JP3035685B2 - Steroid β-sulfate sulfatase, method for producing the same, and method for determination of bile acid 3β-sulfate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a steroid nucleus containing 3β-
The present invention relates to a sulfatase that hydrolyzes a sulfate, a method for producing the same, and a method for determining 3β-sulfate of bile acid.

[0002]

2. Description of the Related Art Serum bile acid concentration increases due to hepatobiliary disease, and at the same time, bile acid (sulfate-conjugated bile acid) in which the hydroxyl group at position 3 is sulfated in urine increases. It is well known. For the determination of bile acids in serum, a simple direct measurement method using an enzyme has been developed and is an important test item for examining liver function in clinical tests. Sulfate esterification of bile acids is considered to be a kind of detoxification mechanism for increasing the water solubility of bile acids and promptly excreting harmful bile acids from the blood to the outside of the body, that is, to urine. It is known that sulfate-conjugated bile acids are present in urine at a concentration similar to or higher than that of bile acids in serum. However, sulfate-conjugated bile acids could not be easily measured because there was no sulfatase that efficiently hydrolyzes the sulfate ester, and the generalization of the test was delayed. The inventor of the present invention has previously identified 3 bile acids.
Finding a specific sulfatase for α-sulfate,
A method for direct colorimetric determination of 3α-sulfate of bile acid by a simple enzymatic method was developed (JP-A-2-145183).

[0003] Although 3α-hydroxy bile acid is a major bile acid, fluctuations in the amount of 3β-hydroxy bile acid, which is referred to as abnormal bile acid, is deeply involved in a specific disease state as research on bile acid progresses. I understand that. For example, the serum of a liver cancer patient has a significantly higher concentration of 3 than that of a healthy person.
β-hydroxy-5-cholenic acid was found to be contained (Record of the 6th meeting of the tumor marker workshop, 254
-256, 1986).

[0004] In addition, 3 in urine of patients with neonatal biliary atresia.
The results of quantification of β-hydroxy-5-cholenic acid show that the urine is highly sulfated in urine, and the measurement of 3β-hydroxy bile acid in urine is similar to that of 3α-hydroxy bile acid. The importance of quantification of the sulfate ester type (sulfate conjugate type) is suggested. However, the existence of an enzyme that efficiently hydrolyzes the 3β-sulfate ester required for establishing a simple method for quantitative determination of the sulfate-conjugated form of 3β-hydroxybile acid was not known.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a reagent for specifically detecting the total amount of 3β-sulfate of 3β-hydroxy bile acid, and a 3β-sulfate of 3β-hydroxy bile acid using the reagent. The aim is to establish a method for measuring the total amount of

[0006]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, have found that a bacterium belonging to the genus Pseudomonas sp.
An enzyme that hydrolyzes a sulfate ester to produce a 3α-hydroxysteroid compound.

That is, the present invention relates to a steroid β-sulfate sulfatase having the following properties: (a) action: acting on a sulfate bound at the β-position to the 3-position of the steroid nucleus to hydrolyze the sulfate moiety; In this case, the bond configuration of the OH group is reversed from the β configuration to the α configuration,
produces an α-hydroxysteroid nucleus; and (b) molecular weight: about 70,000 to 1 as determined by high performance liquid chromatography using a gel filtration column.
50,000 daltons.

[0008] The present invention also provides a steroid β-sulfate which is obtained by culturing a bacterium belonging to the genus Pseudomonas in a medium containing bile acids and collecting the steroid β-sulfate sulfatase of the present invention from the culture. The present invention relates to a method for producing sulfate sulfatase.

Further, the present invention provides a method for preparing
A steroid β-sulfate sulfatase and a 3α-hydroxysteroid dehydrogenase of the present invention are allowed to act on a sample containing 3β-sulfate of bile acid simultaneously or in this order, and the NADH produced at that time is quantified to determine bile acid. It relates to a method for determining 3β-sulfate.

