JPH07115964A - Novel microorganisms, enzymes and protein hydrolysates - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] A novel microorganism that decomposes insoluble proteins such as okara and collagen, which are by-products in food products that are not used industrially, Provided are a protein hydrolyzate obtained by using a proteolytic enzyme, a novel aminopeptidase, and a method for producing the enzyme. [Structure] A novel mutant strain (FERM P-13458) obtained by mutating Bacillus alvei, and a protein hydrolyzate obtained by allowing a plurality of proteolytic enzymes obtained by culturing the microorganism to act on the protein. Degradation product, a protein hydrolyzate obtained by combining a plurality of proteolytic enzymes obtained by culturing the microorganism and an exo-type peptidase to act on a protein, and a novel aminopeptidase derived from Bacillus and the above Bacillus are cultured. A method for producing aminopeptidase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism, a protein hydrolyzate obtained by using a plurality of proteolytic enzymes contained in a culture thereof, a novel aminopeptidase and a method for producing the enzyme. More specifically, the present invention is a microorganism that produces an enzyme having excellent protein degrading ability, a Bacillus alvei bacterium is isolated, and a strain obtained by further mutation treatment (FERM P-13458) Also, by using this strain, underutilized proteins such as okara and collagen, which are by-products after food processing, are decomposed by using multiple proteolytic enzymes such as aminopeptidase contained in the culture of this bacterium. The present invention provides a protein hydrolyzate having a high free amino acid content, a novel aminopeptidase derived from the strain, and a method for producing the same. Furthermore, by decomposing soybean protein, gelatin, etc. using multiple proteolytic enzymes contained in the culture of this bacterium, and further by combining them with exo-type peptidases as necessary, the decomposition rate is high. The present invention provides a protein hydrolyzate having a high content of glutamic acid that exhibits umami or alanine, glycine, and serine that exhibits sweetness.

[0002]

2. Description of the Related Art At present, insoluble proteins such as okara and collagen, which are by-products in processed food products, are not industrially used. However, this insoluble protein is rich in useful amino acids such as glutamic acid. Also,
Since these are wastes after food processing and have become a problem as environmental pollutants, it is being studied to decompose them by using new microbial enzymes (Food industry and enzymes (edited by Eiji Ichishima) p. 107, Asakura Shoten). For example, collagenase, an enzyme that decomposes collagen, has been found in microorganisms such as Pseudomonas, Clostridium, and Streptomyces (Sefter, S. et al., The Enzymes, 3,649- 697 (197
1), Hanada, K. et al., Agric. Biol. Chem., 37, 1771-1781 (1
973), Endo, A. et al., J. Biochem., 102, 163-170 (1987)).
However, few enzymes that decompose insoluble collagen are known. Therefore, the present inventor, Obata et al. Screened microorganisms producing insoluble collagen degrading enzyme from the soil, selected the bacteria having the highest specific activity, and selected the bacteria as Bacillus al.
Identified as vei (Proceedings of the 1st Annual Meeting of the Japan Society for Fermentation Engineering, 1991, p.209).

Further, by using a plurality of proteolytic enzymes contained in the culture of the present bacterium, in addition to soybean protein and gelatin, proteins such as okara and collagen, which are by-products after food processing, are decomposed, and glutamic acid showing a delicious taste. We are also conducting research to provide protein hydrolysates with high free amino acid content such as alanine, glycine, and serine that exhibit sweetness. In general, Bacillus bacteria secrete and produce various degrading enzymes such as endo-type peptidases and amylases outside the cells, and these are widely used for detergents, foods, medicines, etc. (Knowledge of enzyme application, Komaki Toshiaki's work, Kou Shobo), has low activity of exo-type peptidases, especially aminopeptidases, and little is known about Bacillus stearothermophilus (Biochemistry Data Book I, p397, Tokyo Kagaku Dojin) as a bacterium producing the enzyme. . In addition, leucine aminopeptidase, which acts on amino acids except proline with a typical aminopeptidase, has been isolated and purified from each tissue of mammals and various enzymatic properties have been investigated, but its specific activity is low and unstable, Moreover, since only a small amount can be manufactured,
The production cost is high and it is only commercially available as a reagent for determining amino acids (Enzyme Handbook, p531, Asakura Shoten; Agric. Biol. Chem., Vol. 54, 2769 (1990)).

On the other hand, the production of the natural seasonings known so far has been mainly carried out through the decomposition of protein. Among what are called natural seasonings, degradable natural seasonings include acid-decomposable types and enzyme-decomposable types. Acid-decomposed seasonings can be obtained from soybean, wheat, and other vegetable proteins
Hydrolyzed Vegetable Protein (hereinafter sometimes abbreviated as HVP) and Hydrolyzed Animal Protein obtained from animal proteins such as gelatin and milk casein
(Hereinafter, it may be abbreviated as HAP.), And the composition of the amino acid, which is the main component, differs greatly depending on the raw material, and affects the taste and sweetness. However, generally, when obtaining HVP and HAP by acid hydrolysis, the reaction condition is 10
Although it takes 1 to 2 days at 0 ° C., a long-time reaction at high temperature consumes a large amount of energy. Furthermore, while hydrolysis of protein with an acid is simple, it has drawbacks such as generation of offensive odor, excessive decomposition of amino acids, and high salt content due to neutralization.

Therefore, in recent years, a production method utilizing a protease has been proposed in order to effectively utilize the flavor of the raw material. As enzyme-decomposed seasoning, a method for producing a decomposed product of egg white (JP-A-48-68773), a method for producing a seasoning liquid obtained from defatted soybean (JP-A-51-70852), and cheese whey as raw materials have been used so far. And the production method of corn gluten meal hydrolyzate (Japanese Patent Publication No. 2-295437).
No.) etc. have been reported.

By the way, even if the same substrate protein is used,
Since the cleavage site differs depending on the type of proteolytic enzyme, the taste of the degradation product differs. That is, the degree of free amino acid production, the molecular weight of the produced peptide, the amino acid composition,
The taste is different depending on the arrangement order and the like. For example, in the case of soybean protein, it is known that bitterness is generally easily generated when an enzyme having a strong endo-peptidase activity is acted on (Kengo Ishida et al., Food Journal, 28,524 (1976)). Generation of bitterness is the most difficult point in application of endo-type peptidases to food processing, and an enzymatic decomposition method that does not generate bitterness is desired.

[0007] In response to this problem, in studies with milk casein and soybean proteins, various amino acids were released by further treating the bitter peptides produced by endo-type peptidase treatment with exo-type peptidase derived from a microorganism, especially amino acids. Terminal leucine, phenylalanine, carboxyl terminal leucine, phenylalanine,
It is reported that valine etc. is released, bitterness is significantly reduced, and umami is increased (food industry and enzyme, edited by Eiji Ichishima,
p.102, Asakura Shoten).

As described above, amino acids, particularly those having a strong exo-type peptidase activity that liberates hydrophobic amino acids, are effective as the enzyme used for the production of seasonings, and therefore it is desired to provide such exo-type peptidase. . On the other hand, so far, insoluble proteins such as okara and collagen, which are by-products in processed foods, have not been industrially used. However, this insoluble protein is rich in useful amino acids such as glutamic acid. Also,
Since these are wastes after food processing and have become a problem as environmental pollutants, it is considered to decompose them by using new microbial enzymes (food industry and enzymes, edited by Eiji Ichishima, p. 107, Asakura Shoten). However, a method for efficiently decomposing these insoluble proteins has not been found.

