JP2502531B2 - Modified protein manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for modifying a protein. More specifically, the present invention provides a protein having excellent solubility, foaming property, emulsifying property, etc. by deamidating the protein with a protease.

(Prior Art) Various modifications such as acylation, acetylation, phosphorylation, hydrolyzation, deamidation, etc. have been made for protein modification. Among these, many reports have been made on modification of proteins by deamidation. For example, Japanese Patent Application Laid-Open No. 51-139635 discloses a deamidated collagen as a cosmetic agent.
4654, 55-74774, and 58-859 disclose that gluten deamidated by heat hydrolysis is added to food products such as kneaded products. Also, Prikl.Biokhim.Mikrob
Iol., 22 , (2), 198-204, (1986) and other non-enzymatic proteins such as deamidation, Biochemistry, 24 , (26), 7681-
In 7688, (1985), deamidation of cysteine-sulfonated asparagine was reported by Izv.Akad.Nauk Mold.SSR.Ser.Bio.
L. Khim. Nauk, 6 , 61-62, (1983) described the deamidating enzyme in wheat as Proc. Natl. Acad. Sci. USA., 80 , 12, 3599-603,
(1983) deamidation of chemotaxis (chemotaxis), Nahrung, 25 , 4,397-403, (1981) deamidation by alkaline treatment of proteins, Izv, Sev, -Kavk, Nauchn.
entra Vyssh, Shk., Estestv. Naukl, 3 , 90-94, (1980)
Deamidation of proteins is reported in Ann.NYAcad.Sci, 258 , 314-
In 316, (1974), deamidation of glutamine and asparagine, and in Nature, 258 , (5532), 264-266, (1975), deamidation of eye lens proteins, Curr. Top. Cell. Regul. .,
8 , 247-295, (1974), deamidation of glutamine residues and asparagine residues in proteins and peptides was reported in Japan Food Industry Society, 19 , (7), 321-323, (1972). Deamidation of soybean protein and use of deamidated gluten in beverages are reported in US Pat. No. 3,770,452. Of the above, it is known that transglutaminase substitutes the amide group of the glutamic acid residue of the protein to hydrolyze and deamidate as the enzyme deamidation. However, the deamidation in this case is caused by the substitution of α-amino or ε-amino of the protein, and as a result, it has a drawback of causing protein cross-linking.

Also, it has been reported that partial deamidation may occur during soybean protein acylation of N-acetylamine with papain (Sung et al.), But deamidation with protease has been reported elsewhere. Absent.

Papain, trypsin and the like are known to cause deamidation using benzoyl-L-arginine ethyl ester as a substrate, but deamidation of the protein without substantially hydrolyzing it is not known.

The present invention differs from the above-mentioned conventional techniques in that the protein is modified by enzymatically deamidating it with a protease having a peptide-bonding hydrolytic activity without causing cross-linking and usually with very little hydrolysis.

(Problems to be Solved) The present inventors aimed to modify proteins by deamidation. First, we encountered the problem of protein hydrolysis during deamidation with dilute acid. Therefore, we attempted deamidation using protease as an enzymatic means. However, we encountered a problem that the deamidation condition was difficult because the hydrolysis which is a characteristic of protease occurred at the same time.

(Means for Solving the Problem) The inventors of the present invention have conducted diligent research to solve the above-mentioned problems (suppressing hydrolysis and deamidation), and under certain specific conditions (unexpectedly, other than optimum pH for hydrolysis). The pH range and the temperature range other than the optimum temperature for water decomposition of (1), a considerable amount of deamidation can be carried out with almost no hydrolysis, and the knowledge that the protein can be modified was obtained, and the present invention was completed.

That is, the present invention is a method for modifying a protein, characterized in that the protein is deamidated with a protease in a deamidation pH range and a deamidation temperature range.

