JPH0630615B2 - Low molecular peptide composition and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to enzymatically producing a low-molecular-weight peptide composition containing a dipeptide as a main component.

Conventional techniques and problems The absorption of proteins in the intestinal tract is as follows: after the protein is decomposed into oligopeptides by perotiase in the stomach and intestines,
It is considered to be further decomposed by peptidase of intestinal epithelial cells and absorbed. Therefore, there is also a report that absorption of dipeptides and tripeptides is carried out at an extremely early stage as compared with amino acids, which indicates that di- or tripeptides are more preferable than amino acids.

Conventionally, a method for producing a dipeptide is performed by an organic synthetic chemistry method. However, with this method, the cost is high, removal of by-products is difficult, and there is a problem in safety as a food.

In addition, various commercially available protease agents may be allowed to act on the protein, but in this case, they are often amino acidized by the peptidase contained in the enzyme agent. For example, the enzyme agent of Aspergillus oryzae has a strong amino acid-producing ability, and the amino acid content in the soybean protein degradation product resulting from this is as high as 40%.

The present invention relates to a useful di- and tripeptide-based peptide mixture and a new production method thereof.

Means for Solving the Problems The present invention aims to effectively act DPCPase to produce large amounts of di- and tripeptides. More specifically, the point is that DPCPase, endopeptidase and, in some cases, proline-specific endopeptidase are allowed to coexist to decompose the protein. First DPCP
Tell me about ase.

DPCPase is, for example, Bacillus subtilis (Bacill)
us subtilis) HL521 strain (Ministry of Industrial Science, Deposit No. 10005
No.) or Bacillus pumilus HL7
Twenty-one strains (Deposit No. 10006 of Micro Engineering Research Institute) can be produced by culturing by a usual culturing method. The properties of DPCPase are as follows.

I) Action This enzyme acts as Ala-6 (a peptide consisting of 6 alanines is A
It is abbreviated as la-6, and similarly, for example, those consisting of 2 or 3 alanines are abbreviated as Ala-2 and Ala-3. The same shall apply hereinafter), in addition to cleaving less than carboxy into Ala-2s, angiotensin I (Asp-Arg-
Val-Tyr-Ilu-His-Pro-Phe-His-Leu) releases His-Leu from less than carboxy. Also, bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro).
-Phe-Arg) to Arg-Pro-Pro, Gly-Phe, Ser
Decomposed into -Pro and Phe-Arg. That is, this enzyme releases a peptide from the carboxy terminus of a protein in dipeptide units regardless of the type of amino acid. However, it is not cleaved if there is a proline second from the carboxy terminus.

II) Optimum pH and stable pH Bacillus subtilis HL521 strain has pH 6.0.
It is stable between -11.0 and the optimum pH is 7.5.

Bacillus pumilus HL721 strain has a pH of 5.5-
It is stable between 9.0 and the optimum pH is 7.5.

III) Action temperature In both Bacillus subtilis HL521 strain and Bacillus pumilis HL721 strain, the optimum temperature of enzyme action is 50 ° C.
It was stable up to 45 ° C under the condition of H7.0 for 60 minutes.

IV) Molecular weight By the gel filtration method, Bacillus subtilis HL521 strain had 110,000 Bacillus spumils HL7.
The value for the 21 strains was 155,000.

V) Method for measuring activity 10 mM benzoyl-glycyl-alanyl-proline 0.
0.05 ml of 20 mM phosphate buffer (pH 7.0) and 0.05 ml of enzyme solution were added to 1 ml, and the mixture was reacted at 40 ° C. for 15 minutes, and the resulting alanyl-proline was quantified by the ninhydrin method.

In the above reaction, the amount of enzyme that produces 1 μmol of alanyl-proline per minute was defined as 1 unit.

The above is an example of Bacillus subtilis and Bacillus pumilus, but the present invention is not limited thereto. For example, in addition to the above, those derived from the lungs of rabbits, kidneys of pigs, and Esherishia col.
i), Corynebacterium equ
Those of origin such as i) can also be effectively used in the present application.

Next is the endopeptidase, which is a normal endopeptidase except for the proline-specific endopeptidase. Produce a large amount of oligopeptides,
Moreover, it is preferable that the amino acids are not so much produced. The origin of the proline-specific endoprotease is also Flavobacterium meningocepticu.
In addition to m), for example, those derived from sheep kidney can be used.

