JP2000166548A - Bile salt hydrolase protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bile salt hydrolase(BSH) protein, showing BSH activity, capable of lowering human serum cholesterol concentration, and useful for medicines, chemical reagents or the like, by producing it with the aid of a lactic acid bacterium belonging to genus Enterococcus. SOLUTION: This bile salt hydrolase(BSH) protein is produced with the aid of a lactic acid bacterium belonging to genus Enterococcus, e.g. Enterococcus hirae SBT-3482 stock (FERM P-17031) or Enterococcus durans SBT-3481 stock (FERM P-17030). It is preferable that the N-terminal amino acid sequence as the sub-unit of the BSH protein is shown by formula I or II (X is an unidentified amino acid), and that the BSH protein is produced by repeating adsorption and elution of the extract of the body cells belonging to genus Enterococcus using anion-exchanged and cation-exchanged chromatography in this sequence, in order to purify and recover the BSH.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel conjugated bile acid deconjugating enzyme (Bile s) produced by lactic acid bacteria of the genus Enterococcus.
alt hydrolase; hereinafter also referred to as BSH). More specifically, the present invention relates to Enterococcus hirae , and
The present invention relates to a BSH protein produced by lactic acid bacteria such as Enterococcus durans and having a catalytic action to release amino acids from conjugated bile acids. The present invention also relates to a method for producing a BSH protein by culturing these lactic acid bacteria and purifying and collecting such a BSH protein from a cell extract.

[0002]

2. Description of the Related Art Conventionally, it has been suggested that BSH produced by human intestinal bacteria has an effect on human serum lipid metabolism, particularly on cholesterol metabolism. That is, the so-called BSH hypothesis that the conversion of conjugated bile acids present in the human intestinal tract to unconjugated bile acids by the catalysis of BSH lowers the concentration of human serum cholesterol (for example, the report of Smet et al. (Microbial Ecology in Health and D
isease, vol.7, pp.315-329, 1994)).

[0003] The mechanism of action of the BSH hypothesis is formed by the following two mechanisms. First, bile acids secreted from the duodenum are conjugated bile acids, and conjugated bile acids are more capable of forming complex micelles, which are greatly involved in lipid absorption, than unconjugated bile acids. The more bile acids are converted to unconjugated bile acids, the less intestinal uptake of orally ingested lipids, including cholesterol, can be a factor in lowering serum cholesterol levels.
(For example, Sanders report (Advance in Food and Nutri.
Res., Vol.37, pp.67-130, 1993)).

Second, conjugated bile acids secreted into the intestinal tract are reabsorbed mainly from the lower small intestine and transported to the liver by active transport through receptors specific for conjugated bile acids.
There is no active transport system for unconjugated bile acids through the specific receptor in the intestinal tract, and reabsorption of unconjugated bile acids from the intestinal tract depends exclusively on passive transport.
Thus, unconjugated bile acids have a lower rate of intestinal reabsorption than conjugated bile acids. In addition, since bile acids that are not reabsorbed from the intestinal tract are excreted in feces, the total amount of bile acids excreted in feces increases. When the amount of bile acid excreted outside the body increases, the amount of bile acid biosynthesis in the body, that is, the catabolism from cholesterol to bile acid in the liver increases, and at the same time, LDL from the blood via the LDL receptor in the liver increases. Uptake, which is a factor in lowering serum cholesterol (for example, Huis et al. Report (TIBTECH, vol. 12, pp. 6-8, 1994)).

[0005] Among the above reports on the verification of the BSH hypothesis, one report shows that the BSH hypothesis works to lower serum cholesterol levels (for example, the report of Smet et al. (British
J. Nutrition, vol. 79, pp. 185-194, 1998)), but one report also states that the BSH hypothesis does not work, and therefore that BSH does not affect serum cholesterol levels ( For example, a report from Pearce (Int. Dairy
Sci., Vol.6, pp.661-672)).

[0006] By the way, all the previous reports on the effects of BSH on serum cholesterol concentration are directed to experimental animals such as mice, rats or pigs. Although it is known that the mode of cholesterol metabolism varies greatly depending on the animal species, even in the same mammal, an experiment using humans is indispensable to confirm the effect of BSH. However,
No human experiments have been attempted.

Therefore, at present, the BSH hypothesis,
That is, although the conversion of conjugated bile acids into unconjugated bile acids by BSH in the human intestinal tract may lead to a decrease in serum cholesterol concentration, a certain conclusion has not been obtained, but the possibility is fully expected. It is in the situation that it is.

