JPS59183690A - Preparation of allylsulfotransferase - Google Patents

Abstract

PURPOSE:To obtain the titled substance important as an enzyme participating in the detoxication of a phenolic compound, by culturing a microbial strain belonging to Eubacterium genus. CONSTITUTION:A microbial strain capable of producing arylsulfotransferase and belonging to Eubacterium genus, e.g. Eubacterium rectal A-44 (FERM-P No.7007) is cultured preferably in a medium for anaerobe, under anaerobic condition at 25-45 deg.C for 10-40hr, and the produced objective substance is collected from the medium. The productivity of the enzyme can be promoted remarkably by adding an aromatic sulfuric acid ester such as phenolphthalein disulfate, etc. to the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing aryl sulfotransferase using a strain belonging to the genus Eubacterium. Arylsulfotransferase (Arylsulfotran)
sferase (EC2, 8, 2, 1) is an enzyme that catalyzes a reaction that transfers a sulfate group from a sulfate donor to a phenol compound to form a sulfate conjugate of a phenol compound.
It is important as an enzyme involved in the detoxification pathway of phenolic compounds.

Many phenolic compounds are used as laxatives (they have strong effects, but they also irritate the intestines, so it has been found that the phenol group is esterified with sulfuric acid, and picosulfate is used as a liquid laxative). (Pico
sulfate) was born.

It has been thought that picosulfate does not develop its medicinal effects until it is hydrolyzed by enteric bacteria in the large intestine.

The present inventors searched human feces for intestinal bacteria that produce enzymes that decompose aromatic sulfate esters, and succeeded in isolating a strain recognized as Eubacterium phthalate. When this strain was cultivated and the enzyme obtained was examined, it was discovered that the enzyme was not a hydrolase but an allyl sulfotransferase, a type of transferase, based on its properties.

Traditionally, allyl sulfotransferase is used in the liver, small intestine, kidney,
It is known to exist in animal tissues such as the brain and in filamentous fungi. For example, there are four types of aryl sulfotransferases in rat liver [Journal of Biological Chemistry (J. Biol.
Chem, ) Volume 254.

5658 pages, 1979, 'Archives of Biochemistry and High Physics (A
211, page 352, 1981 and the European Journal of Biochemistry (Eur.
, J. Bioche+n, ) Vol. 104, p. 3, 1980], all of which contain 3'-phosphoadenylyl 5''-sulfuric acid (rPAPSJ) as the sulfuric acid donor.
), and the reaction will not proceed without PAPS. Also Aspergillus
The aryl sulfotransferase produced by bacteria of the genus PA
It is known that sulfate groups can be transferred without PS [
The Biochemical Journal (Biochem)
J.), Volume 149, Page 697, 1975].

However, there has been no report that bacteria produce allyl sulfotransferase.

The arylsulfotransferase according to the method of the present invention is PA
It is an enzyme that does not use PS as a sulfate donor, and was newly discovered by the present inventors in a type of enterobacterium, the genus Euhacterium. The screening method and properties of the strain used in the method of the present invention and the production method and properties of the allyl sulfotransferase obtained therefrom will be explained below.

Examples of the Eubacterium genus 5 used in the method of the present invention include Eupacterium phthalate, and more specifically, Eubacterium phthalate A-44, which the present inventors discovered from intestinal bacteria. This strain was obtained using the method of Mitsuma et al. (Clinical Examination, Vol. 23, p. 322, 197).
9). That is, human feces is diluted and EG
Agar plate medium [horse meat exudate (product name Lab-1emco)
powder+ OxoI M) 2.4g.

Proteose peptone Fh3 (Difco)
Log, yeast extract (same) 5g, disodium phosphate 4
g, soluble starch 0.5g, glucose 1.5g, L-
Cystine 0.2g.

