolved in ethanol (final concentration of ethanol was 101) and reacted at 30°C in 1/15M BiU phosphate buffer (pH 8.8).

°　Reacted in a suspended state.

°° It was completely dissolved in the buffer and reacted.

c, Km value (Michaeli constant) The +n value of the enzyme is 5α-antrostane-17β-
All-3-one is 0.29mM and menthone is 14
．． It was 3*M. Both are 0.15mM N A D
3 in 1715M acetate buffer (pH 4,5) in the presence of H
Measured at 0°C.

d, influence of metal ions, etc.Metal ions and other additives (all final concentration 3++
The effect of M) on enzyme activity was investigated. As shown in Table 5, Pa5Cu ions were inhibited.

(>・Person 1: /i, ff!1') Table 5 Effect of metal ions and other additives MgCl2 6H20100° CL15O4・51120 7
6CoSOa・1j120 96
Ri'rikun 105CaC12-
2tlzO95 lysyl lysine 105
ZnSOa・7H2095! /'thiothreitol
99NaC1100β-Meni'hButethanol 96(Nun)gsOa 96
EDTA 100FeSO44H2067 Untwisted 100° Relative value e6 when no addition is taken as 100 Optimum reaction pH k Using menthone as a substrate, the optimal reaction pn was investigated in the presence of NADH. Optimum pH-4.5-5.5 (1/15M
in acetate buffer). Also, in the presence of NAD, 5α
-The optimum pH for the reaction when anthrostan-3α-ol-17-one is used as a substrate is 7.5-8.5 (1/15M
phosphate buffer and Tris-■C1 buffer).

f. Stable pH range The enzyme is stable at pH 6-8 (in 1/15M phosphate buffer) for 30'C524hr. Also pH
50% when left at 7,0,30℃ for 1.100hr
activity remains.

g, stable temperature range 1/15M! Stability was investigated in a hexaphosphate buffer (pH 7.0) at varying temperatures. No deactivation of the enzyme was observed when the enzyme was kept at a temperature of 60° C. or lower for 2 hours.

h. Purification method The purification method is shown in Table 6. After disrupting the bacterial cells, an electrophoretically homogeneous enzyme can be obtained by performing protamine treatment, ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography. The enzyme activity was 0.24mM, 5α-AnFo st-1779.
-ol-3-off, measured in 1/15M acetate buffer (pH 4,5) in the presence of 0.15+gMNA DH. Enzyme titer is 1 Mmol NAD in 11 minutes at 30"C.
The amount by which H was reduced (tracked at 340 nm) was defined as 1 unit.

The enzyme purification results are summarized in Table 10.

(ytT-compound) Table 6 Novel purification method fractions of 3α-H8DI
Fractionation method, condition 1, culture solution ↓ centrifugation, (9,OOOrpm, 15 minutes)
2JI cells ↓ Cell disruption, Inomil (10 minutes) ↓ Centrifugation (1g, 0OOrp+a, 30
3. Cell extract ↓ Protamine treatment (0.25 $) ↓
Ammonium sulfate salting out (20-8o saturation 2) 4, Ammonium sulfate salting out fraction ↓ Dialysis (pH 7,1, l/15M) Lith buffer) 5, Dialysate ↓ DEAE-Seftyloose chromatography 6, Active fraction (0.1M KCl elution fraction) ↓ Blue A. Rio
7) Refill, purified enzyme (0.1M) [CI elution fraction] 1, molecular weight The molecular weight of the enzyme in native polyacrylamide electrophoresis was 98,000. Moreover, the molecular weight in SDS electrophoresis was 24,000. Therefore, the enzyme is thought to consist of four subunits.

j, ultraviolet absorption spectrum The ultraviolet absorption spectrum of the enzyme is shown in FIG.

λmax: 275nm, 3SOnmif:
Arms around 260 rv, around 270 nm, crystal structure, and elemental analysis Up to now, no crystallization has occurred, and therefore elemental analysis cannot be performed.