[0008]

SUMMARY OF THE INVENTION The present invention relates to an Enterococc
An object of the present invention is to provide a novel conjugated bile acid deconjugating enzyme (BSH) protein obtained from a lactic acid bacterium of the genus us and a method for producing the same. As will be described later, the BSH protein of the present invention has enzymatic properties that are convenient for verifying the BSH hypothesis, and is useful not only for this purpose but also as a chemical reagent due to its enzymatic characteristics.

[0009]

SUMMARY OF THE INVENTION The present invention relates to Enterococcus
The present invention relates to a BSH protein produced by a genus Lactobacillus. In the BSH protein of the present invention, there are those in which the N-terminal sequence of the subunit consists of the following A or B. A: (X-Thr-Ser-Ile-Thr-Tyr-Val-Thr-Ser-Asp-His-Tyr
-Phe-Gly-Arg-Asn-Phe-Asp-Tyr-Glu-Ile-Ser-Tyr-Asn-G
lu-Val-Val-Thr-Val-Thr- (X is an unidentified amino acid)). B: (X-Thr-Ser-Ile-Thr-Tyr-X-Thr-Lys-Asp-His-Tyr-
Phe-Gly-Arg-Asn-Leu-Asp-Leu-Glu- (X is an unidentified amino acid)). The above A is produced by Enterococcus hirae and the latter B
Is produced by Enterococcus durans . The present invention also provides an extract and a cation exchange chromatography for an extract of a bacterium of the genus Enterococcus , which can produce a conjugated bile acid deconjugating enzyme (BSH). The present invention relates to a method for producing the BSH protein, which comprises repeatedly collecting and purifying BSH.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As described above, the N-terminal amino acid sequence of the BSH protein of the present invention is X-Thr-Ser-Ile-Thr-Ty
rX-Thr-Lys-Asp-His-Tyr-Phe-Gly-Arg-Asn-Leu-Asp-Le
BSH defined by u-Glu-, for example, derived from Enterococcus durans has properties that are convenient for testing the above-mentioned BSH hypothesis. That is, it has a higher specific activity than BSH derived from other bacteria, and has the ability to deconjugate all six types of conjugated bile acids present in the human intestine. Furthermore, the substrate specificity of BSH derived from Enterococcus durans is expected to be suitable for use in efficiently deconjugating conjugated bile acids in human bile. The reason is that the constituent ratio of conjugated bile acids in human bile is glycine-conjugated bile acid:
The ratio of taurine-conjugated bile acids is 3: 1 whereas En
This is because it has been clarified in the present invention that BSH derived from terococcus durans deconjugates glycine-conjugated bile acid at a rate about three times higher than that of taurine-conjugated bile acid.

Further, since it is possible to easily carry out the cloning of the BSH gene based on the information on the N-terminal amino acid sequence of the BSH protein disclosed in the present invention, the entire structure of the BSH gene was determined. Later, BSH
By creating a mutant that lacks only the activity,
The present invention also provides a method for easily producing a negative control strain essential for testing the BSH hypothesis.

Similarly, the N-terminal amino acid sequence of the BSH protein of the present invention has X-Thr-Ser-Ile-Thr-Tyr-Val-Thr-Ser-
Asp-His-Tyr-Phe-Gly-Arg-Asn-Phe-Asp-Tyr-Glu-Ile-Se
Defined by r-Tyr-Asn-Glu-Val-Val-Thr-Val-Thr-, for example, BSH derived from Enterococcus hirae is also used for experiments to verify the BSH hypothesis from the same viewpoint. Can be used. In this case, B from Enterococcus hirae
Since SH has a higher substrate specificity for glycine-conjugated bile acids than BSH derived from Enterococcus durans , this enzyme should be used in experiments for the purpose of deconjugating only glycine-conjugated bile acids present in the human intestinal tract. Can be.

Therefore, the N-terminal amino acid sequence of the subunit of the BSH protein of the present invention is X-Thr-Ser-Ile-Th
r-Tyr-Val-Thr-Ser-Asp-His-Tyr-Phe-Gly-Arg-Asn-Phe-
Asp-Tyr-Glu-Ile-Ser-Tyr-Asn-Glu-Val-Val-Thr-Val-Th
r- (SEQ ID NO: 1), for example, Enteroco
The N-terminal amino acid sequence of BSH derived from ccus hirae and the subunit of the BSH protein are X-Thr-Ser-Ile-
Thr-Tyr-X-Thr-Lys-Asp-His-Tyr-Phe-Gly-Arg-Asn-Leu-
Asp-Leu-Glu- (SEQ ID NO: 2) defined in, for example,
BSH derived from Enterococcus durans can be used for experiments and the like to verify the BSH hypothesis,
By using the BSH protein of the present invention, BS
If the H hypothesis is verified, the enzyme of the present invention, or Enterococcus hirae having the enzyme activity, and Enterococcus
The possibility of using lactic acid bacteria such as durans as additives for foods, medicines and the like having the ability to regulate human serum cholesterol concentration is born.