15 g of agar (manufactured by Difco) and an appropriate amount of antifoaming agent were dissolved in 950 g of ion-exchanged water, adjusted to pH 7.6, sterilized in an autoclave and brought to 50-60°C, and further added with 0.5 g of L-cysteine hydrochloride and 50 g of horse blood. In addition, the mixture was smeared onto a petri dish and plated, and then cultured for 3 to 5 days in an anaerobic jar (manufactured by Hirayama Seisakusho). Fish out the grown colonies,
Add 0.065% phenolphthalein disulfate (
rPPDsJ) added and agar removed.
After inoculating one liquid medium, it was placed in an anaerobic box (Forma
The cells were cultured at 37°C for 2 days in a 37°C chamber. After that 0.2M
Add 0.5 ml of sodium carbonate to make it alkaline.
A positive strain A-44 with a red color was obtained. A-44 stock is CA
The cells were subcultured into M semi-fluid medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 3- and stored in a low-temperature room.

A-44 strain has the following properties.

(11 Morphological properties ■Shape: Straight to slightly curved rods ■Size: Approximately 0.8 to 3.0μ ■Spores: Not formed ■Durham staining: Positive (2) Growth status on medium ■BL agar plate medium: Growth is good, colonies are raised in a perfect circle, convex circle to hemispherical shape, and the periphery and surface are smooth and grayish-brown ■EC agar plate medium: growth is somewhat poor, colonies are in a perfect circle, convex circle to hemispherical shape Raised, smooth and grayish on the periphery and surface ■ Lactobacillus selective medium: No growth ■ DHL medium: No growth (3) Physiological properties ■ Nitrate reduction: Negative ■ Indole production: Negative ■ Gelatin liquefaction: Negative ■6 .Growth in 5% salt: Negative ■Production of acids and gases from sugars: Produces acids and gases from arabinose, fructose, galactose, raffinose, starch, sucrose, xylose, mannose, glucose, maltose, lactose.Glycerol, Mannitol and Inosit do not produce acids or gases.

■ Esterin degradability: Positive ■ Starch degradability: Positive ■ Lecithinase: Negative ■ Catalase: Negative [Phase] Growth temperature: 25-45°C ■ Growth pH: Best grows around pH 7 @Main fermentation products: lactic acid, butyric acid ■Attitude toward oxygen: Characteristics beyond obligate anaerobic properties include ``Isolation and Identification of the World's Most Anaerobic Enterobacteriaceae'' (written by Tomozoku Mitsuma, published by Soubunsha, 1980) and ``
Bergey'sManua's Manual of Determinative Hacteriology
l of Determinative Bacter
As a result of examination according to J (8th edition, 1974), strain A-44 is an anaerobic Gram-positive non-spore bacillus that produces butyric acid and is therefore classified as a member of the genus Eubacterium.

Additionally, it produces acids from sucrose, raffinose, arabinose and xylose, does not produce acids from glycerol, produces acetic acid but not caproic acid, hydrolyzes starch and grows at 37°C. Eubactrium rectal (Eubactrium rectal)
e) was identified. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Eubacterium lefcul A-44 (FERM P-7007). Is this bacteria 10 per gram of feces? t([lJ
It was the dominant bacterial species in the gastrointestinal tract.
The ability to produce aryl sulfotransferase was investigated for preserved strains of Euhacterium phthalate, but none was observed.

To collect allyl sulfotransferase using the method of the present invention, a strain belonging to the genus Eubacterium that produces allyl sulfotransferase is inoculated into a normal anaerobic culture medium, and cultured under anaerobic conditions at approximately 25 to 45°C. This causes enzymes to accumulate. The culture time may be set at a time when enzyme production is at its maximum, and is usually about 10 to 40 hours. Preferably, when an aromatic sulfate is added to the medium, the production of this enzyme is significantly increased. Examples of aromatic sulfate esters include the above-mentioned PPDS and picosulfate, as well as P-nitrophenyl sulfate (hereinafter referred to as rPNsJ), P-acetylphenyl sulfate, and 4-methylumbelliferyl sulfate. .065% (-/ν)
By adding the specific activity (activity per protein) is approximately 20
Increase twice.

The obtained culture is purified using an appropriate combination of known purification methods. That is, the culture is collected as is or only the bacterial cells are crushed using methods such as ultrasound, French press, and alumina grinding to extract enzymes, followed by salting out, organic solvent precipitation, dialysis, and ultraviolet treatment. It is purified by appropriately combining methods such as filtration, gel filtration, centrifugation, ion exchange chromatography, affinity chromatography, concentration, and freeze drying.