The present inventors have conducted intensive studies and found that bile acid 3β
Succeeded in obtaining a novel steroid β-sulfate sulfatase which hydrolyzes a sulfate portion of sulfuric acid, and conjugated the enzyme with 3α-hydroxysteroid dehydrogenase in the presence of β-NAD to obtain a conventional enzyme method. Of bile acids in blood or urine that could not be measured by
It has been found that β-sulfate can be easily quantified.

[0011] The steroid β-sulfate sulfatase of the present invention acts not only on the 3β-sulfate ester of bile acid but also on other steroid compounds having a 3β-sulfate ester bond, as shown below.

The steroid β-sulfate sulfatase of the present invention (hereinafter referred to as the present enzyme) can be obtained, for example, from a bacterium belonging to the genus Pseudomonas, but as long as it has the above properties, other bacteria, yeasts, fungi, It may be produced by plants or animals. The culturing can be performed by a known method.

The present enzyme is preferably, for example, Pseudomonas testosteron
It is obtained by culturing the bacteria belonging to i).

The bacterium belonging to Pseudomonas testosteroni (hereinafter referred to as the present strain) is not particularly limited.
Known bacteria can be used. Among them, for example, Pseudomonas testosteroni ATCC11996 strain and the like are preferable. This fungus is a commercial product and is available.

As an example, the physicochemical properties of the present enzyme obtained from a bacterium belonging to Pseudomonas testosterony are described in more detail below.

The activity of the present enzyme is measured as follows. That is, 0.1 ml of a 2.5 mM methanol solution of 3β-hydroxy-5-cholenic acid 3-sulfuric acid (manufactured by Sigma) and 1 ml of β-NAD (manufactured by Oriental Yeast Co., Ltd.)
0.2 ml of 5 mM aqueous solution, 0.1 M Tris-HCl buffer (p
H8.0) and 1.55 ml of distilled water were placed in a quartz cell and equilibrated at 30 ° C., and then a 3α-hydroxysteroid dehydrogenase (Nacalai Tesque) solution (1
(0 U / ml) 0.05 ml and the present enzyme solution 0.1 ml are sequentially added and reacted at 30 ° C., and the increase in the initial absorbance at 340 nm is measured. Under these conditions, 1 μmo per minute
The enzyme activity for producing 1 NADH is defined as 1 unit.

(A) Action: Acts on a sulfate ester bonded to the 3-position of the steroid nucleus in a β-configuration to hydrolyze a sulfate ester moiety. At this time, the bond configuration of an OH group is reversed from a β-configuration to an α-configuration. Generates a 3α-hydroxysteroid nucleus.

(B) Substrate specificity: acts on 3β sulfate of a compound having a steroid nucleus. See Table 1 below for details
Shown in

[0019]

[Table 1]

(C) Optimum pH: The optimum pH is 8.5 ± 0.5.
5 and the results are shown in FIG. The sulfatase of the present invention has an optimum pH as described above, but has a pH of about 7 to 9.5.
Shows high activity in a wide range of

(D) Stable pH: The stable pH range is 30 ° C.
The pH is 6.5 to 9.5 after 16 hours of treatment. The results are shown in FIG.

(E) Optimum temperature and thermal stability: The optimum temperature is 35 ° C. ± 3 ° C. pH 7.5, under treatment conditions for 10 minutes, 3
At 5 ° C. or lower, 100% of the activity remains, but at higher temperatures, the activity rapidly decreases to 0% at 45 ° C. The results are shown in FIGS.

(F) Molecular weight: As a result of a high-performance liquid chromatography method using Shim-pack Diol-300 (manufactured by Shimadzu Corporation) of a gel filtration column, a molecular weight of about 70,000- It is about 150,000 daltons.

(G) Km value: Km value for 3β-hydroxy-5-cholenic acid 3-sulfuric acid: 1 × 10 ⁻⁵ M or less.

(H) Inhibition and activation: Hg ²⁺ , Cu ²⁺ , Z
^Inhibited by n ²⁺ , metal chelators (EDT
A, O-phenanthroline, etc.). Further, it is hardly inhibited by SH reagents such as p-chloromercury benzoate and monoiodoacetic acid. Details are shown in Table 2 below.

[0026]

[Table 2]

The enzyme can be obtained, for example, by culturing a bacterium belonging to the genus Pseudomonas, as described above.

