JP3388264B2 - Aminopeptidase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an aminopeptidase and a method for producing the same. Aminopeptidase is
It is an enzyme that acts on the N-terminal side of proteins and peptides and hydrolyzes it in amino acid units, and is utilized in the production of seasoning solutions containing peptides and amino acids as the main components. Further, some of them have an action of removing / reducing the peptidic bitterness. The present invention provides this aminopeptidase and an efficient method for producing the same, and the enzyme is expected to be used in the food field.

[0002]

2. Description of the Related Art In recent years, materials containing hydrolysates of proteins such as seasonings and casein hydrolysates having a calcium absorption promoting action have been developed and used in various food fields. However, the material containing these protein hydrolysates,
Some have a bitter taste due to a bitter peptide rich in hydrophobic amino acids generated when a protein is decomposed by an enzyme or the like. Therefore, it is difficult to commercialize the material.

If this bitterness peptide can be decomposed with a proteolytic enzyme or the like, the bitterness can be reduced or eliminated. However, among proteolytic enzymes, few aminopeptidases that decompose amino acids from the N-terminus are commercially available, and their practical application is also limited.

By the way, it has been known that Maitake ( Grifol a frondosa ), a kind of edible mushroom, has an aminopeptidase activity, but since it is unstable and easily deactivated, it has been separated and purified. There wasn't. Therefore, not only the genetic information of the maitake-derived enzyme but also the characteristics of this enzyme have not been clarified.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to the aminopeptidase derived from Maitake, which has enzymatic properties,
It is an object to provide genetic information and a method for efficiently producing the enzyme.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above-mentioned object, the present inventors refined the extract obtained from the raw material Maitake by hydrophobic chromatography to obtain the target enzyme. It was found that the aminopeptidase having a higher purity can be obtained by efficiently separating and purifying the amino acid, and then by applying various purification means, and thus the present invention has been accomplished.

That is, according to the present invention of claim 1,
Aminopeptidase derived from bamboo and having the following properties
Is. (A) Optimum pH and stable pH range: Optimum pH of this enzyme
Is 8.5, pH 6.0 to 10.5 (45 ° C., 1 hour
Stable). (B) Optimum temperature: The optimum temperature of this enzyme is 65 ° C. (C) Thermostability: The enzyme is stable at 55 ° C or lower. (D) Substrate specificity: the enzyme is leucine and phenyl
Acts on Lanin. (E) Molecular weight: The molecular weight of this enzyme is 30 kDa (SDS-
Polyacrylamide gel electrophoresis). (F) Effect of inhibitor: This enzyme is EDTA and phenane.
It is inactivated by trollin and inhibited by bestatin. (G) Effect of metal ion: The enzyme deactivated by EDTA is
Mn²⁺, Zn²⁺, Cu ²⁺And Co²⁺Activated by the addition of
Turn into. (H) N-terminal amino acid sequence: as shown in SEQ ID NO: 1 in the sequence listing
Has an array.

In the present invention according to claim 2, the extract obtained by extracting from the fruiting body of Maitake mushroom with water or a buffer solution is subjected to a hydrophobic chromatography using a resin having a hydrophobic functional group as a carrier and separated. The method for producing aminopeptidase according to claim 1, wherein the active fraction is collected.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The aminopeptidase of the present invention is derived from Maitake mushroom, and succeeded in obtaining the present enzyme as follows.

First, the fruiting body of Pleurotus cornucopiae is suspended in water or a buffer solution to obtain an extract. Here, as the fruiting body of Maitake, in addition to the fruiting body taken out from Maitake, processed products such as freeze-dried powder thereof can be used. Extraction of the enzyme is carried out by suspending the fruit body of Pleurotus cornucopiae using water or a buffer solution. Various buffers can be used and are not particularly limited. Suitable buffers include, for example, Tris-HC
1 buffer solution (pH 7.5) can be mentioned.

After the fruiting body of Maitake is suspended in water or a buffer solution, the extract (filtrate) is subjected to an appropriate operation such as suction filtration.
To get This is the crude enzyme solution. Then, the extract is repeatedly subjected to operations such as ammonium sulfate precipitation and centrifugation to remove insoluble matter and obtain a supernatant. Instead of the above extraction, it is also possible to use a homogenizer or the like to extract the enzyme while mashing the fruit body of Maitake.