According to the present invention, the N-terminal amino acid sequence of the subunit is X-Thr-Ser-Ile-Thr-Tyr-Val-Thr-Ser-Asp-His-Ty.
r-Phe-Gly-Arg-Asn-Phe-Asp-Tyr-Glu-Ile-Ser-Tyr-Asn-
Glu-Val-Val-Thr-Val-Thr- or X-Thr-Ser-Ile-Th
r-Tyr-X-Thr-Lys-Asp-His-Tyr-Phe-Gly-Arg-Asn-Leu-As
It relates to a BSH protein defined by p-Leu-Glu-. Further, the present invention relates to Enterococcus hirae or Enteroco
It relates to BSH produced by ccus durans . BSH is, Lactobacillus genus of lactic acid bacteria so far, Bifidobacterium
Lactic acid bacteria of the genus, and Clostridium , the so-called intestinal bacteria, such as the genus Bacteroides , its presence is known,
Reports on their purification, gene structure, properties of enzymes, etc. have been made. For lactic acid bacteria of the genus Enterococcus ,
Although the presence of the enzyme has been reported, the properties of the enzyme protein, the nature of the enzyme activity, the gene structure, etc., have not been reported at all, and thus the BSH of the present invention
The protein is BSH isolated and purified for the first time from lactic acid bacteria of the genus Enterococcus .

[0015] The present inventors have produced a high proportion of BSH from lactic acid bacteria, also, in the process that searches an enzyme capable of deconjugation of conjugated bile acids that nature is present in the human intestinal tract, Enterococcus genus Lactic acid bacteria, especially Enterococc
us hirae and also Enterococcus durans produce relatively large amounts of BSH compared to other lactic acid bacteria, and the BSH produced by these lactic acid bacteria has the ability to widely deconjugate the six conjugated bile acids present in humans. After that, the purification of the present enzyme was started, and the properties of the enzyme were examined to complete the present invention.

The lactic acid bacteria belonging to the genus Enterococcus which produce the BSH protein of the present invention are preferably En
Lactobacillus belonging to the genus terococcus hirae or Enterococcus durans and producing a large amount of the enzyme BSH can be mentioned. More specifically, it is separated from dairy products, Enterococcus hirae SBT-3482 strain (FERM P-17)
031) or Enterococcus durans SBT-3481 strain (FER
MP-17030).

Ente producing the BSH protein of the present invention
Among lactic acid bacteria of the genus rococcus , Enterococcus hirae is
The following morphological properties are shown. (1) Cell shape: cocci (2) Motility: no (3) Spores: no (4) Gram stain: positive

Further, the following physiological properties are shown. (1) Catalase negative (2) Gas production: No (3) Growth at 45 ° C: Yes (4) Growth in 4% salt: Yes (5) Growth in 6.5% salt: Yes (6) Arginine Dehydrogenase positive (7) β-glucosidase positive (8) β-galacturonase positive (9) β-glucuronidase negative (10) α-galactosidase positive (11) β-galactosidase positive (12) N-acetyl β-glucosaminidase positive (13) β-mannosidase negative (14) urease Negative (15) Acetoin production: No (16) L (+) lactic acid generated from glucose (17) Peptidoglycan form: A4α, L-Lys-D-Asp (18) Presence or absence of acid production from various saccharides: Ribose + Cyclodextrin-mannitol-glycogen-sorbitol-pullulan-lactose + maltose + trehalose + melibiose + raffinose-meletitose-sucrose-tagatose + L-arabinose-D-arabitose-

Enterococcus durans has the following morphological properties. (1) Cell shape: cocci (2) Motility: no (3) Spores: no (4) Gram stain: positive

The following physiological properties are shown. (1) Catalase negative (2) Gas production: No (3) Growth at 44 ° C: Yes (4) Growth at 45 ° C: No (5) Growth in 4% salt: Yes (6) 6.5% salt Growth in: Yes (7) Arginine dehydrogenase positive (8) β-glucosidase positive (9) β-galacturonase positive (10) β-glucuronidase negative (11) α-galactosidase positive (12) β-galactosidase positive (13) N-acetyl β-glucosaminidase positive (14) β-mannosidase negative (15) Urease negative (16) Acetoin production: Yes (17) L (+) lactic acid is produced from glucose (18) Peptidoglycan form: A4α, L-Lys-D-Asp (19) Various Presence or absence of acid generation from saccharides: ribose + cyclodextrin-mannitol-glycogen-sorbitol-pullulan-lactose + maltose + trehalose + melibiose + raffinose-meletitose-souk Over scan - tagatose + L- arabinose - D- Arabitosu -