By the method of the present invention, Eupacterium phthalum A-44 (
The properties of the allyl sulfotransferase obtained from FERM P-7007) are as follows.

(1) Function: Using an aromatic sulfuric ester as a substrate sulfuric acid donor, catalyzes the reaction of transferring the sulfuric acid group to a phenol compound.

(2) Substrate specificity The amount of tyramine sulfate produced by reacting various aromatic sulfate esters and sulfate conjugates of bile acids as a sulfuric acid donor and tyramine as an acceptor was determined by thin layer chromatography. That is, the reaction solution was spotted on a thin layer plate (silica gel Kieselgel 60 F254, manufactured by Merck & Co., Ltd.), and was mixed with n-butanol:acetic acid:water (25:4:1).
0) and air-dried, a benzene solution of P-dimethylaminobenzaldehyde, which is a coloring reagent for amines, was sprayed and heated at 100° C. for 2 to 3 minutes. Chromatography scanner CS-9 spot with Rf value 0.23
10 (manufactured by Shimabara Seisakusho), the sample wavelength was 220 nm.
, tyramine sulfate was quantified by linear scanning at a blank wavelength of 400 nm. The results are shown in Table 1. This enzyme uses aromatic and sulfate esters as substrate sulfate donors, but showed no activity against aliphatic sulfate esters.

Table 1 and Table 2 show the substrate specificities of various sulfate acceptors when PNS is used as a sulfate donor.

Table 2 No. 2: 4,4'-dihydroxy(2-pyridyl)-diphenylmethane hetanosulfate (3) Optimal p)l Using PNS as the sulfuric acid donor, the optimal p1+ was determined by changing various acceptors. As a result, this enzyme receives tyramine and
When using phenolphthalein or L-tyrosine, pl+about 9.0, α and β-naphthol, 3-(4
-hydroxyphenyl)-2',4'-dihydroxypropiophenone or 2-methyl-3-phenyl-7-
When hydroxychromone was used, there was a pH of about 8.2. (Figures 1 to 3) The buffers used were 0.1M acetate buffer for pH 5 to 7;
pH 7-9 is 0.1M) Liss-HCl buffer, pH 9-1
0.5 is 0.1M glycine-sodium hydroxide buffer.

(4) Stable pl + Range pH After treating at each pH of 5 to 10 for 230 minutes at 37°C, the pH was returned to 9.0 and the activity was measured.
i (around 6.5) was most stable. The buffer used was the same as in (3) above. (Figure 4) (5) Optimum temperature Reaction for 30 minutes at each temperature of 20 to 60°C. When the activity was determined, the optimal temperature was around 40°C.

(6) Temperature stability When the enzyme activity was measured after treatment at pH 6.5 for 10 minutes at each temperature of 5 to 60°C, it was found to be stable up to around 37°C. (Figure 5) (7) Activity measurement method Using PNS as the substrate sulfate donor and tyramine as the acceptor, 50mM PHS 0.03m, 10mM
Tris-HCl buffer containing tyramine (pH 9,0) 0.5
Mix 8- and enzyme solution 0.0'2 me and incubate at 37°C.
Allow to react for 115 minutes. IN sodium hydroxide 0.4m
If the reaction becomes cloudy, centrifuge it and measure the absorbance at 405 nm of the clear supernatant, which is then applied to a standard curve prepared separately using a standard solution of P-nitrophenol for quantification. do. The amount of enzyme that produced 1 μmole of P-nitrophenol per minute was defined as 1 unit.

(8) Substrate affinity When reacting at 37° C. for 5 minutes using PNS as the sulfuric acid donor and tyramine as the acceptor, the Michaelis constant (Km value) for tyramine was 0.39 mM.

(9) Inhibitors Each inhibitor shown in Table 3 and this enzyme were mixed at pH 6.5.
After treatment at 7°C for 10 minutes, activity was measured. Table 3 shows the inhibitor concentration and inhibition rate (%).