[0009]

If the insoluble proteins such as okara and collagen can be decomposed under mild conditions using an enzyme, the present inventors can solve problems such as waste reduction and environmental pollution. At the same time, it was thought that it was possible to obtain a decomposed product containing a large amount of amino acids that exhibited umami and sweetness.

Therefore, at present, there is a long-felt demand for an enzymatic decomposition method for decomposing various insoluble proteins such as okara, collagen, etc., which contain a lot of amino acids exhibiting umami and sweetness under mild conditions. It is also strongly desired to obtain an aminopeptidase having high specific activity and stability from Bacillus bacterium, which is relatively easy to prepare a large amount of enzyme. However, the specific proteolytic enzymes produced by the Bacillus albei wild strain are not sufficient in specific activity and production amount, and there is room for improvement such as improvement in specific activity and productivity on an industrial scale. It is also desired to separate aminopeptidases having excellent specific activity and stability from a plurality of proteolytic enzymes derived from Bacillus albei, clarify their enzymatic properties, and establish a production method thereof. There is.

[0011]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to improve the enzyme specific activity or productivity of the Bacillus albay wild strain, which is the above-mentioned problem. We succeeded in isolating highly mutant strains, which solved the above problems and completed the present invention. Furthermore, as a result of repeated research on the mechanism by which multiple enzyme systems produced by this mutant strain highly degrade various proteins such as collagen, gelatin, okara, and soybean protein, multiple protein degradations obtained by culturing this bacterium The present inventors have completed the present invention by finding that a protein hydrolyzate having a good taste and flavor can be obtained by causing an enzyme to act on a protein. Furthermore, it was found that this bacterium has an extremely high aminopeptidase activity as compared to other Bacillus bacteria, and clarified its enzymatic properties, established a production method, and completed the present invention. Was completed.

That is, the present invention relates to Bacillus albay (B
A new mutant strain (FERM P
-13458) and a plurality of proteases obtained by culturing the microorganism (the bacterium produces a plurality of endo-type and exo-type peptidases in addition to the aminopeptidase). The present invention relates to a protein hydrolyzate, and a protein hydrolyzate obtained by combining a protein hydrolase with an exo-type peptidase to act on a protein. Furthermore, the present invention provides Bacillus albay (FERM P-13
458), and aminopeptidase having the following properties, (1) Action This enzyme hydrolyzes the peptide bond sequentially from the free amino terminus of the peptide to release an amino acid between pH 5 and 9. (2) Substrate specificity This enzyme uses many amino acids such as leucine, cysteine, arginine, and glutamic acid as substrates, but it acts particularly well on leucine. (3) Optimum pH and stable pH range The optimum pH is 6 to 7 when DL-leucine-p-nitroanilide is used as the substrate of this enzyme and a phosphate buffer is used as the buffer. Also, this enzyme is p
It is stable in the range of H4 to 10. (4) Optimum temperature of action When DL-leucine-p-nitroanilide is used as a substrate for this enzyme, the optimum temperature is 30 to 40 ° C. (5) Inhibitor The activity of this enzyme is completely inhibited by EDTA. (6) Molecular Weight This enzyme is a tetramer, and the molecular weight of each subunit is about 50,000 to about 60,000 according to SDS-polyacrylamide gel electrophoresis. In addition, the present invention provides a method for producing aminopeptidase, which comprises culturing Bacillus albei (FERM P-13458) in a medium to produce and accumulate the above aminopeptidase, and collecting this.

The novel microorganism of the present invention, namely Bacillus
The Albay mutant (FERM P-13458) is a cell wall peptidoglycan after being mutated with ethyl methanesulfonic acid (hereinafter abbreviated as EMS), which is a mutagen that has been often used conventionally. It was obtained as a strain resistant to vancomycin, which is known as an antibiotic that inhibits the biosynthesis of Escherichia coli. Mutagenesis can be obtained not only by EMS treatment as described in the following examples, but also by treatment with other chemical substances such as nitrosoguanidine and methylmethanesulfonic acid, irradiation with ultraviolet rays or treatment with a mutagen.
It is also possible to obtain by so-called natural mutation.

Many examples of vancomycin-resistant mutant strains have been known so far as mutant strains that highly produce extracellular enzymes such as amylase, endotype peptidase, and cellulase (Agric. Biol. Chem., Vol. 55). , 2387-2391 (199
1)).

In addition, in order to search for a new exo-type peptidase, the present inventors have found that a bacterium having a persistent peptide-degrading activity remaining in soy sauce, which is a hydrolyzate of soybean protein, is involved in soybean protein degradation. Screening was performed under the assumption that it has a highly active peptidase group. That is, glycylproline, glycylaspartic acid, and glycylglutamic acid (Agr. Biol. Chem., 38 (6), 1185-1194, 1 that have been reported as persistent peptides in soy sauce have been reported.
195-1202, 1974) was cultivated in a medium containing nitrogen as a main nitrogen source to obtain good growth bacteria. After that, the culture-washed cells of the obtained good-growing bacteria were added to the persistent peptide solution as a peptidase source, and the free amino acid of the reaction solution was subjected to thin-layer chromatography and ninhydrin coloration to obtain the persistent peptide-degrading activity. Strains with strong strains were selected. Furthermore, the cultured and washed bacterial cells of the bacteria thus selected are allowed to act on the Bacillus albay degradation solution of soybean protein, and in particular, a strain with a significantly improved amount of glutamic acid is selected, and the peptidase activity has been strong so far. Has discovered a novel peptidase from an unknown microorganism.

Next, the nutrient medium used for culturing the microorganism of the present invention is an ordinary medium comprising a carbon source, a nitrogen source, inorganic salts, cofactors, etc., and the carbon source is glucose, maltose, fructose, choux. Claus, lactose, starch, etc. may be used alone or in combination.
Yeast extract, ammonium sulfate, insoluble collagen, soybean protein, etc. as nitrogen sources, potassium chloride, magnesium sulfate, disodium hydrogen phosphate as inorganic salts,
Potassium dihydrogen phosphate or the like is used, and corn steep liquor or the like is used as a cofactor. Of these ingredients, maltose 0.2%, collagen or soy protein
0.1%, yeast extract 0.1%, ammonium sulfate 0.2%,
A medium containing 0.1% corn steep liquor is preferred.

The culture conditions of the microorganism for producing the aminopeptidase of the present invention may be ordinary aerobic culture conditions, pH 6.0-8.0, preferably pH 6.5-7.5, temperature 20-. 40 ° C., preferably 27-33 ° C., time 4-24
A time, preferably 13-18 hours, is suitable. Also,
The aminopeptidase of the present invention can be used as it is as a peptidase source in a culture, but when it is separated and collected by the method of the present invention, the specific activity of the enzyme is remarkably increased and the stability is also increased. The enzyme such as aminopeptidase of the present invention is not limited to those produced by the above-mentioned method, and recombinant DNA of Escherichia coli, Bacillus subtilis, yeast or the like into which the gene of the enzyme connected to the expression vector has been introduced. It can also be produced by law. Also,
It can also be produced from cells introduced into the chromosome of the mutated gene using homologous recombination. Enzymes produced by either method can be expected to have similar effects. Also, the method for purifying the enzyme is not limited.