[0021] In the present invention, after a predetermined time cultured lactic acid bacteria under anaerobic conditions in Enterococcus genus having bacteriological properties described above, for example cells of Enterococcus hirae, cells of some have <br/> is Enterococcus Durans , A BSH active fraction is extracted from the extract, and then the enzyme can be purified into a single protein from the bacterial cell extract by a method combining two types of column chromatography.

For culturing the cells, any medium can be used as long as it is a medium for anaerobic bacteria. For example, cells can be cultured using a commercially available TPY medium or Briggs Liver liquid medium. The cultivation is preferably performed under anaerobic conditions. For this purpose, a commercially available oxygen adsorbent is used to set the anaerobic conditions in the incubator, or the oxygen in the incubator is forcibly replaced with nitrogen gas or carbon dioxide gas This can create an anaerobic environment suitable for culture. The cultivation may be carried out in a temperature range of 30 to 37 ° C for 10 to 30 hours.

The cultured cells are washed with a buffer having a buffer capacity in the neutral region, and the medium components are sufficiently removed from the cell components. The washed cells are suspended again in a buffer to prepare a cell suspension having an appropriate concentration. Next, ultrasonic treatment or the like is performed to prepare a cell extract (crude enzyme fraction) from the cell suspension. For the method of preparing the cell extract, in addition to the method using an ultrasonic treatment apparatus, a method using a French press,
Examples thereof include a method by freeze-thawing and a method by enzyme treatment with lysozyme, and the like. Preferably, a sonication device is used to prepare a crude enzyme fraction suitable for enzyme purification.

The crude enzyme fraction prepared as described above is
First, fractionation is performed using anion exchange chromatography. Columns for anion exchange chromatography include Pharmacia Mono Q column and DEAE-Sepharose
A column or the like can be used. This chromatography can be performed at around pH 7, where BSH
The active fraction is adsorbed on a column for anion exchange chromatography. The BSH active fraction once adsorbed to the column can then be eluted from the column with a buffer containing 0.25-0.35 M sodium chloride.

The BSH protein can be completely purified by fractionating the BSH active fraction fractionated by anion exchange chromatography and then cation exchange chromatography. As a column for cation exchange chromatography, a Mono S column or a CM-Sepharose column manufactured by Pharmacia can be used. Cation exchange chromatography is preferably carried out when the pH of the buffer used for chromatography is between 4 and 5. If the pH of the buffer exceeds 6, the purification effect by this chromatography cannot be expected,
Further, when the pH of the buffer is adjusted to 4.5 or less, the enzyme is inactivated, and the enzyme activity is lost.

In the cation exchange chromatography under the above conditions, the BSH active fraction is eluted without being retained on the column. On the other hand, since almost all of the contaminating proteins are retained on the column, proteins expressing BSH activity can be isolated and purified as almost pure proteins.

As described above, in the present invention, Enterococcus hirata is obtained by combining two types of fractionation operations by ion exchange chromatography.
The BSH produced by e can be purified relatively easily. Enteroco can also be implemented by using a similar method.
BSH produced by ccus durans can also be easily purified. Further, in the present invention, in order to confirm that the protein purified by the above method shows BSH activity,
The following two types of studies were further performed.

One is a method of subjecting the partially purified enzyme fraction to BSH activity staining, and the other is a method by N-terminal amino acid sequence analysis. As a result, it was revealed that the protein obtained in the present invention was a BSH protein having BSH activity, as will be shown in Examples later.

Furthermore, the En disclosed by the present invention
N-terminal amino acid sequence of BSH produced by terococcus hirae and N-terminal of BSH produced by Enterococcus durans
The terminal amino acid sequence was subjected to sequence homology analysis. As a result, Enterococcus hirae derived from the present invention, and Ente
The N-terminal amino acid sequence of BSH derived from rococcus durans has been registered in any papers and reports such as patent specifications, and in DNA and protein sequence information databases such as EMBL, GenBank, and Swiss-Plot. It is not a novel sequence, and therefore the physical novelty of the two BSH proteins disclosed in the present invention is indisputable.