[Novelty of the Enzyme Core A, Substrate Specificity The enzyme that reduces cyclohexanone is known to be alcohol dehydrogenase derived from horse liver [EC1,1,1,1]. However, while the enzyme has activity against ethanol, it has strong activity against 3α-hydroxysteroids and is similar to the known 3α-H5DI. However, 3α-H3D)I from Pseudomonas testosteronii
(manufactured by Sigma, abbreviated as A enzyme) and 3α-H8DH derived from Pseudomonas putida (abbreviated as B enzyme), there are differences in substrate specificity as shown in Table 7.

Table 7 Comparison of substrate specificity with known enzymes 1.5α-antrostane-3α-ol-17-one too
100 1002, Sodium cholate 1.1 43
−”'3, Theo-sicolic acid
3.1 18 3484
, cholic acid 0.
5 30 3705, 4-methyl-2-°
Tartar 4.9 0.3
-6,5α-γandrostane-17β-ol-3-one 100
100 -7,5α-and0 stun-3,17-sion 44
107 -8,4-ri00ethyl acetoacetate 25 0.2 -
° Sheet' Monas Testosterone ATCC
11996-derived 3α −SD■゛° Shutomonas 7°fri゛ KY4667 (JP-A-53-9
3α-11SDI derived from 9392 (quoted from
l"1OO) No. 6 to 11: Reduction activity of substrate at pH 4, 5 (No. 6-100), : Th, v2. androsterone) oxidation activity to 10
When set to 0, the sodium cholate possessed by the enzyme,
Although the oxidizing activity of cholic acid and deoxyfuric acid is lower than that of enzyme A and enzyme B, the oxidizing activity of 4-methyl-pentanol is higher than that of enzyme A. On the other hand, when the reducing activity of 5α~anthrostan-17β-ol-3-one (4,5-dehydrotestosterone) is set as 100, the ethyl 4-chloroacetoacetate reducing activity of the enzyme is more than 100 times that of enzyme A. expensive. This large difference in substrate specificity strongly suggests that the active sites of each enzyme are different, that is, the amino acid sequences of the enzymes are different, and these enzymes have different structures. it seems to do.

b. Stereoselective reduction) When menthone is reduced with enzyme A, the product has a volume of menthol:d-neomenthol of 1:2. On the other hand, when using the enzyme, menthol=d-neomenthol=9? :3, and the stereoselectivity of the enzyme is higher than that of enzyme A.R=:,! . ：茫′：：こ“ The enzyme was compared with known enzymes in terms of C9 molecular weight, optimum reaction pH, thermostable molecule m, etc. As shown in Table 8, this enzyme is similar to enzyme A and enzyme C. (
3α-II S from Cynidomonas sphaericus
D II) had different properties.

Table 8 Comparison molecular weight with known enzymes 98.000 47,000 16G
, 000 reaction optimum pH 7.5-8.5 11-11.5
10-10.5 Thermal stability"" 100 0
No description・° Derived from Sidomonas testosteroni ° ° Derived from Ha'tilus sphaericus (quoted from Japanese Patent Application Laid-open No. 157894-1983) ° ° ° Numerical value is 1715
M shows the residual activity after incubation at 60°C for 10 minutes in phosphate buffer (pH 7,0).

In other words, the molecular weight differs greatly depending on the king, and the optimum reaction p
Both enzyme A and enzyme C are on the alkaline side compared to the enzyme concerned: 1. Also, the enzyme concerned remains at 100% activity even after heating at 60°C for 110 minutes, but the enzyme A loses its activity at all. Therefore, the thermostability of the enzyme is higher (see Figure 2).

As described above, the enzyme is a known 3α-H3D
5α-Anthrostan-3α-ol-1 compared to H
Although they have a common feature of high activity toward 7-one, they all differ in substrate specificity, stereoselectivity, molecular weight, optimum reaction pH, and thermal stability.

Therefore, it is appropriate to name the enzyme the novel 3α-H5DR.

