JPH08245690A - Disaccharide decomposing enzyme activity-accelerating peptide - Google Patents

Abstract

PURPOSE: To obtain the subject new peptide having a specific molecular weight and bonding ability to an anion exchanger, accelerating disaccharide decomposing enzyme activity existing in small intestine mucos membrane and useful for a food, a medicine and a reagent for biochemistry, etc., by subjecting casein to enzymolysis. CONSTITUTION: The objective new disaccharide decomposing enzyme activity- accelerating peptide is obtained by subjecting casein to enzymolysis and has an N-end amino acid sequence expressed by the formula (Xaa is Gln or Glu), has <=17kDa molecular weight measured by SDS-PAGE, is adsorbed on an anion exchanger, is eluted by a salt solution having >=0.28M concentration, accelerates activities of disaccharide decomposing enzyme lactase and sucrase existing in epithelial cell of small intestine mucos membrane and utilizable for a food, a medicine and a reagent for biochemistry, etc. The peptide is obtained by using casein existing in lactoprotein as a starting substance, decomposing by using carboxypeptidase derived from a genus Rhizopus microorganism, subjecting to ultrafiltration and collecting fraction of <=17kDa and purifying the resultant fraction by high-performance liquid chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having an action of promoting the activity of disaccharide-degrading enzyme present in the small intestinal mucosa. The peptide of the present invention is useful for applications such as foods, pharmaceuticals and biochemical reagents.

[0002]

2. Description of the Related Art Orally ingested food is mainly decomposed mainly by pancreatic juice and comes into contact with the small intestinal mucosa. In addition, sugars such as starch are decomposed into disaccharides such as lactose, sucrose and maltose by saliva and pancreatic juice, but they are further decomposed into monosaccharides by mucosal digestive enzymes on the intestinal mucosa and absorbed into the body. Digestion at this stage is called membrane digestion or terminal digestion. If the activity of the mucosal digestive enzyme can be increased by some method at the stage of this membrane digestion, it can be expected that digestion and absorption will be improved. Infants tend to have poor digestive and absorptive ability due to immature mucosal cells, and elderly people tend to have poor digestive and absorptive ability due to inactive cell metabolism. In such a case, it is possible to compensate for such a decrease in digestive and absorption ability by increasing the enzyme activity in the digestive tract. Therefore, in general, enzyme preparations are administered to compensate for the functional decline.
However, no attempt has been made to improve the function of the intestinal tract in patients with reduced digestive and absorption capacity.
It has been reported that the activity of disaccharide degrading enzyme in the small intestinal mucosa is increased by ingestion of its substrate disaccharide. In order to enhance the absorption of sugars, it is sufficient to ingest foods containing a high content of sugars, especially disaccharides, but in this case, the sugar content per energy (calorie) of foods is large, which is not efficient. . If you can regulate the digestive function of the small intestinal mucosa by ingesting food components such as disaccharides that are involved in the expression of disaccharide-degrading enzyme activity in the small intestinal mucosa, you can supply safe and digestible foods. However, it is the present situation that no report that such components have been separated can be found.

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventors have studied the substance that promotes the activity of the disaccharide-degrading enzyme present in the mucous membrane of the digestive tract, and as a result, by ingesting milk protein, the enzyme For the first time, it was found that the activity was promoted. Furthermore, it was confirmed that this action was strongly induced when milk protein, especially casein, was ingested. It was revealed that this disaccharide-degrading enzyme activity-promoting substance is present in the peptide fraction produced by enzymatic decomposition of casein. An object of the present invention is to provide a novel peptide which is present in a peptide fraction obtained by enzymatically degrading casein and promotes the activity of a disaccharide-degrading enzyme present in the small intestinal mucosa.

[0004]

The present invention provides a peptide obtained by enzymatically degrading casein and promoting the activity of disaccharide-degrading enzyme present in the small intestinal mucosa. One of these peptides is a peptide which has the following characteristics and promotes disaccharide-degrading enzyme activity present in the small intestinal mucosa. N-terminal amino acid sequence: Val-Ala-Pro-Phe-Pro-Xaa-Val-Phe-Gly-Lys-Glu-Lys-Va
l-Asn-Glu-Leu-Ser-Lys-Asp-Ile-Gly-Ser-Glu-Ser-Thr-
Glu-Asp-Gln-Ala-Met- (where Xaa represents either Glu or Gln) Molecular weight: Molecular weight by SDS-PAGE: Approximately 17 kDa Electrical property: Adsorbed on anion exchanger, 0.28M or more Elution with Various Concentrations of Salt Solution: Promoting Lactase and Sucrase Activities of Small Intestinal Mucosal Epithelial Cells