In this way, an insoluble matter-removed extract was obtained, and in order to separate and purify the enzyme from this extract, this was subjected to hydrophobic chromatography using a resin having a hydrophobic functional group as a carrier. Perform fractionation. As the resin having a hydrophobic functional group serving as a carrier, a resin having a hydrophobic functional group such as a phenyl group, a butyl group, an octyl group, an ethyl group or the like can be used. Among these, for example, Phenyl-TOYOPEARL65 having a phenyl group.
0M (manufactured by Tosoh Corporation) and the like are preferable. The fractionation operation was carried out by adding the extract solution to a column packed with a resin equilibrated with a buffer solution to remove non-adsorbing proteins, and then adjusting the ammonium sulfate concentration in the buffer solution to adjust the salt concentration. It can be carried out by gradually lowering to elute the protein adsorbed on the resin.

Since the aminopeptidase of the present invention has a particularly strong affinity with a resin having a hydrophobic functional group, it elutes at a salt concentration of 0M. The specific activity of aminopeptidase obtained as a result of this hydrophobic chromatography was increased by about 20 times as compared with the crude enzyme (see Table 1), and it can be seen that purification is progressing. From this, the inventors of the present invention fractionated the extract obtained by extracting the fruit body of Maitake mushroom with water or a buffer solution by hydrophobic chromatography using a resin having a hydrophobic functional group as a carrier. It was judged that aminopeptidase could be produced by collecting the active fraction.

At the stage where the operation of the hydrophobic chromatography is completed, the enzyme is sufficiently purified and aminopeptidase having a high specific activity is obtained as described above. After that, it is possible to obtain a further purified enzyme by performing a usual purification means such as gel filtration chromatography and anion exchange chromatography.

Gel filtration chromatography can be performed by a conventional method. There is no limitation on the resin packed in the column, and any resin for gel filtration can be used. For example, commercially available products such as Sephadex G25 are suitable. Further, the solvent for elution is not particularly limited, and for example, 3- (N-morpholino) propanesulfonic acid (MOPS, pH 7.5) which is a buffer can be preferably used. Gel filtration chromatography
It is an effective means for purification of aminopeptidase. That is, in the fraction obtained by the hydrophobic chromatography,
Contaminant proteins exist, but this one shows affinity for gel filtration resins, so when gel filtration chromatography is performed, the target enzyme elutes first,
As some contaminating proteins and salts elute later,
Purification of the enzyme is performed.

Next, anion exchange chromatography will be explained. In this purification operation, an ordinary anion exchange resin is used as a stationary phase, and Mono-Q and the like are suitable resins. The column packed with the resin is equilibrated with a buffer solution (for example, MOPS, etc.) in advance, and the adsorbed protein is eluted by increasing the sodium chloride concentration in this buffer with a linear concentration gradient, and the eluate is fractionated. To do. By this operation, the target enzyme can be further purified, and the specific activity is remarkably improved.

A highly purified aminopeptidase is obtained through the above-described series of chromatographic methods. Then, the N-terminal amino acid sequence of this enzyme can be determined by a conventional method. The maitake-derived aminopeptidase obtained by the present invention has the following properties.

(A) Optimum pH and stable pH range: The optimum pH of this enzyme is 8.5, and the pH is 6.0 to 10.5.
It is stable in the range of (45 ° C., 1 hour). (B) Optimum temperature: The optimum temperature of this enzyme is 65 ° C. (C) Thermostability: The enzyme is stable at 55 ° C or lower. (D) Substrate specificity: This enzyme acts on leucine and phenylalanine. (E) Molecular weight: The molecular weight of this enzyme is 30 kDa (SDS-
Polyacrylamide gel electrophoresis).

(F) Effect of inhibitor: The enzyme is EDTA or
And inactivated by phenanthroline, and by bestatin
Be hindered. (G) Effect of metal ion: The enzyme deactivated by EDTA is
Mn²⁺, Zn²⁺, Cu ²⁺And Co²⁺Activated by the addition of
Turn into. (H) N-terminal amino acid sequence: protein sequencer H
For PG1005 type (made by Hewlett-Packard)
The N-terminal amino acid sequence determined by
1 is as described.