The medium used for culturing the organism producing the sulfatase of the present invention is not particularly limited, and an appropriate medium can be used according to various origin organisms.

Hereinafter, culture conditions for the present strain will be described as examples.

As the medium, any medium for bacteria of the genus Pseudomonas can be used. For example, yeast extract, peptone, meat extract and the like as organic nutrients, and ammonium phosphate, ammonium nitrate, potassium phosphate and phosphorus as inorganic nutrients. Those containing sodium acid, magnesium chloride, manganese chloride and the like are used. To further produce the enzyme, bile acids are added to the medium. Examples of bile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, sulfates thereof, salts thereof, and the like, and one or more kinds can be used. The amount of bile acid added is not particularly limited, but may be generally about 0.05 to 1.0% by weight.

The culture time and temperature are not particularly limited.
Usually, it is preferable to culture at about 24-34 ° C. for about 12-24 hours under aerobic conditions. Cultivation for such a period maximizes the enzyme activity.

The present enzyme is extracted from the cells obtained by culturing. The extraction can be performed according to a usual method for extracting enzymes in cells. For example, the cells can be disrupted by a method such as ultrasonic treatment, various mechanical treatments, and enzyme treatments, and the enzyme can be recovered in the supernatant obtained by centrifuging insolubles. By purifying the crude enzyme by appropriately selecting and combining common enzyme purification methods such as nucleic acid removal treatment, ammonium sulfate salting out, ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., the present enzyme can be obtained. it can.

The thus obtained enzyme and 3α-hydroxysteroid dehydrogenase are combined with β-NA
By acting on a sample containing bile acid 3β-sulfate in the presence of D, bile acid 3β-sulfate can be quantified. That is, the enzyme is bile acid 3β-
The sulfate ester is converted to 3α-hydroxy bile acid, which is then converted to 3α-hydroxy steroid dehydrogenase.
While converting α-hydroxy bile acid to 3-oxo bile acid, NAD, which is a coenzyme of the dehydrogenase, is converted to N
Reduce to ADH. Therefore, by determining the amount of conversion from NAD to NADH, bile acid 3β-sulfate in a sample derived from a living body such as urine or blood can be determined.

The 3α-hydroxysteroid dehydrogenase is not particularly limited, and any known one can be used.

The amount of the enzyme to be used is not particularly limited. Usually, the present enzyme is used at about 0.1 to 3.0 units / ml, and 3α-hydroxysteroid dehydrogenase is used at 0.1 to 3.
What is necessary is just to use about 0 unit / ml.

The amount of β-NAD to be used is not particularly limited and may be appropriately selected, but may be usually added so that the β-NAD concentration in the reaction system is about 0.5 to 5 mM. The enzymatic reaction is usually performed at a temperature of about 25 to 40 ° C for 5 minutes.
This is performed for about 30 minutes.

NADH can be quantified according to a known method. As a specific example, UV (3
40 nm) absorption measurement method, fluorescence intensity measurement method, colorimetric quantification method (reduction colorimetric colorimetry method), which is conjugated with a redox dye reaction via diaphorase or phenazine methosulfate, etc.
A method in which NADH oxidase is allowed to act to quantitatively generate hydrogen peroxide from NADH, and the hydrogen peroxide is conjugated with an oxidative condensation coloring system to perform colorimetric determination (oxidative coloring), and the like.

[0038]

According to the present invention, a novel steroid β-
Sulfate sulfatase can be provided. In addition, by conjugating the present enzyme with 3α-hydroxysteroid dehydrogenase in the presence of β-NAD, bile acid 3β-sulfate in urine or blood, which was not possible by the conventional enzymatic method, can be easily quantified. It can be applied to diagnosis of various diseases.

Further, the enzyme of the present invention has an activity of widely hydrolyzing 3β-sulfate ester of a compound having a steroid skeleton having 24 or less carbon atoms. Useful for the determination of sulfate.

[0040]

The following examples are given, but the present invention is not limited to these examples.

In the following examples, “%” means “% by weight”.

