JPH06113883A - Production of cholesterol derivative - Google Patents

Abstract

PURPOSE:To produce a cholesterol derivative in which hydrogen at the 6-position of cholesterols is converted into hydroxyl group with an enzyme. CONSTITUTION:A cholesterol oxidase is made to react with cholesterols, e.g. cholesterol, 4-cholsten-3-one or 5-cholesten-3-one, dihydroxycholesterol, dehydroepiandrosterone, pregnenolone, sitosterol, lanosterol or stigmasterol in the presence of dissolved oxygen to produce the objective 6-hydroxy- cholesterols.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a cholesterol derivative,
For example, 6 such as 4-cholesten-6-ol-3-one
-A novel method for producing hydroxy-cholesterols. More specifically, a novel reaction by cholesterol oxidase, that is, a reaction in which the hydrogen at the 6-position of cholesterols becomes a hydroxyl group, 4-cholestene-6-
It relates to a method for producing 6-hydroxy-cholesterols such as all-3-one.

[0002]

BACKGROUND OF THE INVENTION Conventionally, 4-cholesten-6-all-3
Cholesterol derivatives such as -one are produced by a chemical synthesis method and are sold as research reagents. on the other hand,
Cholesterol oxidase oxidizes cholesterol (5-cholesten-3β-ol) to produce 5-cholesten-3-one as a temporary intermediate metabolite, and further catalyzes an isomerization reaction to 4-cholesten-3-one. It is known that on is the final product. By utilizing this reaction to measure free cholesterol or bound cholesterol in the living body, it is widely used as a diagnostic reagent for diseases such as adult diseases.

[0003]

The present inventors have been investigating the conventional cholesterol metabolism system of cholesterol oxidase, and the enzyme converts a novel reaction, namely, the hydrogen at the 6-position of cholestenone into a hydroxyl group. It was found that the reaction catalyzes a reaction that produces a cholesterol derivative such as 4-cholesten-6-ol-3-one.

[0004]

The present invention provides 6 of cholesterols.
The method for producing a cholesterol derivative is characterized in that hydrogen at the position is converted into a hydroxyl group by cholesterol oxidase.

Cholesterol oxidase used in the present invention is an enzyme produced by microorganisms, plants or animals, for example, Streptomyces genus,
Brevibacterium genus, Pseudomonas genus, Arthrobacter
Genus, Corynebacterium genus, Rhodococcus genus, Nocardia genus, Microbacterium genus, Mycobacterium genus, Actinomyc
Examples include enzymes produced by bacteria such as es) and Schizophyllum. Especially Streptomyces,
Enzymes produced by Brevibacterium and Pseudomonas bacteria are preferred. The cholesterol oxidase used in the present invention may be a commercially available product or a powder of purified enzyme, as well as an unpurified enzyme solution, bacterial cells of a microorganism producing cholesterol oxidase, a crushed extract, or a culture supernatant. it can. The genes derived from the above-mentioned bacteria include those produced by transforming host cells such as Escherichia coli and yeast. The cholesterol oxidase derived from the bacterium may be a bacterium that produces the enzyme. When using the cholesterol oxidase, a purified enzyme or cells may be immobilized on a carrier before use.

The cholesterols used in the present invention are
For example cholesterol, 5-cholesten-3-one, 4
-Cholesten-3-one and the like. Instead of these compounds, if a cholesterol analog that reacts with cholesterol oxidase, it is hydroxylated at the 6-position to give 6-
It is also possible to use a hydroxy derivative. Examples of such cholesterol analogs include dihydroxycholesterol, dehydroepiandrosterone, pregnenolone, sitosterol, lanosterol, stigmasterol and the like.

In the present invention, the desired cholesterol derivative can be produced by reacting the above cholesterols with the above enzyme in the presence of a dissolved enzyme. The hydroxylation reaction is usually carried out in a buffer solution containing the above cholesterols and the above enzymes. The reaction is pH 3-10, preferably pH 5-8
It is suitable to carry out. The reaction temperature is preferably adjusted within the range of 10 to 50 ° C, preferably 30 to 40 ° C. The reaction time is usually 48 hours. It is also possible to add the above-mentioned cholesterols to a culture solution of a microorganism capable of producing cholesterol oxidase, and further convert the cholesterols while culturing.

After completion of the reaction, the product such as 4-cholesten-6-ol-3-one can be extracted with a solvent such as ethyl acetate or methyl isobutyl ketone and isolated by a conventional method. The purification method is performed according to a conventional method.

[0009]

EXAMPLES The present invention will be described below with reference to examples. Example 1 100 ml of a medium containing 1% glucose, 0.3% yeast extract, 0.5% bactopeptone and 0.3% malt extract was transferred to a 500 ml Sakaguchi flask and autoclaved at 121 ° C. for 15 minutes. Streptomyces sp.SA as a cholesterol oxidase-producing bacterium
-Streptomyces lividans transformed with the vector pCO-1 expressing the COO-derived cholesterol oxidase gene (Microtechnology Research Institute No. 8789) (see Japanese Patent Laid-Open No. 62-285789) One platinum loop was inoculated and cultured at 28 ° C for 6 days. After removing the mycelium of the culture solution by centrifugation, ammonium sulfate was added to the supernatant solution so as to be 100% saturated, and then centrifuged to obtain a salted out product. The obtained salted out product was suspended in a phosphate buffer solution and dialyzed several times with the same buffer solution to obtain an enzyme solution. 2 ml of the obtained enzyme solution was mixed with 2 ml of phosphate buffer solution and 0.1 ml of 0.4% cholesterol solution or 0.4% 4-cholesten-3-one solution, and the mixture was incubated at 37 ° C. for 20 hours. Glacial acetic acid was added to the reaction solution to adjust the pH to 2.0, and then 4 ml was extracted three times with ethyl acetate. The extraction solvents were pooled, concentrated under reduced pressure and dried. 100 μl of ethyl acetate was added to the obtained dried solid matter to redissolve it. The redissolved product was an ODS-2 column (4.
6 × 250mm, pore size 5.6μm, GL science
(Manufactured by K.K.) and analyzed by reverse phase HPLC. The monitor was performed at 212 nm. The results are shown in FIGS. 1E and 1F. Figure 1A is cholesterol, B is 4-cholestene-3
-On reverse phase HPLC. In FIG. 1, Ch is cholesterol and CO is 4-cholesten-3-one.