[0020]

EXAMPLES The present invention will now be described in more detail by way of examples, which should not be construed as limiting the invention. Example 1 (Separation and purification of aminopeptidase) 400 g of freeze-dried powder of fruiting body of Maitake mushroom in ice water was used.
The cells were suspended in 16 L of mM Tris-HCl buffer (pH 7.5) and immediately filtered by suction. The obtained filtrate was used as a crude enzyme solution.

Next, ammonium sulfate was added to the filtrate so that it would be 70% saturated, and the mixture was left standing in ice water for 3 hours. The resulting precipitate was collected by centrifugation (6800 rpm, 4 ° C., 30 minutes), and 25 times containing 0.6 M ammonium sulfate in an amount 5 times the weight of the precipitate.
mM MOPS buffer (pH 7.5) (hereinafter referred to as buffer A
Is abbreviated. ) Dissolved in. Centrifugation (18000 rpm, 4 ° C., 30 minutes) was repeated twice on this solution to remove insoluble matter, and a supernatant was obtained.

Example 2 (Chromatographic purification of aminopeptidase) (1) Hydrophobic chromatography The supernatant obtained in Example 1 above was equilibrated with buffer A in advance, Phenyl-TOYOPEARL.
It was subjected to a hydrophobic chromatography using 650M (manufactured by Tosoh Corporation) as a carrier and fractionated and purified. That is, the column diameter is 2.5 cm and the column length is 10 cm.
The supernatant 425 obtained in Example 1 was added to an OPEARL packed column.
After adding non-adsorbed proteins at a flow rate of 3 mL / min, the ammonium sulfate concentration in the buffer solution was changed from 0.6 M to 0.
The adsorbed protein was eluted by reducing with a linear concentration gradient to M, and the eluate was fractionated by 9 mL. Then, the aminopeptidase activity of each fractionated fraction was measured, and the active fractions were collected. The activity of aminopeptidase is
50 μL of each fraction was added to 2 mM leucine-p-nitroanilide dissolved in 50 mM MOPS (pH 7.5),
After reacting at 45 ° C. for 5 minutes, the reaction was stopped with 1.7 M acetic acid, and the absorbance at 405 nm of the reaction solution was detected and measured.

(2) Gel filtration chromatography The active fraction obtained by the above chromatography was subjected to gel filtration chromatography. That is, Sephadex G having a column diameter of 2.5 cm and a column length of 6.1 cm.
A 25 packed column was loaded with 25 mM MOPS buffer (pH 7.
5) (hereinafter abbreviated as buffer solution B) using 1 m flow rate
Elution was performed at L / min. The eluate was fractionated by 1 mL.
The aminopeptidase activity of each of the fractionated fractions was measured, and the active fraction containing no ammonium sulfate was collected.

(3) Anion exchange chromatography The fraction obtained by the above chromatography was subjected to anion exchange chromatography (FPLC; Mono-Q HR5).
/ 5 column, manufactured by Pharmacia). That is, Mono-Q having a column diameter of 5 mm and a column length of 5 cm.
The enzyme solution collected by the gel filtration chromatography was added to the packed column. The column was equilibrated with buffer B in advance and the concentration of sodium chloride in the buffer was adjusted to 0.
The adsorbed protein was eluted by increasing the linear concentration gradient from mM to 100 mM, and the eluate was fractionated by 1 mL. The aminopeptidase activity of each of the fractionated fractions was measured, and the active fractions were collected.

Table 1 shows the amount of enzyme solution, the amount of total protein, the total activity, the specific activity per protein, the yield and the degree of purification in each of the purification steps (1) to (3).

[0026]

[Table 1] Table 1 (Purification of aminopeptidase from Maitake)

From the results shown in Table 1, the following can be seen. The aminopeptidase activity in the fractions obtained by hydrophobic chromatography was the crude enzyme (after ammonium sulfate precipitation).
Purification has advanced up to about 20 times compared to.