[0030]

[Example] BSH derived from Enterococcus hirae is described below.
The present invention will be described in more detail with reference to examples relating to proteins. In addition, BSH derived from Enterococcus durans
The protein may be Enterococcus hi according to the following example.
It can be purified in the same manner as the rae- derived BSH protein.

Measurement of Enzyme Activity In the present invention, BSH activity was measured using the following two methods. In the first method, after reacting an enzyme sample with a conjugated bile acid, the amino acids released from the conjugated bile acid are colorimetrically determined using trinitrobenzenesulfonic acid (TNBS), a modifying reagent for the free amino group. As a result, BSH activity was measured. The reaction solution was composed of 0.18 ml of a 0.1 M sodium phosphate buffer (pH 7.0), 0.01 ml of a substrate solution (200 mM conjugated bile acid mixed solution), and 0.01 ml of an enzyme sample. After mixing the buffer solution and the substrate solution, the reaction was started by adding an enzyme sample, and after reacting at 37 ° C. for 10 minutes, 0.05 ml of the reaction solution was sampled.
The reaction was stopped by adding a 15% (w / v) trichloroacetic acid (TCA) solution. The remaining 0.15 ml of the reaction solution was further allowed to continue the reaction at 37 ° C. and allowed to react for 30 minutes, after which 0.05 ml of the reaction solution was sampled, and 0.05 ml of a 15% TCA solution was added thereto to stop the reaction. Was. The amount of free amino acids in the two samples thus obtained was determined by the TNBS method (Barret, D. and Edwards, B.
F., Methods Enzymol., Vol. 45, pp. 354-373, 1976), and the amount of amino acids released from the conjugated bile acids for 10 to 30 minutes 20 minutes after the start of the reaction was calculated.

The measurement of the amount of free amino acids was performed according to the following procedure. For 0.60 ml of sample solution, 0.30 ml of 4% (w / v) sodium bicarbonate solution and 0.40 ml of 0.1% (w / v) TNBS
The reaction was started by adding the solution, and the mixture was incubated at 50 ° C. for 20 minutes in the dark. After the reaction, 0.25 ml of 2% (w / v) SDS solution and 0.25 ml of 1N hydrochloric acid solution were added to stop the reaction. Within one hour after stopping the reaction, the absorbance at 340 nm of the reaction solution was measured. Glycine at 10-40 nmoles was used as a standard.

Under the above reaction conditions, 1 μm / min
1 unit of enzyme activity required to release amino acids of le
(1 unit). In addition, 200 mM conjugated bile acid mixed solution is sodium taurocholic acid (12.9 mg / ml), sodium tauro
deoxycholic acid (8.4mg / ml), Sodium taurochenodeoxy
cholic acid (12.5mg / ml), Sodium glycocholic acid (2
2.4mg / ml), Sodium glycodeoxycholic acid (15.1mg / m
l), and Sodium glycochenodeoxycholic acid (21.7 mg / m
l). In the TNBS method, the amount of amino acids in a sample subjected to an enzyme reaction in the presence of DTT could not be measured (since DTT reacts with TNBS and the background increases).

Therefore, in the second method, a colorimetric method using ninhydrin (Lee, YP and Takahashi, T., Anal. Bioc.
hem., vol. 14, pp. 71-77, 1966) to determine the amount of amino acids in the sample containing DTT. 0.1 ml
The test tube in which 1.9 ml of the ninhydrin reagent was added to the sample of Example 1 was sufficiently stirred, and then reacted in boiling water (100 ° C.) for 14 minutes.
After the reaction, the test tube was kept in tap water for 3 minutes to cool the reaction solution, and the absorbance at 570 nm of the reaction solution was measured within 1 hour. As a standard sample, 10 to 40 nmoles of glycine was used. When the sample contained TCA and DTT, the same amount of TCA and DTT was added to the standard sample.

Under the above reaction conditions, 1 μm / min
1 unit of enzyme activity required to release amino acids of le
(1 unit). In addition, ninhydrin reagent (1.9 ml)
Is a 0.5 M citrate buffer (pH 5.5) containing 0.5 ml of a 1% (w / v) ninhydrin solution, 1.2 ml of glycerol, and 0.2 ml of glycerol.
Consist of ml of 0.5M citrate buffer (pH 5.5).