Furthermore, one of the peptides of the present invention is
It is a peptide having the following characteristics and promoting the activity of disaccharide-degrading enzyme present in the small intestinal mucosa. N-terminal amino acid sequence: Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro- Molecular weight: Molecular weight by SDS-PAGE: Approximately 15 kDa or 1
0kDa Electrical property: Adsorbed on anion exchanger and eluted with salt solution of 0.28M or higher Action: Promote lactase activity and sucrase activity of small intestinal mucosal epithelial cells

The peptide of the present invention can be obtained by using casein existing in milk protein as a starting material and decomposing it with a protease or peptidase. As the enzyme used for decomposition, pepsin, trypsin, animal protease such as chymotrypsin, protease derived from microorganisms,
It can be carried out using a peptidase such as carboxypeptidase. It is preferable to use a peptidase derived from a microorganism such as peptidase R (trade name, Amano Pharmaceutical Co., Ltd.). Peptidase R is an enzyme containing peptidase, a proteinase derived from Rhizopus. In the enzyme treatment, casein and the enzyme are reacted at a reaction temperature of 25 to 50 ° C. at a pH of 3 to 8 for 1 to 24 hours to obtain an enzymatic decomposition product of casein, and then the enzyme reaction is subjected to an appropriate method such as heat treatment or acid treatment. Then, the enzyme and unreacted casein are removed by precipitation. In order to purify the peptide, a fraction containing the desired peptide having a molecular weight of 17,000 or less is obtained by a method such as solvent extraction or ultrafiltration, and the fraction is further purified by a method such as high performance liquid chromatography. You can The peptide of the present invention is adsorbed on an anion exchanger and is eluted with an aqueous salt solution having a concentration of 0.28 M or more. Therefore, for example, M
A column packed with an ion exchanger such as ono Q column (Pharmacia) can adsorb the active fraction and elute with a salt concentration gradient solution or the like. The obtained active fraction is subjected to dialysis treatment to remove salt, and the target active fraction is separated by reverse phase column chromatography or the like.

The N-terminal amino acid sequence of the obtained purified peptide can be confirmed by the Edman degradation method or the like to identify the peptide of the present invention.

The obtained peptide can be pulverized by a treatment such as freeze-drying if necessary. The peptide may also be a pharmaceutically acceptable salt such as hydrochloride, sulfate, succinate, citrate, tartrate, sodium salt, potassium salt or calcium salt.

The peptide of the present invention can be orally administered as it is, or by mixing it with a food or the like. In administration, it depends on the subject, but usually,
Adults can administer 100 mg or more. The peptide of the present invention is present in enzymatically decomposed casein and is highly safe.

The action of the peptide of the present invention, that is, the action of promoting the activity of disaccharide-degrading enzyme in the small intestinal mucosa, is described by Miller (Mille
r, The American Journal of Clinical Nutriton, Vo
l.34, pp.1153-1170, 1981). (1) Preparation of substrate With 0.1 M sodium malonate buffer (pH 6.0),
Lactose and sucrose were prepared to 0.28M concentration,
Keep frozen until just before use. (2) TG
Preparation of O reagent 100 ml of 0.5 M Tris-HCl buffer (pH 7.0)
A solution of 4 mg of glucose oxidase in 9
A solution of 20 g of Triton X in 80 g of 5% ethanol, 1.0 ml of a solution, 1 ml of 3,3′-dimethoxybenzidine 1% in ethanol, and 0.5 ml of a 0.1% peroxidase aqueous solution were mixed to prepare a TGO reagent. It was stored refrigerated. (3) Measurement method Human-derived cultured cell line intestine 407 (AT
CC CCL6) or rat small intestinal mucosa-derived cultured epithelial cells IEC18 (ATCC CRL1589), PBS
And wash twice. Then, about 1 mg of the cells was put into a 10 ml test tube, and 300 μl of the substrate was further added to each test tube.
Add at intervals of 2 seconds and react at 37 ° C. for 1 hour. Then TGO
Add 2 ml of reagent to each test tube at 15 second intervals and
After reacting for 15 minutes, the substrate solution added to the cells is used as a blank, and the absorbance at 450 nm is measured using water as a control. The amount of glucose released by the calibration curve obtained in advance is obtained, and the amount of cellular protein is determined by the dye binding method (Bio-Rad, trade name: Protein Assay Kit), and the enzyme activity per cellular protein is measured.
The method for measuring the enzyme activity is determined by the following formula (1).