It is also clear that the aminopeptidase can be further purified by gel filtration chromatography. The column carrier used for gel filtration chromatography has no affinity for aminopeptidase, but has affinity for contaminants, so that not only desalting but also sufficient purification effect can be obtained by the chromatography.

Further, by performing anion exchange chromatography, it can be highly purified up to about 3.5 times that after gel filtration chromatography. In this way, by going through each chromatographic method,
It is clear that the specific activity increases as the enzyme purification proceeds.

Example 3 (Optimum pH of aminopeptidase)
And stable pH range) (1) Optimum pH pH was adjusted from 4.0 to 11.0 at 0.5 intervals (p.
H4.0-5.5 acetate buffer; pH 5.0-7.0
MES buffer; pH 6.5-8.0 MOPS buffer;
pH 7.5-9.0 TAPS buffer solution; pH 8.5-1
0.0 CHES buffer; pH 9.5 to 11.0 CA
Example 2 using 2 mM leucine-p-nitroanilide (Leu-pNA), each using PS buffer.
The activity of the purified aminopeptidase obtained in (3) was 10%.
It was made to react for minutes and measured. The results are shown in Fig. 1. The horizontal axis in the figure represents the reaction pH, and the vertical axis represents the relative activity (%) when the maximum activity value is 100. Also, ▽ is acetate buffer, ● is MES buffer, ▲ is MOPS buffer, and ◆ is T.
APS buffer, □ is CHES buffer, ○ is CAPS
Each buffer is shown. From FIG. 1, it is clear that the optimum pH of this aminopeptidase is 8.5.

(2) Stable pH range The purified aminopeptidase solution obtained in the above Example 2 (3) was adjusted from pH 4.0 to 13.0 at 0.5 intervals (pH 4.0 to 5.5 acetic acid). Buffer; pH 5.0-
7.0 MES buffer; pH 6.5-7.5 MOPS
Buffer; pH 7.5-8.5 TAPS buffer; pH
8.5-10.0 CHES buffer; pH 9.5-1
1.0 CAPS buffer solution; pH 11.0 to 13.0 piperidine buffer solution were used respectively), and the mixture was incubated at 45 ° C. for 1 hour and then left in ice water for several minutes. Then, the aminopeptidase activity of each pH-treated solution was measured. The results are shown in Figure 2. The horizontal axis in the figure represents the treatment pH, and the vertical axis represents p.
The relative activity (%) when the activity in H8.5 (MOPS buffer) is set to 100 is shown. ▽ is acetate buffer, ● is MES buffer, ▲ is MOPS buffer, and ◆ is T.
APS buffer, □ is CHES buffer, ○ is CAPS
Buffers and Δ indicate piperidine buffers, respectively. The stable pH range of this aminopeptidase is about 6.0-1.
It is clear that it is 0.5.

Example 4 (Optimum temperature and thermal stability of aminopeptidase) (1) Optimum temperature The purified aminopeptidase activity obtained in the above Example 2 (3) was measured under various temperature conditions (0, 20, 30). , 40, 50,
55, 60, 65, 70, 75, 80, 90, 100
It was made to react for 10 minutes under (° C) and measured. The results are shown in Fig. 3. In the figure, the horizontal axis represents the reaction temperature (° C), and the vertical axis represents the relative activity (%) when the maximum activity value is 100. Figure 3
From this, it is clear that the optimum temperature of this aminopeptidase is 65 ° C.

(2) Thermostability The purified aminopeptidase activity obtained in the above-mentioned Example 2 (3) was measured under various temperature conditions (0, 20, 30, 40, 50,
55, 60, 65, 70, 75, 80, 90, 100
After incubating for 30 minutes at (° C.), it was left for several minutes in ice water. Then, the aminopeptidase activity of each temperature-treated solution was measured. The results are shown in Fig. 4. In the figure, the horizontal axis represents the treatment temperature (° C), and the vertical axis represents the relative activity (%) when the maximum activity value is 100. As is clear from FIG.
The aminopeptidase is stable up to 55 ° C.

Example 5 (Substrate specificity of aminopeptidase) p-nitroanilide derivatives of various amino acids (Leu-
pNA, Phe-pNA, Val-pNA, Ala-p
NA, Lys-pNA and Pro-pNA) as substrates, the activity of the purified aminopeptidase obtained in Example 2 (3) above was measured, and the substrate specificity of this aminopeptidase was examined. Is shown in Table 2.