Example 1 Monoammonium phosphate 0.1%, diammonium phosphate 0.1%, monopotassium phosphate 0.2%, magnesium chloride 0.01%, manganese chloride 0.01% and yeast extract 1 300 ml of a medium (pH 6.9) containing 2.0%
The flask was placed in an Erlenmeyer flask and sterilized in an autoclave. This includes Pseudomonas testosterony ATCC11
996 was inoculated and cultured with shaking at 28 ° C. for 24 hours. This seed culture solution was inoculated into 30 L of a sterilized medium having the same composition as above in a 50 L jar fermenter, and at the same time, 1.2 L of a 10% aqueous sodium cholate solution which had been separately sterilized was added aseptically. And cultured for 15 hours with aeration and stirring.

The culture solution was centrifuged, and the obtained cells
mM phosphate buffer (pH 7.2), and this suspension was applied to a mini-lab cell disrupter (manufactured by Lanie) to obtain a suspension.
The cells were disrupted. This was centrifuged to remove the precipitate to obtain a crude enzyme solution. The crude enzyme solution was subjected to a nucleic acid removal treatment with protamine sulfate, and then subjected to salting out treatment with ammonium sulfate.
It was dialyzed against M Tris-HCl buffer (pH 8.0). This dialysate is added to a 10 mM Tris-HCl buffer (pH 8.0)
The solution was passed through a column of DEAE Sepharose CL-6B (manufactured by Pharmacia) equilibrated in the above. The obtained non-adsorbed fraction was subjected to 70% saturated salting out with ammonium sulfate, and the precipitated fraction was collected and dissolved in a 50 mM phosphate buffer (pH 6.8). The solution was passed through an octyl sepharose CL-4B (Pharmacia) column equilibrated with 50 mM phosphate buffer containing 30% saturation. The obtained non-adsorbed fraction was subjected to 70% saturated salting out with ammonium sulfate, and the precipitated fraction
10 mM phosphate buffer containing 0 mM sodium chloride (p
H6.4), dialyzed against the same buffer, passed through a CM Sepharose Fast Flow (Pharmacia) column equilibrated with the same buffer to adsorb the active fraction.
This column was developed by a gradient elution method in which the concentration of sodium chloride in a 10 mM phosphate buffer (pH 6.4) was continuously increased from 50 mM to 300 mM, and the active fraction was eluted. This was concentrated by a salting-out treatment with ammonium sulfate, and 50 mM containing 0.15 M sodium chloride.
Gel filtration was performed through an Ultrogel ACA34 (manufactured by LKB) column equilibrated with a phosphate buffer (pH 6.8). The active fraction was desalted and concentrated by ultrafiltration to obtain 250 units of steroid β-sulfate sulfatase. The purified sample showed a single band in slab gel electrophoresis using a polyacrylamide electrophoresis PAG plate 4/15 (Daiichi Pure Chemicals).

The molecular weight of the purified sample was determined by measuring the molecular weight of Shim-Pakdiol-300 (manufactured by Shimadzu Corporation) in a gel filtration column.
As a result of measurement by a high performance liquid chromatography method using, it was about 95,000 daltons.

Example 2 Using the steroid β-sulfate sulfatase obtained in Example 1, a sulfate ester of 3β-hydroxy-5-cholenic acid, a typical abnormal bile acid having a double bond in the steroid nucleus ( 3β-hydroxy-5-cholenic acid 3-sulfuric acid) was quantified. β-NAD (Oriental Yeast Industry)
3 μmol, 0.5 unit of 3α-hydroxysteroid dehydrogenase (manufactured by Nacalai Tesque), steroid β
-35 mM pyrophosphate buffer containing 0.4 units of sulfate sulfatase and 0.5% hydrazine monohydrate (pH 8.
9) To 2.9 ml of a substrate, 0.1 ml of a methanol solution of 3β-hydroxy-5-cholenic acid 3-sulfuric acid (manufactured by Sigma) having a different concentration was added as a substrate, reacted at 30 ° C. for 10 minutes, and then subjected to absorbance at 340 nm. Was measured. Fig. 5 shows the results.
Shown in From FIG. 5, it is found that the substrate concentration and the absorbance have a positive correlation, and therefore, the quantification of 3β-hydroxy-5-cholenic acid 3-sulfuric acid is possible.