The protein used in the present invention includes soybean protein, oil seed protein such as peanut protein, cereal protein such as rice protein and wheat protein, other plant protein, animal protein such as casein and the like (including meat storage, fish meat protein and the like). Well-known edible proteins such as leaf proteins and microbial proteins, physiologically active proteins such as enzymes, and so-called amino acid-containing peptide conjugates such as peptides are suitable. A protein soluble in the pH range of deamidation is preferable because it is easily deamidated by a protease.

The protease used in the present invention is preferably an endo-type protease, and a so-called acidic protease or neutral protease having an optimum pH for hydrolyzing in the acidic or neutral range is preferable. For example, any protease such as a protease originating from a plant such as papain, a protease originating from a microorganism such as pronase, a protease originating from an animal such as pepsin, chymotrypsin and trypsin, and a protease produced by other means such as biotechnology can be used. .

The present invention has a deamidation pH range (usually a pH higher than the optimum pH for water dissolution).
Region), and a deamidation temperature region (a temperature region lower than the optimum temperature for water dissolution), which is characterized by treating the protein with a protease.

Usually, protease has been used at the optimum pH and the optimum temperature for water hydrolysis. Even if provisional deamidation occurs in such pH range and temperature range, it is extremely small. However, surprisingly, there is almost no hydrolysis in the deamidation pH range and deamidation temperature range, which are different from the optimum pH for hydrolysis and the optimum temperature for hydrolysis, and even if hydrolysis occurs, the degree of hydrolysis does not occur at a very low level. Can be amide. Usually, the deamidation pH region is higher than the hydrolyzed pH region and depends on the type of enzyme used. For example, in the case of papain, the optimum pH for water dissolution is 7, and the suitable pH range for deamidation is 7.5 or higher, preferably 8 or higher.
Although the pH is 8 or less, a suitable pH range for deamidation is 8 or higher, and in the case of pronase, the optimum pH for hydrolysis is 8 and a suitable pH range for deamidation is 8 or higher, preferably 9 or higher. In the case of trypsin, the optimum pH for hydrolysis is 8, and the suitable pH range for deamidation is 9 or higher. For pepsin, the optimum pH for hydrolysis is around 2, and the optimum pH range for deamidation is 4 Approximately 5 is appropriate, and usually pH for optimum water dissolution
A pH range of 0.5 or more, preferably 1 or more, more preferably 1.5 or more is suitable. When the pH is in the optimum pH range for hydrolysis, the protease causes more hydrolysis than deamidation, which is not preferable. If the pH and temperature are too high, formation of lysinoalanine or racemization of amino acids may occur, which is not preferable. The deamidation temperature range is usually lower than the optimum temperature for water hydrolysis, and depends on the type of enzyme used. For example, in the case of papain, the optimum temperature for water dissolution exceeds 50 ° C, but the suitable temperature range for deamidation is less than 50 ° C. In the case of chymotrypsin, the optimum temperature for water dissolution exceeds 50 ° C, but A suitable temperature range is 50 ° C. or lower, and in the case of pronase or chymotrypsin, the optimum temperature for water dissolution is 40 ° C., and a suitable temperature range for deamidation is 40 ° C. or lower. 5 ° C or less, preferably 10 ° C, below the optimum temperature range for water digestion of commonly used enzymes
Below, more preferably below 15 ° C is suitable.

The modification of the protein by deamidation is more effective when the deamidation rate is higher than the hydrolyzation rate, and usually the (deamidation rate / hydrolysis rate) ratio is preferably 2 or more, more preferably 4 or more. (The deamidation rate and hydrolyzation rate will be described later) The properties of the protein deamidated as described above are improved such that some or all of the properties such as solubility, foaming property and emulsifying power are improved. It is speculated that this is because deamidation changes the charge of the protein and increases the hydrophobicity and flexibility of the protein molecule. Therefore, the modified protein is widely used for foods (eg, kneaded product material, confectionery material, emulsified food material, beverage, etc.), cosmetics, medical products, etc., which are required to have solubility, foaming property, emulsifying property, etc. be able to.