The Bacillus albay mutant of the present invention (FERM
P-13458) produces various proteolytic enzymes in addition to aminopeptidase, so when the culture of this bacterium is allowed to act on proteins, a protein hydrolyzate with good taste and flavor can be obtained. In addition to the aminopeptidase produced by this bacterium, other exo-type peptidase may be supplementarily added. Now, as the peptidase used in the present invention, in addition to the above-mentioned ones, leucine aminopeptidase, aminopeptidase M, carboxypeptidase A, and Y, which are widely distributed in the plant, animal, and microbial kingdoms, amino acids sequentially from any end of the peptide chain Exo-type peptidases that release one by one can be optionally used. It is generally known that when a food protein such as milk casein or soybean protein is treated with an endo-type peptidase, a bitter peptide having leucine at the terminal is produced and the flavor is remarkably reduced. Therefore, by decomposing this bitter peptide with various exo-type peptidases, attempts have been made to improve the taste by releasing an amino acid from the amino terminus or the carboxyl terminus (Ishida et al., Food Journal, Volume 23, 524-530 (1976)).

In the present invention, various proteins are decomposed into peptides and even amino acids by a plurality of proteolytic enzymes contained in the culture broth of Bacillus albay mutants. At that time, if necessary, the amount of released amino acids is improved by allowing a combination of other exo-type peptidases other than the aminopeptidase produced by the present bacterium to act. Therefore, a significant improvement in taste can be expected by releasing glutamic acid, which originally has a large amount of soybean protein or collagen, and which has an umami taste, and amino acids such as glycine and alanine, which have a sweet taste.

The protein raw material used in the present invention is not particularly limited in preparation before the enzyme treatment, but it is preferably degreased with a surfactant or an organic solvent. Raw materials such as raw okara and insoluble collagen (Type I, manufactured by Sigma), which are by-produced during food processing and are not effectively used, obtained from the manufacturing process of tofu, and defatted soybean as a vegetable protein raw material. , Isolated soybean protein, and water-soluble gelatin as a raw material for animal protein are used alone or in combination of two or more kinds. Of course, protein raw materials other than the above may be used.

Next, the conditions for the protein decomposition reaction by the novel microorganism of the present invention will be described. For example, a protein such as raw materials such as okara, collagen, defatted soybean, isolated soybean protein and gelatin is added to the culture solution of the novel microorganism of the present invention. 10 to 6
The reaction may be performed at 0 ° C, preferably 30 to 50 ° C for 6 hours to 8 days, preferably 12 hours to 4 days. At this time, it is desirable to adjust the pH of the culture medium containing the proteolytic enzyme to 5 to 9 with a buffer solution. The enzyme may be either a purified one or a crude product.

Subsequently, the present inventors conducted extensive research to further increase the degradation rate, and as the exo-type peptidase screened as described above, for example, in addition to the aminopeptidase derived from the Bacillus albay mutant strain of the present invention,
Candida lipolytica (ATCC 8661), Geotricham candidum (FERM P-13734), Candida tropicalis (FERM P-13732),
Philobassium capsulighenum (ATCC221
79), Candida Guilimondi (ATCC 905)
8), Debaryomyces Hanseni (FERM P-1
3735), Torrlopsis versatilis (FERM)
P-13733), Serratia Marcesans (ATC
C14226), Lactobacillus plantarum (FE
RM P-13737), Pediococcus pentosaceus (FERM P-13736), Lactobacillus casei subspecies casei (FERM P-
13738), a fraction containing a peptidase activity or a protease M (trade name, Amano Pharmaceutical Co., Ltd.)
0.1 to 10% is added to the reaction solution for 12 hours to 8 hours.
The hydrolyzate is obtained by reacting at 10 to 60 ° C., preferably 30 to 50 ° C., pH 3 to 8 for a day, preferably 1 to 6 days. During the reaction, salt or ethanol may be added for the purpose of preserving.

After completion of the reaction, insoluble matter such as unreacted raw material protein may be removed by a conventional separation method such as centrifugation or filtration. For each proteolytic solution produced, amino acid analysis, ninhydrin quantification, total nitrogen content, free peptide content by formal nitrogen quantification, free amino acid content, degradation rate measurement, and weight degradation rate measurement by weight comparison before and after enzyme treatment, etc. Analysis of.

[0024]

[Examples] The following examples show the isolation of the Bacillus albei high-resolution mutant strain of the present invention, the enzymatic chemical properties of the enzyme derived from the microorganism, the method for producing the same, and the method for producing various protein hydrolysates. Etc. will be shown. Example 1 (Isolation of Bacillus albei Mutant and Properties of Enzyme Derived from the Microorganism) First, a carbon source in the medium as a production medium for various proteolytic enzymes such as collagenase of Bacillus alvei DC-1 wild strain. As a result of examining the effects of nitrogen source and cofactors, ammonium sulfate 0.2%, maltose 0.2
%, Yeast extract 0.1%, and corn steep liquor 0.1% were found to be effective. Since then, maltose 0.2
%, Collagen (Type I, manufactured by Sigma) 0.1%, corn steep liquor 0.1%, yeast extract 0.1%, potassium chloride 0.05%, ammonium sulfate 0.2%, magnesium sulfate 0.05% , A medium containing 0.1% of disodium hydrogen phosphate and 0.1% of potassium dihydrogen phosphate was used as a production medium.

Broth medium (meat extract 10 g, peptone 10
g, 5 g of sodium chloride, 10 ml of pH 7.2) were inoculated with the above microorganism, precultured at 30 ° C. for 24 hours and inoculated into the production medium as a seed culture (1%), and cultured at 30 ° C. for 12 hours. . Then, EMS was added to the above culture solution so as to be 0, 1.0, 2.0, 3.0%, and shaken at 30 ° C. for 30 minutes. In addition, it was previously confirmed that the resistance of this strain to vancomycin was 20 μg / ml or less. Therefore, 100% solution of EMS-treated culture medium diluted to the same dilution ratio (10 ^-5 ) was applied to the medium coated on the plate medium so that vancomycin was 25 μg / ml.
μl was applied. As a result, the number of colonies was as shown in Table 1, and the colonies were designated as vancomycin resistant mutants.

[0026]

[Table 1] Table 1 Number of colonies and survival rate of wild strain of Bacillus albay EMS treated ──────────────────────────── ─ EMS concentration 3% 2% 1% 0% Number of colonies 287 1,706 2,536 2,648 Survival rate (%) 10.8 64.4 95.8 100 ──────────────────────── ────

Several vancomycin-resistant strains obtained by screening after EMS treatment were treated with collagen (type I).
After culturing as a carbon source, the ammonium sulfate precipitation fraction (0-80%) of the culture supernatant was prepared as a crude enzyme. The specific activity of this crude enzyme fraction for collagen and dried soybean insoluble protein was determined by the Mandle method (Enzyme Data Sheet Library (Seikagaku Corporation)
Code No. 100370). Specifically, 14
To 0 ml of 1M calcium chloride, 10.5 ml of 1M trishydroxymethylaminomethane was added, and the pH was adjusted to 7 with hydrochloric acid.
Adjust to 4 and add 10 ml of the mixture to 90 ml of 0.85%
Added to sodium chloride. Next, 25 mg of collagen or dried soybean insoluble protein was taken into a test tube, 4.9 ml of buffer solution was added, and preincubated at 37 ° C. for 5 minutes.