Example 3 Reagent 1: 50 units of steroid β-sulfate sulfatase,
20 mg of bovine serum albumin, 400 mg of saccharose, and 600 mg of Tween 20 were dissolved in 20 ml of 100 mM Tris-HCl buffer (pH 7.5).

Reagent 2: 130 mg of β-NAD (manufactured by Oriental Yeast), 30 mg of nitroblue tetrazolium (manufactured by Nacalai Tesque), 100 units of 3α-hydroxysteroid dehydrogenase, 400 units of diaphorase (manufactured by Oriental Yeast), 250 m of bovine serum albumin
g and 1 g of saccharose were dissolved in 50 ml of 200 mM phosphate buffer (pH 7.0).

Reagent 3: 2 M aqueous citric acid solution Measurement: Various concentrations of 3β-hydroxy-5-cholenic acid 3-
0.4 ml of reagent 1 was added to 0.2 ml of an aqueous solution of sulfuric acid, and the mixture was kept at 30 ° C. for 10 minutes. After desulfation reaction was performed, 0.5 ml of reagent 2 was added, mixed, and kept at 30 ° C. for 10 minutes. After the color was developed, 0.1 ml of Reagent 3 was added to stop the reaction, and the absorbance at 540 nm was measured. As a result, good results were obtained as shown in FIG.

Example 4 3β-hydroxy-5-cholenic acid 3-sulfuric acid (3β-CA
Diluting the urine to which S) has been added to various concentrations with no urine added;
Test urine was used.

Reagent 1 of Example 3 in 0.2 ml of test urine
0.4 ml was added and kept at 30 ° C. for 10 minutes. to this,
0.5 ml of the reagent 3 of Example 3 was added, mixed, and kept at 30 ° C. for exactly 10 minutes to develop a color.
After stopping the reaction by adding 0.1 ml, the absorbance at 540 nm was measured. Table 3 shows the results.

[0051]

[Table 3]

From the results, it was found that the substrate concentration and the absorbance had a positive correlation.

Example 5 3β-hydroxy-5-cholenic acid 3-sulfuric acid (3β-CA
The serum to which S) was added was diluted to various concentrations with serum without addition, and used as a test serum.

Reagent 1 of Example 3 in 0.2 ml of test serum
0.4 ml, and then 3β-CA
S was quantified. Table 4 shows the results.

[0055]

[Table 4]

From the results, it was found that the substrate concentration and the absorbance had a positive correlation.

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the present enzyme.

FIG. 2 is a graph showing a stable pH of the present enzyme.

FIG. 3 is a graph showing the optimum temperature of the present enzyme.

FIG. 4 is a graph showing the thermostability of the present enzyme.

FIG. 5 shows a substrate (bile acid 3β) when the method of the present invention is carried out.
-Graph showing the relationship between (sulfuric acid) concentration and absorbance.

FIG. 6 shows a substrate (bile acid 3β) when the method of the present invention is carried out.
-Graph showing the relationship between (sulfuric acid) concentration and absorbance.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 C12Q 1/00-1/70 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A steroid β-sulfate sulfatase having the following properties: (a) action: acting on a sulfate bound at the β-position to the 3-position of the steroid nucleus to hydrolyze the sulfate moiety, whereby OH The bond configuration of the group is reversed from the β configuration to the α configuration, and 3
produces an α-hydroxysteroid nucleus; and (b) molecular weight: about 70,000 to 1 as determined by high performance liquid chromatography using a gel filtration column.
50,000 daltons. 2. A steroid β-sulfate, wherein a bacterium belonging to the genus Pseudomonas is cultured in a medium containing bile acids, and the steroid β-sulfate sulfatase according to claim 1 is collected from the culture. A method for producing sulfatase. 3. A sample containing bile acid 3β-sulfate in the presence of β-NAD, the steroid β-sulfate sulfatase and 3α-hydroxysteroid dehydrogenase according to claim 1 act simultaneously or in this order. And a method for quantifying 3β-sulfate of bile acid, which comprises quantifying NADH produced at that time.