(Examples) The embodiments of the present invention will be described with reference to the following examples.

Example 1 (Preparation of ovalbumin) Kekwick and Cannan method (Biochem. J, 30 , 277-28)
0, (1936)) was used to obtain ovalbumin from fresh egg white by recrystallization 5 times in a sodium sulfate solution.

(Preparation of lysozyme) Alderton and Fevold method (J. Biol. Chem., 164 , 1
-5, (1946)) was used to obtain lysozyme by direct crystallization from fresh egg white and recrystallization five times.

(Preparation of α-casein and κ-casein) α-casein and κ-casein were obtained from fresh milk by the method of Zittle and Custer (J. Dairy Sci., 46 , 1183-1185. (1963)).

(Preparation of soybean 11S, 7S globulin) Soybean 11S, 7S globulin can be prepared by Than et al.
ol., 56 , 19-22, (1975)).

Trypsin is manufactured by Sigma, α-chymotrypsin is manufactured by Miles Laboratory, and pronase is manufactured by Kaken Kagaku Co., Ltd.
Papain (Merck) is the method of Kimmel and Smith (J. Bio
I. Chem., 207 , 515, (1954)) was used.

Direct measurement of detached ammonia during the protease reaction.

Conway and 0'Malley's Microdiffusion Method (Bi
ochem.J., 36 , 655-661, (1942)) was used. That is, 2 including methyl red and bromocresol green
1% of borate buffer solution was placed in the central tank of the micro-diffusion device and dissolved in 0.05M carbonate buffer solution (pH 10.0) at 0.5%
4 ml of the protein solution was put in the outer tank of the micro diffusion device, and 0.1 ml of 0.2% enzyme solution was separately put in the outer tank of the micro diffusion device so as not to mix with the protein solution. Immediately after closing the lid of the micro diffusion device and sealing it with gum arabic, the protein solution and the enzyme solution in the outer tank of the micro diffusion device were mixed and reacted at 30 ° C. for 36 hours to release ammonia gas.

Deamidation% = 100 x [(Titration value of original protein)-(Titration value of enzyme-treated protein]] / (Titration value of original protein) (Titration value is 1/200 N of sulfuric acid generated by sulfuric acid) (Titration value) The results of the above implementation are shown below.

(Direct measurement of detached ammonia) Table 1 shows the amount of detached ammonia by chymotrypsin and papain treatment at pH 10.

In the table, CONT is a 0.05 M carbonate buffer (pH 10.0) protein solution left at 30 ° C for 36 hours, CHYMO is chymotrypsin treatment, PAPAI
N is papain treatment, OA is ovalbumin, LYZ is lysozyme, 7S is soybean 7S globulin, 11S is soybean 11S globulin,
αCAS represents α-casein, and κCAS represents κ-casein.

It can be seen that the protein is deamidated by chymotrypsin and papain.

Example 2 Ovalbumin was prepared in the same manner as in Example 1, treated with papain in the same manner as in Example 1, and deamidated and hydrolyzed. Figure 1 shows the time course of deamidation and proteolysis of ovalbumin by papain at pH 10.0 and 20 ° C. Deamidation reaches a maximum in 2 hours. It can be seen that deamidation does not occur in the absence of papain.

Example 3 Ovalbumin was prepared in the same manner as in Example 1, treated with papain in the same manner as in Example 1, deamidated and hydrolyzed, and the effect of pH was examined. Fig. 2 shows ovalbumin 20
The effect of pH on deamidation and proteolysis by papain treatment at ℃ for 2 hours is shown. Deamidation starts around pH 6.5-7 and pH is 1
Compared with reaching the maximum around 0, hydrolyzation reaches the maximum at pH 7.
It can be seen that as the pH increases, it hardly happens.