To this solution was added 0.1 ml of enzyme solution, and 3
Incubated for 18 hours at 7 ° C. Furthermore, 0.5 ml of the reaction solution was added to a mixed solution of 1 ml of a 5% pyridine solution and 1 ml of a 1% ninhydrin solution, and the mixture was boiled for 25 minutes. After cooling, 3 ml of distilled water was added and the absorbance at 570 nm was measured. The enzyme activity was calculated by multiplying the absorbance at 570 nm by 1,000 and dividing it by the amount of enzyme (mg) in 0.5 ml of ninhydrin-pyridine mixed solution as a Mandle unit.

Among several resistant strains, Bacillus alvei VR, which is a resistant strain showing increased activity in the neutral and acidic regions with respect to the substrate dry soybean insoluble protein
300 were isolated as high resolution mutants. This bacterium has been deposited as FERM P-13458 at the Institute of Biotechnology, Institute of Biotechnology, AIST. The mycological properties of this bacterium are shown below.

(A) Morphological properties Cell size and shape: 0.6 to 0.65 × 1.1 to 1.2 μ
Presence / absence of polymorphism in bacillus cell of m: None Motility: None Spore presence: Spore formation Gram stainability: Gram positive

(B) Physiological properties (1) Reduction of nitrate: negative (2) MR test: positive (3) VP test: positive (4) Indole production: positive (5) Hydrogen sulfide production: negative (6) ) Hydrolysis of starch, casein and gelatin: Positive (7) Pigment formation: The colonies are yellow, but no pigment formation outside the cells is observed. (8) Urease: Positive

(9) Oxidase: Positive (10) Catalase: Positive (11) Range of growth pH: Grow at pH 6-9. It shows good growth at pH 7-8. It shows good growth at a temperature of 30 ° C. It does not grow at 40 ° C. (12) Attitude toward oxygen: aerobic (13) OF test: produces acid aerobically and anaerobically than glucose by the method of Hugh and Leifson. (14) Generation of acid and gas from saccharides: Acid and gas are generated from D-glucose. Not produced from L-arabinose, D-xylose or D-mannose. (15) Generation of dihydroxyacetone: Positive (c) DNA base composition: GC content is 45% (d) Separation source: soil

[0033] The above-mentioned mycological properties are described in Bergey's Manual of D
The bacterium was identified as Bacillus albay by searching the Bacillus taxonomy of eterminative Bacteriology. Next, this mutant strain was cultured using collagen (type I) or insoluble soybean protein as a carbon source, and an ammonium sulfate precipitation fraction (0 to 80%) of the culture supernatant was prepared as a crude enzyme. PH of this crude enzyme for dry insoluble soybean protein degradation
As a result of examining the effect of the above, two activity peaks were obtained at pH 5.5 and pH 7.5. Both activity peaks were endopeptidase activities. The effects of various protease inhibitors on both enzyme activities were investigated. As a result, as shown in Table 2, it was shown that both enzymes were one of metalloproteases because they were highly sensitive to sodium sulfide.

[0034]

[Table 2] Table 2 Effect of protease inhibitors on dry insoluble soybean degrading activity ─────────────────────────────── Inhibitor pH Specific activity (Mandle U / mg) Relative activity (%) ────────────────────────────── Untreated 7.5 32 1 × 10 ⁵ 100 5.5 295 × 10 ⁵ 100 N-ethylmale 7.5 290 × 10 ⁵ 90 Imide 5.5 234 × 10 ⁵ 79 Sodium sulfide 7.5 112 × 10 ⁵ 35 5.5 134 × 10 ⁵ 45 6-Amino-n 7.5 353 × 10 ⁵ 110-Capron Acid 5.5 245 × 10 ⁵ 83 ───────────────────────────────

Further, the influence of various metal ions on both enzyme activities was examined. As a result, as shown in Table 3, the activity was exhibited in the presence of calcium ion and magnesium ion, but the activity was markedly inhibited by zinc ion, iron ion, copper ion and the like. In addition, manganese ion is p
The activity of the fraction having H7.5 activity was inhibited, but the pH was 5.
The fraction with activity at 5 was not affected.

[0036]

[Table 3] Table 3 Effect of various metal ions on dry insoluble soybean decomposition activity ────────────────────────────── Metal ion pH Specific activity (Mandle U / mg) Relative activity (%) ────────────────────────────── Untreated 7.5 1337 × 10 ⁵ 100 Ca 1111 83 Mg 1327 99 Mn 521 39 Zn 65 5 Fe 404 30 Cu 0 0 ─────────────────────────────── None Treatment 5.5 368 100 Ca 368 100 Mg 416 113 Mn 400 109 Zn 182 49 Fe 211 57 Cu 45 12 ──────────────────────────── ──

Example 2 (Degradation of various proteins by Bacillus albay mutant) Bacillus albay mutant (FERM described in Example 1 above)
P-13458) was used to measure the degradation activity of various proteins. First of all, the collagenase activity in the culture supernatant obtained by changing the carbon source during the culture was measured by Mandle
The measurement results were shown in Table 4. As is clear from the table, high collagenase activity was induced by dry insoluble soybean powder and collagen.

[0038]

[Table 4] Table 4 Effect of Collagenase-Induced Substrate during Culture of Bacillus albei Mutant ──────────────────────── Induced Substrate Specific Activity (Mandle U / mg) ──────────────────────── Collagen 42.8 × 10 ⁵ Dry insoluble soybean powder 58.1 × 10 ⁵ Okara 23.1 × 10 ⁵ ──── ────────────────────

Next, the activity of degrading each substrate by the enzyme in the culture supernatant after culturing with various induction substrates was measured by the ninhydrin method. The enzymatic reaction was carried out under the conditions of pH 7.4, 18 hours, and 37 ° C. As a substrate for the enzyme reaction, collagen, dry insoluble soybean powder, and okara were used. The results are shown in Table 5. As is clear from the table, high activity was induced and various insoluble proteins were decomposed by using each inducing substrate.

[0040]

[Table 5] Table 5 Degradation activity of various substrates by the culture supernatant after culturing with various induction substrates (collagen / relative activity with collagen being 100) ────────────────── ───────────── Inducing substrate Enzyme substrate (%) Collagen Dry insoluble soybean protein Okara ──────────────────────── ────── Collagen 100 133 72 Dry insoluble soybean protein 136 86 57 Okara 72 NT 156 ────────────────────────────── ─

Furthermore, various substrates were decomposed using the ammonium sulfate precipitation fraction of the culture supernatant after induction culture with collagen.
Then, the amount of free peptide and free amino acid was quantified by back counting from the ninhydrin quantification. The results are shown in Table 6.

[0042]

[Table 6] Table 6 Free peptide and free amino acid amounts after various substrate decomposition reactions ───────────────────────────────── ── Enzyme substrate Collagen Dry insoluble soybean protein Okara 18hr 66hr 18hr 66hr 18hr 66hr ─────────────────────────────────── Specific activity 25.5 57.6 35.1 112.0 28.4 24.6 (Mandle U / mg) Peptide 2,182 5,116 3,205 10,440 3,175 3,722 (nmol / l) Amino acid 194 336 310 1,468 149 83 (nmol / l) ──────────── ──────────────────────

The results of amino acid analysis are shown in Table 7.
Although the value varies depending on each substrate protein, various amino acids such as glutamic acid, alanine, glycine, methionine, lysine, tyrosine, and threonine were released in a large amount.