[0011]

## EQU1 ## Glucose (μg) / cell protein amount (mg) released in 60 minutes = E (μg / mg) (1)

The acceleration of the enzyme activity is obtained by adding a measurement sample to each test tube, measuring the test tube containing this sample by the above-mentioned method to obtain the amount of free glucose, and determining it by the following formula (2).

[0013]

(Equation 2) (Activity Es of sample addition test tube / Activity Ec-1 of control test tube) / Protein amount of sample (mg) × 100 = S (Unit) (2)

The present invention will be described in more detail below with reference to examples.

Example 1 Human-derived cultured cell line intestine
407 (ATCC CCL6) or rat intestinal mucosa-derived cultured epithelial cells IEC18 (ATCC CRL158)
9) to give a bottom area of 1 × 10 ⁴ cells / cm ²
The cells were seeded on a 24-well multiwell plate (Coaster) and cultured at 37 ° C. in a 5% CO ₂ atmosphere. When the cells reached confluence or subconfluence, a sample was added to the culture medium so that the maximum amount was 20%, and the cells were cultured for 1 to 6 days. For the membrane digestive enzyme, lactase and sucrase activities were measured. Lactase and sucrase activities were determined by the Miller modified Dahlqvist method described above. That is, the culture solution was removed from the culture vessel, the cell surface was lightly washed with PBS (phosphate buffer solution), and then a lactose and sucrose solution was added as a substrate. After reacting at 37 ° C. for 1 hour, TGO reagent (0.004% glucose oxidase, 0.0005
% Peroxidase, 0.01% 3,3'-dimethoxybenzidine, 0.2% Triton X-100 0.5
M Tris-hydrochloric acid, pH 7.0) was added, color was developed for 1 hour and 15 minutes, and the reaction solution was measured for absorbance at a wavelength of 450 nm. The activity accelerating action of the sample was determined by the calculation formula shown above as a relative ratio to the control without addition. When the promoting action of milk on the small intestinal mucosa disaccharide-degrading enzyme was examined by this measuring method, it was confirmed that it had an action of enhancing lactase and sucrase activities. Further, whole milk was fractionated into skim milk and cream by a conventional method, and the cream was further fractionated into butter and buttermilk. There was an enhancing effect on skim milk and buttermilk.

Skim milk is microfiltered with a fine diameter of 0.1 μm (M
When it was fractionated into a casein fraction and a whey fraction in F), a promoting action was observed in the casein fraction. In FIG. 1, the amount of casein added was 100 μl (1.5 mg) and 300 μl (4.
5 mg) and the measurement results are shown. The intestinal mucosa disaccharide-degrading enzyme promoting action was dose-dependent. Since the casein also contained a large amount of casein, it was considered that the active substance in the buttermilk was also due to casein.

Example 2 Production of a substance promoting small intestinal mucosa disaccharide-degrading enzyme activity When casein was digested with a proteolytic enzyme, the enhancing action remained in some enzymatic degradation products. Therefore, it was revealed that the enhancing action is due to the specific structure of the casein molecule. Therefore, in order to clarify the amino acid sequence in the casein molecule that exerts its action, casein was enzymatically decomposed with peptidase R (Amano Pharmaceutical Co., Ltd.). 1 kg of lactate casein (New Zealand Daily Board) was suspended in 10 liters of water, and the pH was adjusted to 7.0 with 1N sodium hydroxide to obtain a casein solution. After keeping the solution at 37 ° C., 20 g of peptidase R enzyme powder was added and reacted for 1 hour. The reaction was stopped by holding it at 100 ° C. for 30 minutes. The reaction solution was fractionated into a permeate and a concentrate by ultrafiltration (UF; Asahi Kasei ACP-1010) having a molecular weight cut off of 13,000. 40% for 200 ml of the obtained permeate
Ammonium sulfate was added to adjust the concentration to 15,000
It was separated into a precipitate fraction and a supernatant fraction by centrifugation at × g for 30 minutes. Both fractions were dialyzed against distilled water. Three
7 ml of precipitate and 360 ml of supernatant were obtained. The protein concentrations were 18.2 mg / ml and 2.7 mg / ml, respectively. Among these fractions, the activity of promoting the enzyme activity was found in the supernatant.