[0035]

Table 2 Table 2 (Substrate specificity of aminopeptidase for N-terminal amino acid)

From Table 2, it is clear that the aminopeptidase of the present invention shows high reactivity with leucine (Leu) and phenylalanine (Phe).

Example 6 (SDS- of aminopeptidase)
Molecular weight measurement by polyacrylamide gel electrophoresis) SDS-polyacrylamide gel electrophoresis (SDS-P)
AGE method (Laemmli, UK, Nature, 227, 680 (197
The molecular weight of aminopeptidase was measured in (0)). That is, 5% sodium dodecyl sulfate (SDS) equivalent to the purified aminopeptidase obtained in Example 2 (3) above,
A sample treatment solution containing 5% mercaptoethanol and 50% glycerin was mixed and incubated at 100 ° C for 2 minutes. Next, SDS-polyacrylamide gel (separation gel 12.5) on a slab electrophoresis apparatus (manufactured by ATTO).
%, Concentrated gel 3%), add 30 μL of this solution,
It was run at mA. 10 kDa P as a molecular weight marker
rotation Ladder (Gibco BRL L
If Technologies was used), and electrophoresis was performed at the same time.

After the completion of the electrophoresis, it was stained with Coomassie Brilliant Blue and the molecular weight of the present aminopeptidase was measured from the relative migration distance between the present aminopeptidase and the molecular weight marker. Results are shown in FIG. The left lane is a molecular weight marker, and the right lane is the aminopeptidase. As is clear from FIG. 5, this aminopeptidase showed a single band and had a molecular weight of about 30 kDa.

Example 7 (Effect of Inhibitor) The present aminopeptidase was treated with various inhibitors, and its effect was investigated. That is, EDTA (1 mM and 10 m
M), phenanthroline (0.1 mM and 10 m
M) and bestatin (0.05 mM and 0.5 mM) each inhibitor treated with the purified aminopeptidase obtained in the above Example 2 (3) (4 ° C., 30 minutes) and 2 mM of the treated solution. 50 including leucine-p-nitroanilide
Aminopeptidase activity was measured in mM TAPS buffer (pH 8.5). The results are shown in Table 3.

[0040]

[Table 3] Table 3 (Influence of inhibitors)

As is clear from Table 3, this aminopeptidase is inactivated by the chelating agents EDTA and phenanthroline and inhibited by bestatin.

Example 8 (Influence of Metal Ion) The activity when a metal salt was added to the aminopeptidase inactivated by EDTA was measured. That is, 50 μm of the purified aminopeptidase obtained in Example 2 (3) above
50 μL of 0.5 M TAPS buffer (pH 8.5) for L
And 50 μL of 0.1 mM EDTA were added, and the mixture was left in ice water for 1 hour. Then, 0.1 mM metal salt (M
gCl ₂ , MnCl ₂ , ZnCl ₂ , CuCl ₂ , CaCl
₂ , BaCl ₂ , CoCl ₂ , FeCl ₂ ) was added and the mixture was allowed to stand in ice water for 1 hour. To this solution, 1 mM leucine-
200 μL of p-nitroanilide was added (final concentration 2 m
M), aminopeptidase activity was measured. The fourth result
Shown in the table.

[0043]

[Table 4] Table 4 (Influence of metal ions)

From the results shown in Table 4, the activity of aminopeptidase inactivated by EDTA was confirmed to be MnCl ₂ , ZnC.
It is apparent that it is activated by the addition of l ₂ , CuCl ₂ and CoCl ₂ .

Example 9 (Determination of N-terminal amino acid sequence) The N-terminal amino acid sequence of the purified aminopeptidase obtained in Example 2 (3) was used as a protein sequencer H.
It was determined using PG1005 type (manufactured by Hewlett Packard). The determined N-terminal amino acid sequence is as shown in SEQ ID NO: 1 in the sequence listing, and is 65 from the N-terminal.
The amino acid sequence of the residue could be deciphered.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, a purified aminopeptidase derived from Maitake and a method for producing the same are provided. The aminopeptidase according to the present invention has an action of removing / reducing the peptidic bitterness, and is highly safe because it is derived from edible maitake mushroom which has been conventionally eaten. Therefore, this enzyme is expected to be widely used in seasonings and other food fields.