In addition to those listed in the table, inhibition by ethylenediaminetetraacetic acid (5mM) was 30%, and iodoacetic acid, N-ethylmaleimide, and diisopropylfluorophosphate (10-3M each)
No inhibition was observed.

Table 3 (9) Ampholine with isoelectric point pt + range 2.5 to 4
ne.

The isoelectric point of this enzyme was determined to be 3.8 as measured by sucrose density gradient isoelectric focusing using LKB (manufactured by LKB).

00) Molecular weight: Approximately 240.0 as measured by gel filtration using Sephadex G-200 (manufactured by Pharmacia)
It was 00.

Next, the method of the present invention will be specifically explained using test examples and examples.

Test example PNS decomposition activity of human feces Using 12 cases of human feces, suspend 1 g of each in double-distilled water, bind the total amount with 10-ml, and add 0.1'M containing this and 50mM PH3.
Tris-HCl buffer (pH 9,0) 0.5 rne was mixed and reacted at 37°C for 12 hours. After adding IN sodium hydroxide to stop the reaction, centrifuge and collect the supernatant with 405n
P-nitrophenol is quantified from the absorbance at m,
Activity (μmole/h) was determined. As shown in Table 4, PNS decomposition activity was observed in all samples.

Table 4 Example 1 Tributicase (manufactured by BBL) Log, proteose peptone Na3 (manufactured by Difco) Log, yeast extract (same) 5g1 glucose 3g1 potassium phosphate 2
g, sodium chloride 3g, soluble starch 5g, PP D
After dissolving 65 g of SO, 0.3 g of L-cysteine hydrochloride, and 0.3 g of sodium thioglycolate in 1,000 liters of ion-exchanged water and sterilizing it, Eubacterium phthalate A-44 ( FE
20 m of seed culture solution obtained by culturing RM P-7007)
1 and inoculated 37 in an anaerobic box (manufactured by Forma).
The cells were cultured at ℃ for 24 hours. The culture solution was centrifuged to collect the bacterial cells, which were crushed using a French press, and the supernatant was collected by centrifugation to obtain a crude enzyme extract. Streptomycin was added to this to remove nucleic acids, and a 40-60% ammonium sulfate saturated fraction was collected, dissolved in 0.1M #acid buffer (pH 5.5), and dialyzed against the same tE solution overnight. Next, the enzyme was adsorbed through a DEAE Sephadex (Pharmacia) column (2.9 x 45 cm) equilibrated with the same buffer, and the active fraction was eluted with a gradient of 0 to 0.5 M potassium chloride. Collected. The same chromatography was performed again to obtain a purified enzyme preparation. FIG. 6 shows the elution vector of DEAE Sephadex column chromatography, and Table 5 shows the results of the purification process. The amount of protein is Fol 1n-Loivry.
Bovine serum albumin (fraction ■) was measured using the method as a standard.

Table 5

[Brief explanation of the drawing]

Figures 1 to 3 are diagrams showing the optimum pH of aryl sulfotransferase obtained from Euhacterium phthalate A-44 (FERM P-7007>) by the method of the present invention, and Figure 4 is a diagram showing the optimum pH of aryl sulfotransferase obtained from Euhacterium phthalate A-44 (FERM P-7007). Range, FIG. 5 is a diagram representing temperature stability. Figure 6 shows the enzyme DEAE-
It is a figure showing the elution pattern in Sephadenx column chromatography. Patent applicant Amano Pharmaceutical Co., Ltd. Figure 1 (i, 7 8 9 1
0. 11th 2nd field 5 6 7 8 1
0 11 Figure 3 () 7 'd 9 10-'4
゛I'17 8 9 IQ 1
1

Claims (1)

[Scope of Claims] 1. A method for producing allyl sulfotransferase, which comprises culturing an allyl sulfotransferase-producing bacterium belonging to the genus Euhacterium in a nutrient medium to produce and accumulate allyl sulfotransferase, which is then collected. 2. The method for producing allyl sulfotransferase according to claim 1, wherein the allyl sulfotransferase-producing bacterium belonging to the genus Eubacterium is Eupacterium phthalate. 3 Yuuhacterium lephthal is Yuuhacterium
A method for producing aryl sulfotransferase according to claim 2, which is phthalate A-44.