[0044]

[Table 7] Table 7 Amount of free amino acid (nmol) by amino acid analysis after various substrate decomposition reactions ────────────────────────────── ──── Amino Acid Collagen Dry Insoluble Soy Protein Okara 18hr 66hr 18hr 66hr 18hr 66hr ────────────────────────────────── Asp-4.1 1.9 17.9 1.1 2.9 Thr 1.2 6.2 10.3 94.9 1.1-Ser 0.7 2.3 4.9 53.9 1.1-Glu-3.2 16.9 99.8 0.5 29.4 Gly 2.3 13.1 13.4 54.3-4.6 Ala 9.5 5.0 7.6 82.6-11.3 Val-5.4 1.5 46.0 15.5-Met 2.8 15.6 1.8 178.1 16.3-Lys 89.5 126.7-266.0 86.4 31.4 Arg 79.3 34.1 117.4 39.3-0.8 Cys 2.4 1.2-91.5-2.7 Tyr-93.5-162.1--──────────────── ─────────────────

Further, the weight decomposition rate of each substrate was measured using the culture supernatant or ammonium sulfate fraction. The enzyme reaction was carried out at 37 ° C. for 18 hours at pH 7.4 and the optimum acidic pH of each substrate (collagen: pH 4.5, dry insoluble soybean protein: pH 4.5,
Okara: pH 5.5). The protein concentration used in the reaction was 26.1 μg / ml in the supernatant and 642 μg / ml in the ammonium sulfate fraction. The results are shown in Table 8. In addition, the acidity in the table (supernatant)
Indicates the reaction in the supernatant of the optimum acidic pH.

[0046]

[Table 8] Table 8 Weight decomposition rate of each substrate by culture supernatant or ammonium sulfate fraction ─────────────────────────────── ── Substrate Weight decomposition rate (%) (w / v) pH 7.4 (supernatant) pH 7.4 (ammonium sulfate) Acidic (supernatant) ────────────────────── ────────── Collagen 10.0 34.7 2.4 Dry insoluble soybean powder NT 34.7 NT Okara 23.6 NT 8.2 ───────────────────────── ───────

Example 3 (Degradation test of gelatin by Bacillus albei mutant) Water-soluble gelatin HA-J (trade name, manufactured by Nitta Gelatin Co., Ltd.)
Maltose 0.2% in 4 g, soy protein isolate Adipron SU
0.1%, corn steep liquor 0.1%, yeast extract 0.1
%, Potassium chloride 0.05%, ammonium sulfate 0.2%,
Magnesium sulfate 0.05%, disodium hydrogen phosphate 0.
Medium containing 1% and 0.1% potassium dihydrogen phosphate (pH
7.5 ml of Bacillus albay mutant strain (FERM P-13458) cultured at 30 ° C for 18 hours at 30 ° C (300 units as protease activity), 12.5 ml of 10 mM calcium phosphate buffer (pH 7.0) And ethanol (3%) were added, and the mixture was reacted aseptically at 40 ° C. for 3 days. Further, after adjusting the pH to 5, Protease M (trade name, manufactured by Amano Pharmaceutical Co., Ltd.) was added as an exo-type peptidase to 80
mg was added, and the mixture was decomposed at 40 ° C. for 6 days. Decomposed 10
After heating at 0 ° C. for 10 minutes to inactivate the enzyme, centrifugation was performed to obtain a decomposition filtrate. Table 9 shows the analysis values of this decomposition filtrate.
Shown in.

As is apparent from the table, the decomposition rate shown as formol nitrogen / total nitrogen, the amount of glutamic acid exhibiting umami, the amount of alanine having sweetness, and the amount of glycine were significantly increased as compared with the case where no enzyme was treated. . Therefore, the obtained decomposition filtrate can be used as it is as an excellent amino acid seasoning liquid.

[0049]

[Table 9] Table 9 ───────────────────────────── Analysis item Analysis value (enzyme-free section in parentheses) ── ─────────────────────────── Total nitrogen (g / dl) 2.81 (2.92) Formol nitrogen (g / dl) 1.32 (0.11) ) Formol nitrogen / total nitrogen (%) 47 (3.6) Glutamic acid (g / dl) 0.69 (0.012) Glutamic acid / total nitrogen 0.25 (0.0039) Glycine (g / dl) 1.12 (0.01) Glycine / total nitrogen 0.40 (0.003) ) Alanine (g / dl) 1.27 (0.003) Alanine / total nitrogen 0.45 (0.001) ──────────────────────────────

Example 4 (Decomposition test of soybean protein by Bacillus albay mutant) Isolated soybean protein Adipron SU (trade name, manufactured by Ajinomoto Co., Inc.)
Maltose 0.2%, isolated soy protein adipron SU 0.1%, corn steep liquor in 16 ml (0.8 g) of 5% suspension
0.1%, yeast extract 0.1%, potassium chloride 0.05%, ammonium sulfate 0.2%, magnesium sulfate 0.05%, disodium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%.
2.5 ml (100 units as protease activity) of a Bacillus albay mutant strain (FERM P-13458) cultured in a medium (pH 7.0) containing 1% at 30 ° C. for 18 hours, 10 mM calcium phosphate buffer (pH 7.0) 17.
5 ml and ethanol (3%) were added, and the mixture was aseptically reacted at 40 ° C. for 3 days. After adjusting the pH to 5, 80 mg of Protease M (trade name, manufactured by Amano Pharmaceutical Co., Ltd.) was added as an exo-type peptidase, and decomposed at 40 ° C. for 6 days. Then, the decomposition product is heated at 100 ° C. for 10 minutes,
After deactivating the enzyme, centrifugation was performed to obtain a decomposition filtrate. Table 10 shows the analytical values of this decomposition filtrate.

As is clear from the table, the raw material protein was almost completely decomposed, as estimated from the decomposition rate shown as formol nitrogen / total nitrogen and the amount of glutamic acid exhibiting umami, as compared with the case of no enzyme treatment. Therefore, the obtained decomposition filtrate can be used as it is as an excellent amino acid seasoning liquid.

[0052]

[Table 10] Table 10 ───────────────────────────── Analysis item Analysis value (enzyme-free section in parentheses) ── ─────────────────────────── Total nitrogen (g / dl) 0.56 (0.53) Formol nitrogen (g / dl) 0.42 (0.063) ) Formol nitrogen / total nitrogen (%) 75 (11) Glutamic acid (g / dl) 0.58 (0.012) Glutamic acid / total nitrogen 1.04 (0.023) ───────────────── ─────────────

Example 5 (Screening for Peptidase-Producing Bacteria) As a primary screening, yeast 22 possessed by Ajinomoto Co., Inc.
0 strains, 366 strains of bacteria, and 22 strains of lactic acid bacteria are each grown by culturing in a medium containing a main nitrogen source of a persistent peptide such as glycylproline, glycylaspartic acid and glycylglutamic acid shown in Tables 11 to 13. Good bacteria were selected as primary selection bacteria.