[0036]

Example 1 BSH protein from Enterococcus hirae
After culturing the purified Enterococcus hirae strain SBT-3482 (FERM P-17031), the culture was centrifuged to collect the cells.
The collected cells were washed with a 0.1 M sodium phosphate buffer (pH 7.0) and then collected again by centrifugation. This washing operation was performed twice. After washing, the cells were resuspended in 0.1 M sodium phosphate buffer (pH 7.0), and the A600 value of the suspension was _adjusted to 15%.
Was adjusted. Next, the suspension was sonicated on ice to prepare a cell extract. The crushing conditions were determined by the equipment (Model VC375;
Sonics and Materials Inc.) output is 5, Duty cycle is
50% and the processing time was 3 minutes. Add the cell suspension after disruption to 2
The mixture was centrifuged at 5,000 × g for 10 minutes, and the supernatant was used as a bacterial cell extract. The cell extract was stored at −20 ° C. before use.

As an initial step of purifying the BSH protein, anion exchange chromatography was performed using a Mono Q column (0.5 × 5 cm, manufactured by Pharmacia). The buffer is 0.1 M sodium phosphate buffer (pH 7.0)
It was used. For the elution of the protein adsorbed on the column, a buffer obtained by adding 1.0 M sodium chloride to the above buffer was used. The flow rate was 1.0 ml / min. After the cell extract was added to the column, the column was washed with 10 ml of buffer. Next, a concentration gradient of sodium chloride was formed using an elution buffer, and the protein adsorbed on the column was eluted. The BSH active fraction was eluted from the Mono Q column by a buffer containing about 0.3 M sodium chloride.

The BSH active fraction obtained by anion exchange chromatography was then subjected to cation exchange chromatography using a Mono S column (0.5 × 5 cm, manufactured by Pharmacia). As the buffer, a 0.05 M sodium acetate buffer (pH 5.0) was used. For the elution of the protein adsorbed on the column, a buffer obtained by adding 1.0 M sodium chloride to the above buffer was used. The flow rate was 1.0 ml / min.

The BSH active fraction obtained by Mono Q column chromatography was diluted with 9 times the volume of 0.05 M sodium acetate buffer (pH 5.0), and the pH of the solution was adjusted to 5.0 with a 0.05 M acetic acid solution. To obtain a sample to be added to the column. After the sample was added to the column, the column was washed with 10 ml of the above buffer. Next, a concentration gradient of sodium chloride was prepared using an elution buffer, and the protein adsorbed on the column was eluted. Under these conditions, the BSH active fraction was Mono S
The column was eluted without passing through the column.

As described above, the cell extract obtained by sonicating the washed cells is fractionated using two types of ion-exchange chromatography, so that BS can be relatively easily separated.
The H protein could be purified. Purification was started from 150 ml of the culture solution, and 0.06 mg of purified BSH protein was obtained. The activity recovery was 27%, and the specific activity of the final purified sample was 137 U / mg.

[0041]

Example 2 By using the same method as in Example 1, Enterococcus durans strain SBT-3481 (FERM P-17030)
The BSH protein produced by was purified. Purification was started from 150 ml of the culture solution, and 0.01 mg of purified BSH protein was obtained. The activity recovery was 12%, and the specific activity of the final purified sample was 98 U / mg.

[0042]

Example 3 The protein purified in Example 1 is BSH
Confirmation of existence: SDS-PAGE- activity staining method After separating proteins contained in the target sample by SDS-PAGE method, the method of Coleman and Hudson (Appl. Environ. Microb
iol., vol.61, pp.2514-2520, 1995), only the BSH protein was actively stained on the gel. This method uses SD
The BSH protein denatured by S-PAGE was converted to Triton X-100.
Reconstitution by treatment, followed by incubating the gel in an acidic buffer containing a conjugated bile acid solution,
This is a method of forming a white precipitate (precipitation of unconjugated bile acid) on the SH protein band.

After separating the proteins in the sample by SDS-PAGE using a 10% acrylamide gel, the gel was incubated overnight at room temperature in a reconstitution buffer. Thereafter, the gel was transferred to a substrate solution and incubated at room temperature for 1 to several hours. The composition of the reconstitution buffer was 0.1 M sodium phosphate buffer (pH 7.0), 1% (v / v) Triton X-100, and 10 mM DT.
T, the composition of the substrate solution is 0.5 M sodium acetate buffer (pH 5.5), 10 mM DTT, 1 mM EDTA, and 10 mM sodium
glycodeoxycholic acid. The partially purified fraction after Mono Q column chromatography was subjected to SDS-PAGE and then subjected to an activity staining method. As a result, purified BSH protein and SD
A white precipitate formed on the protein band with the same mobility on S-PAGE. This result indicated that the protein purified by the present invention had BSH activity.