Example 4 Ovalbumin was prepared in the same manner as in Example 1, treated with papain in the same manner as in Example 1, deamidated and hydrolyzed, and the effect of temperature was examined. Figure 3 shows ovalbumin
The effect of temperature on deamidation and proteolysis by papain treatment at pH 10.0 for 2 hours is shown. It can be seen that deamidation occurs at 50 ° C or lower compared to the optimum temperature for water dissolution exceeding 50 ° C.

Example 5 Ovalbumin, lysozyme and 7S soy globulin prepared in the same manner as in Example 1 were treated with papain in the same manner as in Example 1. Table 2 shows the rates of deamidation and enzymatic degradation of various proteins by papain at pH 10, 0.2 hours and 20 ° C.

It can be seen that 7S soy globulin is more easily deamidated than ovalbumin or lysozyme, but all proteins are deamidated in a low hydrolyzed state.

Example 6 Ovalbumin prepared in the same manner as in Example 1 was treated with pronase, chymotrypsin and trypsin for deamidation.
The effect of pH was investigated. Results are shown in FIG.

Chymotrypsin (Fig. 4A) has a pH of 8 or higher, pronase (Fig. 4B) has a pH of 7 or higher, and trypsin (Fig. 4C).
It can be seen that the deamidation occurs at pH 9 and above. Pronase and chymotrypsin showed deamidation similar to papain, and trypsin had lower deamidation activity than papain.

Example 7 Ovalbumin prepared in the same manner as in Example 1 was treated with pronase, chymotrypsin, and trypsin, and the effect of deamidation temperature was examined. Results are shown in FIG.

It can be seen that chymotrypsin (Fig. 5A), pronase (Fig. 5B) and trypsin (Fig. 5C) all undergo deamidation at 5 ° C or higher. The optimum temperature for water dissolution was 50 ℃ or higher, 40 ℃, or 40 ℃, which were higher than the temperature range suitable for co-deamidation of each enzyme.

Example 8 0.5% of chymotrypsin prepared in the same manner as in Example 1 was used.
% 7S soy globulin was added to 4 ml and reacted at pH 10 and 20 ° C. for 60 minutes to examine the effect of enzyme concentration on deamidation.

Results are shown in FIG. It can be seen that deamidation is proportional to enzyme (chymotrypsin) concentration.

Example 9 (Preparation of gluten) Wheat flour (manufactured by Torikoshi Flour Milling Co., Ltd.) was washed with water, dialyzed and lyophilized to prepare.

As a control, the same treatment was performed without using an enzyme.

(Identification of Ammonia) Deamidation was performed in a Samberg test tube (20 ° C. under vacuum). 5 ml of 0.5% gluten solution containing 0.25 mg of chymotrypsin (pH 10.0) was put into the main tube, and 2.0 ml of 0.1 N sulfuric acid was put into the side tube as an ammonia absorbent and kept warm to obtain the sulfuric acid.
1 ml was diluted with 9 ml deammonified water, and 100 μl thereof was applied to a Toyo Soda amino acid analyzer (HLC-805).

(Identification of Deamidation Rate) The method of Chibnall et al (Biochem.j., 68, 122, 1958) was used. That is, freeze-dried gluten was used with 2N hydrochloric acid.
Complete deamidation was carried out by hydrolyzing under vacuum at ℃ for 2 hours. The released ammonia was measured using the microdiffusion method.

(Identification of Hydrolysis Rate) 3 ml of the enzyme-treated gluten solution was mixed with an equal amount of 20% trichloroacetic acid (TCA), filtered, and the OD280 nm of the filtrate was measured. The hydrolyzation rate depends on whether the enzyme is treated or not.
It was expressed as the OD280nm ratio.

(Gel filtration) 40 mg of gluten treated with enzyme for 2 hours was dissolved in 10 ml of 10 mM Tris-glycine buffer (pH 8.3) containing 1% SDS containing 2-mercaptoethanol and left overnight. 12000 rpm
After centrifugation for 10 minutes, 5 ml of the supernatant was applied to a Toyopearl HW65F column (2 × 90 cm) equilibrated with 10 mM Tris-glycine buffer (pH 8.3) containing 0.1% SDS. Flow rate 12 ml / H
r, fraction volume 4 ml, OD280nm of each fraction
Was measured.