[0054]

[Table 11] Table 11 Medium composition used for culturing yeast ───────────────────────── Medium composition Concentration (%) ───── ──────────────────── glucose _{1.0 KH 2 PO 4 0.5 MgSO 4} · 7H 2 O 0.05 glycyl-proline 0.3 glycyl aspartate 0.3 Glycylglutamic acid 0.3 (pH 6.0) ─────────────────────────

[0055]

[Table 12] Table 12 Medium composition used for culture of bacteria ───────────────────────── Medium composition Concentration (%) ───── ──────────────────── glucose _{0.5 K 2 HPO 4 0.2 MgSO 4} · 7H 2 O 0.1 yeast extract 0.025 glycyl proline 0. 3 Glycyl aspartic acid 0.3 Glycyl glutamic acid 0.3 (pH 7.0) ──────────────────────────

[0056]

[Table 13] Table 13 Medium composition used for culturing lactic acid bacteria ───────────────────────── Medium composition concentration (%) ───── ──────────────────── glucose 2.0 K ₂ HPO ₄ 0.2 sodium acetate _{0.5 MgSO 4 · 7H 2 O 0.02} MnSO 4 · 4H 2 O 0.005 Yeast extract 0.025 Glycylproline 0.3 Glycyl aspartic acid 0.3 Glycylglutamic acid 0.3 (pH 6.5) ────────────────── ────────

Further, the secondary screening was carried out as follows. Cultivation of primary selection bacteria as a source of exo-type peptidase in 0.1% final concentration glycylproline, glycylaspartic acid, and glycylglutamic acid solutions dissolved in 100 mM potassium hydrogen phosphate buffer (pH 7.0) Washed cells were added and reacted at 30 ° C. for 24 hours. After that, the centrifugal supernatant of the reaction solution was developed by thin layer chromatography using a developing solvent of ethyl acetate: ethanol: acetic acid: water = 5: 2: 1: 2, and then ninhydrin was developed to detect those dipeptide degrading activities. The strong strain was used as the secondary selection strain.

Next, the final screening was carried out as follows. Soy protein isolate Adipron SU (trade name, manufactured by Ajinomoto Co., Inc.) 5% suspension 16 ml (0.8g), maltose 0.2%, soy protein isolate Adipron SU 0.1%, corn steep liquor 0.1% Contains yeast extract 0.1%, potassium chloride 0.05%, ammonium sulfate 0.2%, magnesium sulfate 0.05%, disodium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1% 2.5 ml (100 units as protease activity) of a culture broth of Bacillus albay mutant (FERM P-13458) cultivated in a medium (pH 7.0) at 30 ° C. for 16 hours, 10 mM potassium phosphate buffer (pH 7. 0) (1.5 ml) and ethanol (3%) were added, and the mixture was aseptically reacted at 40 ° C. for 3 days. In addition, 10% of washed and washed cells of the secondary selection bacteria
The mixture was added and decomposed at 40 ° C. for 6 days, and the total amount of nitrogen, formal nitrogen and glutamic acid in the centrifuged supernatant was quantified, and a strain with a significantly improved amount of glutamic acid was used as the third selection bacterium. The above screening results are shown in Table 14,1.
5 shows. In Table 15, the glutamic acid / total nitrogen value shows a significant difference if there is a difference of 0.1 or more as compared with the value without addition.

[0059]

[Table 14] Table 14 Number of strains selected by screening ───────────────────────────────────── Bacterial species Total number Primary selection number Secondary selection number Third selection number ───────────────────────────────── ─── Yeast 220 134 34 7 Bacteria 366 27 15 1 Lactic acid bacteria 22 5 5 3 ──────────────────────────────── ────

[0060]

[Table 15] Table 15 Glutamic acid / total nitrogen values when the third selection bacteria were used ─────────────────────────────── ───── Strain name Glutamic acid / Total nitrogen ─────────────────────────────────── Additive-free 0. 67 Candida lipolytica 0.84 Geotricham candidam 0.77 Candida tropicalis 0.83 Philobassium capsulighenum 0.78 Candida guilimondi 0.71 Debarriomyces hansenii 0.79 Torrlopsis versatiris 0.86 Serratia Marcesans 0.85 Lactobacillus plantarum 0.78 Pediococcus pentosaceus 0.79 Lactobacillus casei subspecies casei 0.80 ────────────────── ──────────────────

Example 6 (Test for Degrading Soybean Protein by Combining Yeast-Derived Exo-Peptidase with Bacillus albei Mutant) As in Example 5, isolated soybean protein Adipron SU (trade name, manufactured by Ajinomoto Co., Inc.) was used. A Bacillus albay digestion solution was prepared, and then Candida lipolytica (ATCC866
1), Geotricham Candi Dam (FERM P-1)
3734), Candida tropicalis (FERM P-
13732), Firobacillium capslighenum (ATCC22179), Candida guilimondi (ATCC9058), Debaryomyces hanseni (FERM P-13735), and 10% of culture-washed cells of Torulopsis versatilis (FERM P-13733). Then, after decomposing at 40 ° C. for 6 days, the total amount of nitrogen and formol nitrogen in the centrifuged supernatant and the amino acid analysis were performed. The analysis values are shown in Tables 16 to 23.

[0062]

[Table 16] Table 16 When using Candida lipolytica (ATCC 8661) ─────────────────────────────────── Analysis Item Analytical value (enzyme-free area in parentheses) ──────────────────────────────────── Total nitrogen ( g / dl) 0.50 (0.45) Formol nitrogen (g / dl) 0.33 (0.059) Formol nitrogen / total nitrogen (%) 66 (13) Glutamic acid (g / dl) 0 .42 (0.0009) Glutamic acid / total nitrogen 0.84 (0.020) ─────────────────────────────── ────

[0063]

[Table 17] Table 17 When using Geotricham Candidam (FERM P-13734) ────────────────────────────────── ── Analysis item Analysis value (enzyme-free area in parentheses) ──────────────────────────────────── ─ Total nitrogen (g / dl) 0.49 (0.45) Formol nitrogen (g / dl) 0.33 (0.059) Formol nitrogen / total nitrogen (%) 67 (13) Glutamic acid (g / Dl) 0.38 (0.009) Glutamic acid / total nitrogen 0.77 (0.020) ──────────────────────────── ────────

[0064]

[Table 18] Table 18 When using Candida tropicalis (FERM P-13732) ──────────────────────────────── ──── Analysis item Analysis value (enzyme-free area in parentheses) ────────────────────────────────── ─── Total nitrogen (g / dl) 0.51 (0.45) Formol nitrogen (g / dl) 0.33 (0.059) Formol nitrogen / total nitrogen (%) 65 (13) Glutamic acid (G / dl) 0.42 (0.009) Glutamic acid / total nitrogen 0.83 (0.020) ────────────────────────── ───────────

[0065]

[Table 19] Table 19 When using Philobassium capsulighenum (ATCC22179) ────────────────────────────────── ─── Analysis item Analysis value (enzyme-free area in parentheses) ─────────────────────────────────── ── Total nitrogen (g / dl) 0.48 (0.45) Formol nitrogen (g / dl) 0.32 (0.059) Formol nitrogen / total nitrogen (%) 67 (13) Glutamic acid ( g / dl) 0.37 (0.009) Glutamic acid / total nitrogen 0.78 (0.020) ────────────────────────── ──────────

[0066]

[Table 20] Table 20 When using Candida guilimondi (ATCC 9058) ───────────────────────────────────── Analysis item Analysis value (enzyme-free area in parentheses) ──────────────────────────────────── Total Nitrogen (g / dl) 0.50 (0.45) Formol Nitrogen (g / dl) 0.33 (0.059) Formol Nitrogen / Total Nitrogen (%) 66 (13) Glutamic Acid (g / dl) ) 0.41 (0.009) Glutamic acid / total nitrogen 0.81 (0.020) ───────────────────────────── ───────

[0067]

[Table 21] Table 21 When using Debaryomyces Hanseni (FERM P-13735) ────────────────────────────────── ─── Analysis item Analysis value (enzyme-free area in parentheses) ─────────────────────────────────── ── Total nitrogen (g / dl) 0.46 (0.45) Formol nitrogen (g / dl) 0.30 (0.059) Formol nitrogen / total nitrogen (%) 65 (13) Glutamic acid ( g / dl) 0.36 (0.009) Glutamic acid / total nitrogen 0.79 (0.020) ────────────────────────── ──────────