Further, the supernatant was separated by an anion exchange column. 10 ml of the supernatant was added to Mono Q (Pharmacia, FPLC system) equilibrated with 0.02 M Tris-HCl buffer, pH 7.2, and the peak eluted with a sodium chloride concentration of 0 to 1 M was separated. I took it. The monitor was performed by absorption at 280 nm. This operation was repeated and a total of 50 ml of the supernatant was added to the column. With respect to the obtained fractions, the small intestinal mucosa disaccharide degrading enzyme activity promoting action was measured by the above method. The measurement results are shown in FIG. The enzyme activity promoting activity was found in the fourth fraction (Fr. IV, FIG. 3) of the fractions fractionated by Mono Q. In addition, this fraction is SD
When electrophoresis was performed using S polyacrylamide gel, three bands of 10 kDa, 15 kDa, and 17 kDa were observed. Next, Fr. The IV was further separated and purified by reverse phase chromatography. C18 column (218D52 manufactured by Bidac, 0.21 x 25c
m) was used and the Fr. IV 2
0.2 ml of a linear concentration gradient of 0-65% of acetonitrile containing 0.1% trifluoroacetic acid loaded with 60 μg.
Elution was performed at a flow rate of / min. The elution pattern by absorption at 220 nm is shown in FIG. Collect each fraction,
Electrophoresis on an SDS polyacrylamide gel revealed a 17 kDa band in peak 9 and a 10 kDa and 15 kDa band in peak 7.
In FIG. 5, casein, Fr. The electrophoresis patterns of SDS polyacrylamide gel of IV, peak 7 and peak 9 are shown.

Using the fractions of peaks 7 and 9, Fr. The amino acid sequence of the acting peptide contained in IV was determined by Edman degradation. The sequences were analyzed by a gas phase automatic sequencer of 477A type and 120A type (manufactured by Applied Biosystems Incorporated), and as a result, the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 in Sequence Listing were N
It was revealed that it was included as a terminal amino acid sequence. This N-terminal amino acid sequence corresponds to residues 25 to 54 of known αs-1 casein in the case of 17 kDa peptide, and 1 to 9 in the known β casein in the case of 10 or 15 kDa peptide. Was found to match the residue. In addition, in the amino acid sequence of this 17 kDa peptide, the sixth residue was Glu, but there is also one that becomes Gln due to gene mutation.
It became clear from the A sequence.

[0019]

INDUSTRIAL APPLICABILITY The present invention provides a peptide which promotes the activity of disaccharide-degrading enzyme present in the small intestinal mucosa. The peptide of the present invention can be used as a food or a drug.

[0020]

[Sequence listing] SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide sequence Val Ala Pro Phe Pro Glu Val Phe Gly Lys Glu Lys Val Asn Glu Leu 1 5 10 15 Ser Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Met 20 25 30

SEQ ID NO: 2 Sequence length: 9 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide

[0022]

[Brief description of drawings]

FIG. 1 shows the results confirmed that casein promotes lactase activity in the small intestinal mucosa.

FIG. 2 shows the results of confirmation that casein and an enzymatic degradation product of casein promote lactase activity.

FIG. 3 shows a chromatography pattern obtained by fractionating an enzymatic degradation product of casein with an anion exchanger.

FIG. 4 shows a pattern obtained by fractionating an active fraction present in an enzymatic degradation product of casein by reverse phase chromatography.

FIG. 5 shows an SDS polyacrylamide gel electrophoresis pattern of active fractions. In the figure, 1 is a molecular weight standard, 2 is casein, 3 is Fr. IV, 4 is peak 7, 5 is peak 9,
6 indicates a molecular weight standard, respectively.

Claims (3)

[Claims] 1. A peptide having a molecular weight of 17 kDa or less, which is obtained by enzymatically degrading casein and accelerates the activity of a disaccharide-degrading enzyme present in the small intestinal mucosa. 2. A peptide having the following characteristics, which promotes the activity of disaccharide-degrading enzyme present in the small intestinal mucosa. (1) N-terminal amino acid sequence Val-Ala-Pro-Phe-Pro-Xaa-Val-Phe-Gly-Lys-Glu-Lys-Va
l-Asn-Glu-Leu-Ser-Lys-Asp-Ile-Gly-Ser-Glu-Ser-Thr-
Glu-Asp-Gln-Ala-Met- (where Xaa represents either Gln or Glu) (2) Molecular weight Molecular weight by SDS-PAGE: Approx. 17 kD (3) Adsorbed on anion exchanger, 0.28 M or more (4) Promoting lactase activity and sucrase activity of small intestinal mucosal epithelial cells eluted with salt solution of various concentrations 3. A peptide having the following characteristics, which promotes the activity of a disaccharide-degrading enzyme present in the small intestinal mucosa. (1) N-terminal amino acid sequence Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro- (2) Molecular weight Molecular weight by SDS-PAGE: about 15 kDa or 1
0kDa (3) Adsorbed to anion exchanger and eluted with a salt solution of 0.28M or higher (4) Promoting lactase activity and sucrase activity of small intestinal mucosal epithelial cells