[0044]

Example 4 The protein purified in Example 1 is BSH
Confirmation: After performing BDS protein purified by N-terminal amino acid sequence analysis on SDS-PAGE, Matsudai
ra method (J. Biol. Chem., vol.262, pp.10035-10038,
1987), the BSH protein in the gel was transferred to a PVDF membrane and used as a sample for N-terminal amino acid sequence analysis. When transferring, use Towbin buffer at 150 V constant pressure.
Energized for 30 minutes. After the transfer operation, the PVDF membrane is stained with CBB,
The BSH protein band of interest was cut out. This sample was directly transferred to Applied Biosystem's Protein Sequencer Mo.
Analyzed with del 476A. As a result, Enterococcus hirae
The N-terminal amino acid sequence of the BSH protein produced by
It was determined as follows. X-Thr-Ser-Ile-Thr-Tyr-Val-Thr-Ser-Asp-His-Tyr-Phe-
Gly-Arg-Asn-Phe-Asp-Tyr-Glu-Ile-Ser-Tyr-Asn-Glu-Va
l-Val-Thr-Val-Thr- (X is an unidentified amino acid).

[0045]

Example 5 The protein purified in Example 2 is BSH
Confirmation: N-terminal amino acid sequence analysis BSH derived from Enterococcus durans obtained in Example 2
The N-terminal amino acid sequence of the protein was determined using the same method as in Example 4 as follows. X-Thr-Ser-Ile-Thr-Tyr-X-Thr-Lys-Asp-His-Tyr-Phe-Gl
y-Arg-Asn-Leu-Asp-Leu-Glu- (X is an unidentified amino acid).

Based on the sequence determined this time, GenBank and EM
A homology search was performed on the BL DNA database and the Swiss-Plot protein database. As a result, the N-terminal amino acid sequence of the BSH protein derived from Enterococcus hirae was determined by the previously reported Lact
N estimated from BSH gene of obacillus plantarum
Terminal amino acid sequence (EMBL Accession No.S51638, (Chris
tiaens, H., Leer, RJ, Pouwels, PH and Verstraete,
W., Appl.Environ.Microbiol., Vol.58, pp.3792-379
8, 1992)) with 72% homology to Clos
N estimated from BSH gene of tridium perfringens
It showed 41% homology to the terminal amino acid sequence (EMBL Accession No. U20191). In addition, BSH of Lactobacillus delbruekii registered in the DNA database
N-terminal amino acid sequence (EMBL Acce
ssion No. U90066).
In addition, Lactob registered in the DNA database
It also showed 45% homology to the N-terminal amino acid sequence (EMBL Accession No. AF054971) deduced from the BSH gene of acillus johnsonii . Also, N-protein of BSH protein of Lactobacillus spec . Registered in Swiss-Plot
Terminal amino acid sequence (Swiss-Plot Accession No.G259955,
(18)) showed 52% homology.

Also, BSH derived from Enterococcus durans
The N-terminal amino acid sequence of the protein shows 58% homology to the N-terminal amino acid sequence deduced from the previously reported BSH gene of Lactobacillus plantarum , and the N-terminal amino acid sequence of Clostridium perfringens It showed 58% homology to the deduced N-terminal amino acid sequence. Further, it showed 47% homology to the N-terminal amino acid sequence deduced from the BSH gene of Lactobacillus delbruekii registered in the DNA database. Furthermore, the N-terminal amino acid sequence of the BSH protein deduced from the BSH gene of Lactobacillus johnsonii registered in the DNA database is
It showed 47% homology. In addition, it showed 68% homology to the N-terminal amino acid sequence of the BSH protein of Lactobacillus spec . Registered in Swiss-Plot.

From the results of the above sequence analysis and homology search, the N-terminal amino acid sequence of the protein derived from Enterococcus hirae and the protein derived from Enterococcus durans purified according to the present invention were compared with those of other BSH proteins reported so far. Showed homology to the N-terminal amino acid sequence. Therefore, it was further confirmed that the protein purified in this study was a BSH protein.

[0049]

Example 6 Measurement of Molecular Weight of BSH Protein The molecular weight of BSH protein in a denatured state was measured by ordinary SDS-PAGE analysis using a 10% acrylamide gel. The result is shown in FIG. According to this, Enterococc
The molecular weight of the BSH protein from us hirae is 38 kDa
It was estimated to be ± 2 kDa. Similarly, the molecular weight of BSH protein derived from Enterococcus durans was also 38 kDa ± 2.
It was estimated to be kDa.

Next, Enteroco was purified using the usual gel filtration method.
The molecular weight of the BSH protein derived from ccus hirae in a non-denatured state was measured. Gel filtration conditions were a Pharmacia Superose 12 column, and the mobile phase was 0.1 M sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride.
And the flow rate was 0.5 ml / min. The molecular weight measurement standard used was a molecular weight measurement kit (MW-1000) manufactured by Sigma. As a result, the molecular weight of the BSH protein derived from Enterococcus hirae in a non-denatured state was 138 kDa ± 5 kDa.
It was speculated.