Comparative Example 1 Vector p that does not encode cholesterol oxidase
Streptomyce transformed with UC702 (J. Ferment. Biotech., (1992) 72, 268).
S. lividans) in the same manner as in Example 1,
The protein was purified. 2 ml of the obtained purified protein solution was added with 2 ml of phosphate buffer solution and 0.4 m of 0.4% cholesterol solution or 0.4% 4-cholesten-3-one solution.
1 was mixed and incubated at 37 ° C. for 20 hours. Glacial acetic acid was added to the reaction solution to adjust the pH to 2.0, and then 4 ml was extracted three times with ethyl acetate. The extraction solvents were pooled, concentrated under reduced pressure and dried. 100 μl of ethyl acetate was added to the obtained dried solid matter to redissolve it. The redissolved product was an ODS-2 column (4.6 × 250 mm, pore size 5.6 μm, GL sc
reverse phase HPLC (manufactured by ience). The monitor was performed at 212 nm. The result is shown in Figure 1.
Shown in C and D. As is clear from FIG. 1, peak IV was observed in the cholesterol oxidase of Example 1.
No peak IV was observed in the produced protein solution of Comparative Example 1. Further, as shown in FIG. 2, peak IV (A in FIG. 2)
4-cholesten-6-all-3-one (Steraloids
(Manufactured by Inc.) (B in FIG. 2) and the retention times in reverse phase HPLC were the same, and both were the same in the mass spectrum, IR, and H-NMR. The chemical structure of the substance of peak IV is shown in FIG.

Example 2 Sequence encoding the cholesterol oxidase gene of cholesterol expression plasmid pCO-1 (J. Baterio
l. (1990), 172 (7) 3644) and the E.coRI site was introduced by site-directed mutagenesis to excise only the resulting fragment.
It was cut out with oRI and BamHI and inserted into the downstream of the tac promoter of pKK223-3 to obtain the expression vector pCO117. E. coli XL1 Bl transformed with pCO117
The ue strain was cultured by a conventional method, and the cells obtained by centrifugation were disrupted by ultrasonic treatment to prepare a cell-free extract. Ammonium sulfate was added 100% to the obtained cell-free extract, the salted out product was redissolved in a phosphate buffer, and dialyzed several times with the same buffer to prepare a purified enzyme solution. The obtained enzyme solution was treated in the same manner as in Example 1 with choleslerol or 4-cholesten-3-.
Reacted with on. When the reactants were analyzed in the same manner as in Example 1, it was confirmed that 4-cholesten-6-ol-3-one was produced in each case.

Example 3 Commercially available cholesterol oxidase, Streptomyces-derived COO311 (Toyobo), Brevibacterium-derived cholesterol oxidase (Kyowa Fermentation) and Pseudomonas-derived cholesterol oxidase
(Manufactured by Wako Pure Chemical Industries, Ltd.) For each 100 units, in the same manner as in Example 1,
Reacted with cholesterol or 4-cholesten-3-one. The result is shown in FIG. It was confirmed that 4-cholesten-6-ol-3-one was produced by all the enzymes.

[0013]

Industrial Applicability According to the present invention, a cholesterol derivative in which hydrogen at the 6-position of cholesterols is converted into a hydroxyl group can be produced. The present invention, compared to conventional synthetic methods,
It enabled the production of 6-hydroxy-cholesterols under the condition that the production of by-products was small.

[Brief description of drawings]

FIG. 1 shows a reverse-phase HPLC chromatography pattern of products produced by the action of each enzyme solution.

[Explanation of symbols]

 Ch Cholesterol CO 4-Cholesten-3-one

FIG. 2: Reversed phase HPL of the product of cholesterol oxidase and a standard of 4-cholesten-6-ol-3-one.
3 shows the C chromatographic pattern.

FIG. 3 shows the chemical structure of the substance of peak IV.

FIG. 4 shows a reverse phase HPLC chromatographic pattern of the products of the action of commercial cholesterol oxidase.

Claims (3)

[Claims] 1. A method for producing a cholesterol derivative, which comprises converting hydrogen at the 6-position of cholesterols into a hydroxyl group by cholesterol oxidase. 2. Cholesterol is cholesterol, 4
The method for producing a cholesterol derivative according to claim 1, which is -cholesten-3-one or 5-cholesten-3-one. 3. The method for producing a cholesterol derivative according to claim 1, wherein the cholesterols are dihydroxycholesterol, dehydroepiandrosterone, pregnenolone, sitosterol, lanosterol or stigmasterol.