(containing 100 mL of soil) and cultured for 48 hours. During culture, menthone (0.05 mL) was added in two portions as an induction substrate (the added menthone was mixed with ethanol in a volume ratio of 1=1). 2L of this culture solution
Transfer to a jar fermenter (containing the same medium as IL),
48' at rotation speed 40Orpm, ventilation fio, 05vvm
An hr culture was performed.

Menthone was added in the same manner as flask culture.

Next, the cultured solution was transferred to a 3OL jar fermentor (containing 2OL of the same medium) and heated at 200 rpm, i. Cultivation was further carried out for 50 hours at 0.5 yvm. During the culture, add 100 mL of 25% D-arabinose to
I attached a batch. In addition, 25 mL of a mixture of menthone and ethanol was added four times as in the pre-culture. Using this cultured bacterial cell, the enzyme was purified by the method shown in Table 6. The purification process is summarized in Table 10.

Table 9 Medium composition NHaNO30, 1g Fe5Oa, 7H2010n
gNa2HPO4,12H200,9g Na2Mo
O* 0.6vagKI12PO490mg
Mn5Oa, 6H200, 6mgMg5Oa, '1H2
00.1g Yeast Extract 1.0
gCaCl2.2H2050mg D-7 Rahinose D, 5g distilled water 100ml,
pH7,2 Table 10 Enzyme purification result fraction Total activity Specific activity Recovery rate unit (
υl,,% Cell extract 19300 0.40 1
00 Protamine treatment solution 16900 0.48
88 ammonium sulfate fractionated liquid 15600 0.6! l
81DEAE/Cef7o4 elution fraction 9750
15.3 51BIueA gel elution fraction
8470 20.4 44 Example 2 The enzyme can regenerate NADH when methylisobutylcarbinol is used as a substrate. Therefore, we produced menthol (from tomentone) in the presence of a coenzyme regeneration system.

The purified enzyme prepared in Example 1 was added to 0.11 units (one unit is the amount of enzyme that produces 1 μmol of menthol per minute in 1/15M acetate buffer at 30°C and pH 4.5). and NADHo, 1μiO, 1IllL (7)
Dissolved in 1/15M phosphate buffer (pH 7,0) and A, Mento:! 'O', '1lllL (578 μmol) and methylisobutylcarbinol O, 1mL (
775 μmol) and reacted at 30'C for 4 hours.

0.6 mL of hexane was added to the reaction solution, and the hexane phase was analyzed by gas chromatography ( )IR-1
0M column, 100℃, helium 50mL/+nfn
). In the reaction solution, 16.7 g mole of menthol and 8 μmol of 20 methyl isobutyl ketone were detected, corresponding to approximately 170 times the mole of NADH added.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the enzyme. Second
The figure shows the enzyme (←・) and the known 3α-H5DH (L o-befu from S. testosterini).
) indicates the stable temperature range. At each temperature, 1715
The residual activity after incubation for 10 minutes in M phosphate buffer (pH 7,0) is shown.

Claims (2)

[Claims] (1) Highly stable 3α-hydroxysteroid dehydrogenase having the following properties (a) It has the ability to reduce steroid compounds having an oxo group at the 3-position and carbonyl group-containing compounds. (b) The molecular weight measured by native polyacrylamide electrophoresis is 98,000, and the molecular weight measured by SDS electrophoresis is 24,000. (c) 30 at pH 6-8 (in 1/15M phosphate buffer)
℃, 24 hours and pH 7 at 60℃, 2 hours
All are stable when left alone. (2) 3α belonging to the genus Cellulomonas and having the following characteristics
- A method for producing 3α-hydroxysteroid dehydrogenase, which comprises culturing a microorganism capable of producing hydroxysteroid dehydrogenase and collecting 3α-hydroxysteroid dehydrogenase from the culture. (a) It has the ability to reduce steroid compounds and carbonyl group-containing compounds having an oxo group at the 3-position. (b) The molecular weight measured by native polyacrylamide electrophoresis is 98,000, and the molecular weight measured by SDS electrophoresis is 24,000. (c) 30 at pH 6-8 (in 1/15M phosphate buffer)
℃, 24 hours and pH 7 at 60℃, 2 hours
All are stable when left alone.