(Physical property identification) Solubility measurement: 0.05% gluten solution is adjusted to a predetermined pH with 0.1 N hydrochloric acid or caustic soda, and stirred,
After centrifugation at 12000 rpm for 10 minutes, the supernatant OD280nm was measured. OD of 0.05% gluten solution dissolved in 0.1N caustic soda
The ratio with 280 nm was taken as the solubility.

Measurement of foamability: 0.1% dissolved in 1 / 15M sodium carbonate buffer (pH 9.5) using the method of J. Food Sci., 48, 62, 1983.
Air was blown into 5 ml of the gluten solution to measure the electric conductivity of the formed foam.

Measurement of emulsifying property: 3 ml of 0.1% gluten solution dissolved in 1/15 M sodium carbonate buffer (pH 9.5) and 1 ml of corn oil were used at 20 ° C and 12000 rpm using Ultra Turrax (manufactured by Hansen).
Homogenize for 1 minute, take the bottom 50 μl emulsion,
It diluted with 5 ml of 0.1% SDS solution, and measured OD500nm.

Table 3 shows the amount of ammonia released with time from gluten decomposed by chymotrypsin at pH 10 and 20 ° C.

However, the treatment is gluten chymotrypsin treatment and the non-treatment is chymotrypsin treatment.

It can be seen that gluten is deamidated by chymotrypsin treatment.

Example 10 Gluten prepared in the same manner as in Example 9 was treated at pH 10 and 20 ° C.
After treatment with chymotrypsin, changes in deamidation and hydrolysis were examined with time. The results are shown in FIG.

The deamidation rate reaches about 25% and the hydrolyzation reaches about 14% in 2 hours. When it was examined by gel filtration whether or not it was derived from alkali, deamidation was not observed without the addition of chymotrypsin, but the TCA-soluble fraction was observed to the same extent as when chymotrypsin was added.

Example 11 Undenatured gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten obtained in the same manner as in Example 9 (the same treatment without chymotrypsin) were subjected to SDS gel filtration in the presence of 2-mercaptoethanol described in Example 9. .

The results are shown in Fig. 8. Fig. 8A shows unmodified gluten,
The peaks of glutenin and gliadin are shown, and FIG. 8B shows the SDS gel filtration pattern of chymotrypsin-treated gluten and FIG. 8C shows the chymotrypsin-untreated gluten. Whether treated with chymotrypsin in the alkaline region or not treated with chymotrypsin shows another peak after gliadin, which seems to be the dissociation of glutenin. 8B and 8C
From the results, it can be seen that under the conditions of Example 10 at pH 10 and 20 ° C., hydrolysis by chymotrypsin treatment hardly occurred, and the TCA-soluble fraction was dissociated by alkali treatment.

Example 12 The solubility of unmodified gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten prepared in the same manner as in Example 9 was examined.

The results are shown in Fig. 9. Solubility by deamidation (especially pH
5 or more) increases.

Example 13 The emulsifiability of unmodified gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten obtained in the same manner as in Example 9 was examined.

The results are shown in Fig. 10. It can be seen that the chymotrypsin-treated gluten has excellent emulsifying properties and is stable.

Example 14 Deamidated proteins (ovalbumin, lysozyme, 7S soy globulin, 11S soy globulin) were obtained in the same manner as in Example 1.

(Measurement of foaming property and foaming stability) 0.1M carbonate buffer solution (pH) in a glass column (2.4 × 30 cm)
The electric conductivity of bubbles formed by blowing air into 5 ml of a 0.1% protein solution dissolved in 9.5) at a flow rate of 90 cm / min was measured (J.
Fod Sci., 48, 623, 1983). The foaming stability was represented by the foam disappearance time.