[0068]

[Table 22] Table 22 When using Torulopsis persatilis (FERM P-13733) ────────────────────────────────── ─── Analysis item Analysis value (enzyme-free area in parentheses) ─────────────────────────────────── ── Total nitrogen (g / dl) 0.48 (0.45) Formol nitrogen (g / dl) 0.32 (0.059) Formol nitrogen / total nitrogen (%) 67 (13) Glutamic acid ( g / dl) 0.41 (0.009) Glutamic acid / total nitrogen 0.86 (0.020) ────────────────────────── ──────────

[0069]

[Table 23]

The numbers in the table are exo-type peptidase-free, Candida lipolytica (ATCC 8661),
Geotricham Candi Dam (FERM P-137
34), Candida tropicalis (FERM P-1
3732), Philobasidium capsuligenum (ATCC22179), Candida guilimondi (ATCC9058), Debaryomyces hansenii (FERM P-13735), and Torropsis versatiris (FERM P-13733) -derived exo-type peptidases.

Example 7 (Decomposition test of soybean protein by combination of Bacillus albei mutant and exo-type peptidase derived from bacteria) As in Example 5, decomposition of soybean protein Adipron SU (trade name, manufactured by Ajinomoto Co., Inc.) A Bacillus albaea decomposition solution was prepared and then Serratia marcescens (ATCC142
10% of the cultured and washed bacterial cells of 26) was added, the mixture was decomposed at 40 ° C. for 6 days, and then the total amount of nitrogen and formol nitrogen in the centrifuged supernatant was quantified and the amino acid was analyzed. The analysis values are shown in Table 2.
4,25.

[0072]

[Table 24] Table 24 ──────────────────────────────────── Analysis item Analysis value (in parentheses Enzyme-free zone) ──────────────────────────────────── Total nitrogen (g / dl) 0. 49 (0.45) Formol nitrogen (g / dl) 0.32 (0.059) Formol nitrogen / total nitrogen (%) 65 (13) Glutamic acid (g / dl) 0.42 (0.009) ) Glutamic acid / total nitrogen 0.85 (0.020) ─────────────────────────────────────

[0073]

[Table 25]

The numbers in the table correspond to the exo-type peptidase-free group and the exo-type peptidase-derived group derived from Serratia marcesans.

Example 8 (Decomposition test of soybean protein by combination of Bacillus albei mutant strain and exo-type peptidase derived from lactic acid bacterium) As in Example 5, isolated soybean protein Adipron SU (trade name, manufactured by Ajinomoto Co., Inc.) was used. A Bacillus albay digestion solution is prepared, and then Lactobacillus plantarum (FERM
P-13737), Pediococcus pentosaceus (FERM P-13736), and Lactobacillus casei (FERM P-13738) 10% each of the washed culture cells were added, and the mixture was decomposed at 40 ° C for 6 days, and then centrifuged. The total nitrogen and formol nitrogen in the supernatant were quantified and amino acids were analyzed. The analyzed values are shown in Tables 26 to 29.

[0076]

[Table 26] Table 26 When using Lactobacillus plantarum (FERM P-13737) ─────────────────────────────── ───── Analysis item Analysis value (enzyme-free section in parentheses) ───────────────────────────────── ──── Total Nitrogen (g / dl) 0.48 (0.45) Formol Nitrogen (g / dl) 0.31 (0.059) Formol Nitrogen / Total Nitrogen (%) 65 (13) Glutamic acid (g / dl) 0.38 (0.0009) Glutamic acid / total nitrogen 0.78 (0.020) ───────────────────────── ─────────────

[0077]

[Table 27] Table 27 When using Pediococcus pentosaceus (FERM P-13736) ───────────────────────────────── ───── Analysis item Analysis value (enzyme-free area in parentheses) ───────────────────────────────── ──── Total Nitrogen (g / dl) 0.49 (0.45) Formol Nitrogen (g / dl) 0.32 (0.059) Formol Nitrogen / Total Nitrogen (%) 65 (13) Glutamic acid (g / dl) 0.39 (0.009) Glutamic acid / total nitrogen 0.79 (0.020) ───────────────────────── ─────────────

[0078]

[Table 28] Table 28 Using Lactobacillus casei Subspecies Casei (FERM P- 13738) ──────────────────────────── ───────── Analysis item Analysis value (enzyme-free area in parentheses) ───────────────────────────── ──────── Total nitrogen (g / dl) 0.48 (0.45) Formol nitrogen (g / dl) 0.30 (0.059) Formol nitrogen / total nitrogen (%) 63 (13) Glutamic acid (g / dl) 0.39 (0.009) Glutamic acid / total nitrogen 0.80 (0.020) ───────────────────── ───────────────

[0079]

[Table 29]

The numbers in the table refer to Lactobacillus plantarum without addition of exo-type peptidase (FERM P
-13737), Pediococcus pentosaceus (FERM P-13736), Lactobacillus casei subspecies casei (FERM P-13)
738) -derived exo-peptidase.

As is clear from the above Examples 6 to 8,
By combining Bacillus albei and the exo-type peptidase derived from the strains in the table, the raw material protein was almost completely degraded. In addition, any strain can further improve the decomposition rate and amino acid release rate by examining the culture conditions and medium composition. Therefore, the obtained decomposition filtrate can be used as it is as an excellent amino acid seasoning liquid.

Example 9 Maltose 0.2%, Collagen (Type I, Sigma) 0.1%, Corn Steep Liquor 0.1%, Yeast Extract 0.1%, Potassium Chloride 0.05%, Ammonium Sulfate 0% .
Bacillus albey (FERM P-13) in a medium (pH 7.0) containing 2%, magnesium sulfate 0.05%, disodium hydrogen phosphate 0.1% and potassium dihydrogen phosphate 0.1%.
458) was incubated at 30 ° C. for 16 hours. Then, the culture was harvested by centrifugation, and the cells were suspended in 50 mM Tris-HCl buffer (pH 7.5). The cell suspension was ultrasonically disrupted and then subjected to ultracentrifugation (105,000 xg) to obtain a crude extract fraction. When ammonium sulfate precipitation was carried out using this crude extract fraction as a starting material, the desired enzyme activity was present in the 50 to 70% precipitate fraction.

The obtained fraction was subjected to anion exchange chromatography (DEAE-Toyopearl 650M), and the adsorbed activity peak was collected. Then, two types of gel filtration chromatography (FPLC Superose 6 and FPLC Phenyl-Suporos) were performed.
The target enzyme fraction was prepared by fractionating in step e). After passing through each of these separation steps, the specific activity of the aminopeptidase of the present invention was 0.6 unit / mg in the starting crude extract fraction, but after the treatment by gel filtration chromatography in the final step, This was a dramatic increase to 58.5 units / mg. As a result, this peptidase has a significantly higher specific activity than previously reported aminopeptidases from various mammalian tissues, and is a new aminopeptidase found in Bacillus bacteria that has been rarely reported so far. It has been found.