[Sequence Listing] SEQUENCE LISTING <110> Director of National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries <120> Aminopeptidase and its production method <130> P111081K <160> 1 <210> 1 <211> 65 <212> PRL <213> Grifola frondosa <400> 1 Arg Thr Gln Asp Asn Ala Pro Trp Gly Leu Asn Arg Ile Ser Gln Gly 1 5 10 15 Pro Pro Leu Ala Asn Gln Asn Pro Phe Ala Thr Asn Phe Val Tyr Thr 20 25 30 Tyr Asn Asn Asn Asn Pro Gly Tyr Gly Val Asp Ile Tyr Val Met Asp Thr 35 40 45 Gly Val Leu Thr Ser His Thr Glu Phe Phe Gly Arg Ala Thr Asp Gly 50 55 60 Tyr 65

[Brief description of drawings]

FIG. 1 is a graph showing the relative activity of aminopeptidase activity at each pH.

FIG. 2 shows aminopeptidase at 45 ° C. under each pH condition.
2 is a graph showing the relative activity of the enzyme activity measured after 1 hour of incubation at.

FIG. 3 is a graph showing the relative activity of aminopeptidase activity at each temperature.

FIG. 4 is a graph showing the relative activity of the enzyme activity measured after incubating aminopeptidase for 30 minutes at each temperature.

FIG. 5 is an electrophoretic photograph of aminopeptidase.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/48 C12R 1: 645) (C12N 15/09 ZNA C12R 1: 645) (72) Inventor Motomitsu Kitaoka Tsukuba, Ibaraki Prefecture 1-34-16 City Kannondai Luminous Kannondai Ichibankan 403 (72) Inventor Satoshi Yoshimizu 1584 Gosuda, Kamo City, Niigata Prefecture (72) Inventor Michio Furuta 2-3 May, Satsuki Town, Kameda Town, Nakakabara District, Niigata Prefecture 41 (56) Reference JP-A-59-34885 (JP, A) J. Biol. Chem. , USA, November 28, 1997, Vol. 272, No. 48, p. 30032-30039 J. Biochem. , Japan, July 1, 1998, Vol. 124, p. 157-162 J. Biochem. , Japan, November 1, 1995, Vol. 118, p. 1014-1020 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 ZNA C12N 9/48-9/86 BIOSIS (DIALOG) JISST file (JOIS) WPI (DIALOG) PubMed SwissProt / PIR / GeneS eq GeneBank / EMBL / DDBJ / Gene Seq

Claims (2)

(57) [Claims] 1. Derived from Maitake mushrooms and has the following properties
Aminopeptidase. (A) Optimum pH and stable pH range: Optimum pH of this enzyme
Is 8.5, pH 6.0 to 10.5 (45 ° C., 1 hour
Stable). (B) Optimum temperature: The optimum temperature of this enzyme is 65 ° C. (C) Thermostability: The enzyme is stable at 55 ° C or lower. (D) Substrate specificity: the enzyme is leucine and phenyl
Acts on Lanin. (E) Molecular weight: The molecular weight of this enzyme is 30 kDa (SDS-
Polyacrylamide gel electrophoresis). (F) Effect of inhibitor: This enzyme is EDTA and phenane.
It is inactivated by trollin and inhibited by bestatin. (G) Effect of metal ion: The enzyme deactivated by EDTA is
Mn²⁺, Zn²⁺, Cu ²⁺And Co²⁺Activated by the addition of
Turn into. (H) N-terminal amino acid sequence: as shown in SEQ ID NO: 1 in the sequence listing
Has an array. 2. An extract obtained by extracting water from a fruiting body of Pleurotus cornucopiae with water or a buffer solution is subjected to a hydrophobic chromatography using a resin having a hydrophobic functional group as a carrier to fractionate the active fraction. The method for producing aminopeptidase according to claim 1, wherein the aminopeptidase is collected.