Both endopeptidase and DPCPase have no particular restriction on the action temperature, action time, amount used (potency), pH, etc. It may be set appropriately within the operable range.

The order of action of endopeptidase, proline-specific endopeptidase and DPCPase is arbitrary, but generally, endopeptidase, proline-specific endopeptidase and DPCPase are usually acted in this order.

Any protein can be used as the substrate regardless of its origin, variety, etc. For example, soy protein, wheat protein, milk protein or egg white.

Action The DPCPase produced by Bacillus subtilis and Bacillus pumilus has been clarified by the present inventors to act in units of two amino acids from the carboxy terminus to produce a dipeptide. That is, as described as the action of the enzyme in the section of means for solving the above-mentioned problems, A
La-6 is cleaved into 3 Ala-2s and bradykinin is cleaved into 3 dipeptides and 1 tripeptide. However, the reaction does not proceed in the presence of the second proline from the carboxy terminus, as in the case of angiotensin I. This is a general property because it does not act on Ala-Ala-Pro-Ala.

In order to overcome the inhibition of this reaction, the present inventors have found through various studies that the action of DPCPase begins again when a proline-specific endopeptidase typified by Flavobacterium meningocepticum is allowed to coexist or act in advance. did.

As described above, if DPCPase and proline-specific endopeptidase are allowed to cooperate with each other, theoretically all proteins can be decomposed into dipeptides. However, when actually acting on various peptides and high-molecular-weight proteins such as casein, the action is significantly inferior to that of peptides, and in some cases, it hardly acts. As a result of various studies on the cause of this, it was revealed that di- and tripeptides were efficiently produced by reacting endoprotease to form an oligopeptide and then reacting with DPCPase and proline-specific endopeptidase.

Example 1 A medium containing 1% peptone, 0.5% yeast extract and 0.5% sodium chloride was adjusted to pH 7.3, and after sterilization, Bacillus subtilis HL521 strain was inoculated and cultured at 37 ° C. for 16 hours. After culturing, cells were obtained by centrifugation and suspended in 10 mM phosphate buffer and sonicated to disrupt the cells. Further, the disrupted cells were removed by centrifugation.

The supernatant contained 0.03 U / ml DPCPase. The supernatant was purified by various chromatographies such as Q-sepharose, hydroxyapatite, TSK-gel G3000SWXL. The purified enzyme thus obtained behaved as a single protein by gel electrophoresis. The yield was about 1%.

Example 2 The Bacillus pumilus strain HL721 was cultured in the medium having the same composition as in Example 1 under the same conditions. Under the same purification conditions, an enzyme showing the same action could be obtained. The yield was 1.5%.

Example 3 1 g of ovalbumin was dissolved in 100 ml of water, 0.1 g of Bacillus subtilis endopeptidase was added, and the mixture was added at 40 ° C.
・ We let you act for 4 hours. After heating to 100 ° C. to inactivate the endopeptidase, 5 U of proline-specific endopeptidase was added and allowed to act at 40 ° C. for 16 hours. After heating to 100 ° C. to inactivate the proline-specific endopeptidase, 0.5 U of DPCPase was added, and the mixture was further reacted at 40 ° C. for 16 hours.

Table 1 shows the ratio of the molecular weight of the reaction product at each stage by the gel filtration method.

As described above, a composition containing a low molecular weight peptide having a molecular weight of 1000 or less as a main component could be obtained.

Effect of the present invention From the specificity of the enzyme used in the present invention, since the low-molecular composition of the present invention contains a dipeptide as a main component, when orally administered, it is likely to be rapidly absorbed in the intestinal tract,
It is excellent nutritionally.

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Claims (5)

[Claims] 1. An aqueous solution of protein is treated with endopeptidase and dipeptidyl carboxypeptidase (hereinafter referred to as DPC).
Pase) and a low-molecular-weight peptide composition. 2. A method for producing a low-molecular-weight peptide composition, which comprises adding endopeptidase and DPCPase to an aqueous protein solution and hydrolyzing the same. 3. A low-molecular-weight peptide composition according to claim 1 or a method for producing the same, wherein a proline-specific endopeptidase is used as the endopeptidase. 4. A hydrolysis condition of a temperature of 25 to 60 ° C.
The low molecular weight peptide composition according to the claims or the method for producing the same, which is 8 to 72 hours. 5. The low molecular weight peptide composition according to claim 1 or the method for producing the same, wherein a normal endopeptidase and a proline-specific endopeptidase are used in combination as the endopeptidase.