(Emulsification measurement) Pearce and Kinsella (J.Agric.Food Chem., 26,716,
1983), 1 ml of corn oil and 3 ml of protein solution were homogenized in the same manner as in Example 1, and OD500nm was measured.

(Results) Figs. 11 and 12 show the foaming property and emulsifying property of proteins treated with chymotrypsin at pH 10 and 20 ° C.

11 and 12 show that deamidation improves the foaming property and emulsifying property of the protein. It is speculated that this is due to the increase in hydrophobicity of the protein molecule surface due to deamidation and the increase in flexibility of the protein.

(Effect) As described in detail above, the present invention enables deamidation of a protein enzymatically without causing cross-linking and with almost no hydrolyzation. (Emulsification, foamability, etc.) and functional changes of enzymes and the like are facilitated.

[Brief description of drawings]

FIG. 1 shows the time course of deamidation of ovalbumin by papain and hydrolysis at pH10. ● represents deamidation, ○ represents hydrolysis, and ■ represents deamidation in the absence of papain. FIG. 2 shows the effect of pH on deamidation and hydrolysis of ovalbumin by papain. ● represents deamidation, and ○ represents hydrolysis. FIG. 3 shows the influence of the temperature of deamidation and hydrolysis of ovalbumin by papain. ● represents deamidation, and ○ represents hydrolysis. FIG. 4 shows the effect of pH on deamidation and hydrolysis of ovalbumin by chymotrypsin, pronase and trypsin. A represents pronase, B represents chymotrypsin, C represents trypsin, ● represents deamidation, and ◯ represents hydrolysis. FIG. 5 shows the effect of deamidation of ovalbumin by chymotrypsin, pronase and trypsin and the temperature of hydrolysis. A represents pronase, B represents chymotrypsin, C represents trypsin, ● represents deamidation, and ◯ represents hydrolysis. FIG. 6 shows the effect of enzyme concentration on protein deamidation. Add chymotrypsin to 4 ml of 0.5% 7S globulin, pH 10,
The reaction was carried out at 20 ° C for 60 minutes. FIG. 7 shows the time course of amide deamidation and hydrolysis of gluten by chymotrypsin at pH 10.0 and 20 ° C. O indicates deamidation, Δ indicates hydrolyzation, O and Δ indicate chymotrypsin treatment, and ● and ▲ indicate chymotrypsin non-treatment. FIG. 8 shows SDS gel filtration patterns of native gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten in the presence of 2-mercaptoethanol. A represents unmodified gluten, B represents chymotrypsin-treated gluten, and C represents chymotrypsin-untreated gluten. Figure 9 shows the effect of pH on native gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten.
Represents the effect of. ○ represents unmodified gluten, ● represents chymotrypsin-treated gluten, and Δ represents chymotrypsin-untreated gluten. FIG. 10 shows the emulsifying properties of native gluten, chymotrypsin-treated gluten, and chymotrypsin-untreated gluten. ○ represents unmodified gluten, ● represents chymotrypsin-treated gluten, and Δ represents chymotrypsin-untreated gluten. FIG. 11 shows the foaming property of the deamidated protein. a represents ovalbumin, b represents lysozyme, c represents 7S soybean globulin, d represents 11S soybean globulin, ■ represents deamidated protein, and □ represents undenatured protein. Figure 12 shows the emulsifying properties of deamidated proteins. a represents ovalbumin, b represents lysozyme, c represents 7S soybean globulin, d represents 11S soybean globulin, ■ represents deamidated protein, and □ represents undenatured protein.

Claims (2)

(57) [Claims] 1. A pH higher than the optimum pH for hydrolysis of protease.
A method for producing a modified protein, which comprises deamidating a protein under conditions in which the rate of deamidation is higher than the rate of hydrolysis, using a protease in a region lower than the optimum temperature for hydrolysis of the region and protease. 2. The production method according to claim 1, wherein the (deamidation rate / hydrolysis rate) ratio is 2 or more.