Next, various properties of the aminopeptidase of the present invention were examined using the obtained enzyme fraction at the final stage. The properties of the novel aminopeptidase of the present invention are as follows. (1) Action The enzyme is an aminopeptidase that hydrolyzes the peptide bond sequentially from the free amino terminus of the peptide between pH 5 and 9 to release the amino acid. (2) Substrate specificity Table 30 shows the substrate specificity when a low-molecular synthetic substrate is used. The substrate used was DL-Leucine-p-nitroanilide (Leu-pNA
Abbreviated. ), DL-Cysteine-p-nitroanilide (abbreviated as Cys-pNA), DL-Arginine-p-nitroanilide (abbreviated as Arg-pNA) and DL-Glutamic acid-p-nitroanilide (Glu-pN).
Abbreviated as A. ).

[0085]

[Table 30]

The enzyme is considered to be essentially an aminopeptidase because it does not act on a substrate having a free α-amino group protected. Further, it is a leucine aminopeptidase because it has the highest decomposition activity when leucine has an amino terminal. (3) Optimum pH of action The optimum pH is 6 to 7 when Leu-pNA is used as a typical substrate of this enzyme and a phosphate buffer is used as a buffer.

(4) pH stability It is stable at pH 4 to 10. (5) Optimum temperature of action The optimum temperature when Leu-pNA is used as a typical substrate of this enzyme is 30 to 40 ° C. (6) Temperature stability After incubating at pH 6.5 for 15 minutes or 45 minutes at each temperature, the residual activity was measured using Leu-pNA as a substrate, and the results shown in Table 31 were obtained. It is fairly stable near the optimum temperature, but deactivates rapidly at 50 ° C or higher.

[0088]

[Table 31] Table 31 Thermal stability at each temperature ─────────────────────────── Temperature (℃) Residual activity (%) 15 After 45 minutes ─────────────────────────── 35 100 45 50 50 55 27 27 55 55 55 36 ────────── ──────────────────

(7) Effect of metal ion The present enzyme was added to Leu in the presence of 1 mM of each metal ion.
-Relative activity was measured using pNA as the substrate. The results are shown in Table 32. This enzyme was strongly activated by Fe ²⁺ , but its activity was almost lost by Cu ²⁺ and Sn ²⁺ .

[0090]

[Table 32] Table 32 Effect of metal ion on enzyme activity ─────────────────────── Metal ion (1 mM) Relative activity (%) ─── ──────────────────── Additive-free 100 FeCl ₂ 378 CaCl ₂ 103 MgCl ₂ 100 MnCl ₂ 89 ZnCl ₂ 57 CoCl ₂ 51 CuCl ₂ 27 SnCl ₂ 0 ─── ────────────────────

(8) Inhibitor In the presence of each protease inhibitor of the present enzyme, Leu
-Relative activity was measured using pNA as the substrate. The results are shown in Table 33. This enzyme is a kind of metalloprotease because its activity is completely inhibited by a chelating agent such as EDTA.

[0092]

[Table 33] Table 33 Effect of protease inhibitors on activity ────────────────────────── Inhibitor (1 mM) Relative activity ( %) ────────────────────────── Additive-free 100 Dithiothreitol 100 Benzamidine 38 EDTA 0 ──────────── ───────────────

(9) Molecular weight The present enzyme is a tetramer, and the molecular weight of each subunit is about 50,000 to about 60,000 as measured by SDS-polyacrylamide gel electrophoresis. (10) Titer measurement method and titer display Using the above-mentioned Leu-pNA as a substrate, the amount of paranitroaniline released by the enzymatic action is colorimetrically determined at 410 nm. That is, 50 mM potassium phosphate buffer (pH
Leu-pNA was dissolved in 6.5) to 1 mM,
To 600 μl thereof, 20 μl of enzyme solution and 20 μl of the same buffer solution
Were mixed and reacted at 37 ° C. for 20 minutes, 200 μl of 40% acetic acid was added to stop the reaction, and
The absorbance at 0 nm is measured. By this method, the amount of enzyme that produces 1 μmol of paranitroaniline per minute is 1
Defined as a unit. According to this method, the titer of this enzyme is about 60 units / mg.

[0094]

INDUSTRIAL APPLICABILITY According to the present invention, a novel protein which has a low utility value and is difficult to be decomposed by conventional enzymes can be produced under neutral mild conditions to produce a plurality of enzymes having excellent proteolytic activity. A Bacillus albei mutant Bacillus albei mutant (FERM P-13458) is used in a culture medium, and if necessary, it is combined with an exo-type peptidase to decompose it, resulting in a high decomposition rate and containing glutamic acid, alanine, glycine, etc. It is possible to obtain a protein hydrolyzate having a high rate and excellent taste. At the same time, it is possible to solve the problems of disposal of processing by-products and the conventional problems associated with the acid decomposition method. Furthermore, the novel aminopeptidase obtained by the present invention is efficiently obtained from a Bacillus bacterium that has not been reported previously, and has a significantly higher specific activity and stability than those known. Therefore, the use of this enzyme is not limited to reagents, and it is widely used not only in food processing but also in medicine and other fields.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area // (C12N 1/20 C12R 1:07) (C12N 9/48 C12R 1:07) (72) Inventor Hideki Okamura 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Food Research Institute

Claims (8)

[Claims] 1. A new mutant strain obtained by mutating Bacillus alvei (FERM P-13458, Institute of Biotechnology, Institute of Biotechnology). 2. An aminopeptidase derived from Bacillus albay (FERM P-13458) having the following properties. (1) Action This enzyme hydrolyzes the peptide bond sequentially from the free amino terminus of the peptide between pH 5 and 9 to release the amino acid. (2) Substrate specificity This enzyme acts particularly on leucine. (3) Optimum pH and stable pH range The optimum pH is 6 to 7 when DL-leucine-p-nitroanilide is used as the substrate of this enzyme and a phosphate buffer is used as the buffer. Also, this enzyme is p
It is stable in the range of H4 to 10. (4) Optimum temperature of action When DL-leucine-p-nitroanilide is used as a substrate for this enzyme, the optimum temperature is 30 to 40 ° C. (5) Inhibitor The activity of this enzyme is completely inhibited by EDTA. (6) Molecular Weight This enzyme is a tetramer, and the molecular weight of each subunit is about 50,000 to about 60,000 according to SDS-polyacrylamide gel electrophoresis. 3. The aminopepti according to claim 2, wherein Bacillus albay (FERM P-13458) is cultured in a medium to produce and accumulate the aminopeptidase according to claim 2, and the aminopeptidase is collected. Method for producing tase. 4. A protein hydrolyzate obtained by reacting a protein with a proteolytic enzyme obtained by culturing the microorganism according to claim 1. 5. The protein hydrolyzate according to claim 4, wherein the protein is at least one selected from collagen, okara, defatted soybean, soybean protein isolate and gelatin. 6. A protein hydrolyzate obtained by combining a protein degrading enzyme according to claim 4 and an exo-type peptidase to act on a protein. 7. The exo-type peptidase is the aminopeptidase or Candida lipolytica (A) according to claim 2.
TCC8661), Geotricham Candi Dam (FE
RM P-13734), Candida tropicalis (F
ERM P-13732), Firobacidium capsuligenum (ATCC22179), Candida guilimondi (ATCC9058), Debaryomyces hansenii (FERM P-13735), Torlopsis
Versatiris (FERM P-13733), Serratia marcessans (ATCC 14226), Lactobacillus plantarum (FERM P-13737), Pediococcus pentosaceus (FERM P-13)
736) and Lactobacillus casei subspecies casei (FERM P-13738). 8. The protein hydrolyzate according to claim 6, wherein the protein is at least one selected from collagen, okara, defatted soybean, soybean protein isolate and gelatin.