[0051]

Example 7 Substrate characteristics of BSH derived from Enterococcus hirae
Under the conditions of isomer substrate saturation (final concentration of substrate is 10 mM), Enterococcus hira against six major conjugated bile acids in humans.
The substrate specificity of e- derived BSH was examined. In addition, BSH
The activity was measured in the presence of 10 mM DTT according to the ninhydrin colorimetric method described above. Table 1 shows the results. This BSH showed higher activity for glycine-conjugated bile acids than for taurine-conjugated bile acids. Among the taurine-conjugated bile acids, the activity against taurodeoxycholic acid was the highest. From this result, Enteroco
BSH produced by ccus hirae was found to be highly specific for glycine-conjugated bile acids.

Substrate of BSH from Enterococcus durans
Under the conditions of specific substrate saturation, the substrate specificity of BSH derived from Enterococcus durans for the six main types of conjugated bile acids in humans was examined. Table 1 shows the results. This BSH is Entero
Like BSH produced by coccus hirae , it showed higher activity on glycine-conjugated bile acids than on taurine-conjugated bile acids. However, the activity of this BSH against taurine-conjugated bile acids was higher than the activity of BSH derived from Enterococcus hirae . The ratio of the activity value of BSH to glycine-conjugated bile acid to the activity value to taurine-conjugated bile acid was found to be about 3: 1.

[0053]

[Table 1] BSH and Ente derived from Enterococcus hirae
Substrate specificity of BSH from rococcus durans The relative activity value is shown assuming that the activity value for glycodeoxycholic acid is 100.

[Sequence List] SEQUENCE LISTING <110> YUKIJIRUSHI NYUGYO KABUSHIKI GAISHA <120> Bile salt hydrolase protein <130> SNMFP98385 <160> 2 <210> 1 <211> 30 <212> PRT <213> Enterococcus hirae <400> Thr Ser Ile Thr Tyr Val Thr Ser Asp His Tyr Phe Gly Arg Asn 1 5 10 15 Phe Asp Tyr Glu Ile Ser Tyr Asn Glu Val Val Thr Val Thr 20 25 30 <210> 2 <211> 20 <212> PRT <213 〉 Enterococcus durans 〈400〉 2 Xaa Thr Ser Ile Thr Tyr Xaa Thr Lys Asp His Tyr Phe Gly Arg Asn 1 5 10 15 Leu Asp Leu Glu 20

[Brief description of the drawings]

FIG. 1. BSH from Enterococcus hirae of Example 6.
Shows the molecular weight of the protein.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Igo Mielau Groningen The Netherlands 9728 H.N.M. Creestraat 167 (72) Inventor Atsushi Serizawa 3-4-1-704 Minamidai, Kawagoe-shi, Saitama F-term (reference) 4B050 CC01 DD02 FF11C LL01 LL10

Claims (6)

[Claims] 1. Bile salt hydrolase (Bile salt hydrolase) produced by a lactic acid bacterium of the genus Enterococcus.
BSH protein having activity (referred to as SH). 2. The N-terminal amino acid sequence of the subunit of the BSH protein is X-Thr-Ser-Ile-Thr-Tyr-Val-Thr-Ser-
Asp-His-Tyr-Phe-Gly-Arg-Asn-Phe-Asp-Tyr-Glu-Ile-Se
The BSH protein according to claim 1, which is defined by r-Tyr-Asn-Glu-Val-Val-Thr-Val-Thr- (X is an unidentified amino acid). 3. The BSH protein according to claim 2, which is produced by Enterococcus hirae . 4. The N-terminal amino acid sequence of the BSH protein subunit is X-Thr-Ser-Ile-Thr-Tyr-X-Thr-Lys-As.
The BSH according to claim 1, wherein the BSH is defined by p-His-Tyr-Phe-Gly-Arg-Asn-Leu-Asp-Leu-Glu- (X is an unidentified amino acid).
protein. 5. The BSH protein according to claim 4, which is produced by Enterococcus durans . 6. An extract and a cation exchange chromatography of an extract of a bacterium of the genus Enterococcus capable of producing a conjugated bile acid deconjugating enzyme (BSH) are sequentially treated by anion exchange chromatography and cation exchange chromatography to repeat adsorption and elution. B
The method for producing a BSH protein according to claim 1, wherein the SH is purified and collected.