JPH0215192B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an iminopeptidase obtained from edible fungi and a method for producing the same, and more particularly to an iminopeptidase obtained from the fruiting bodies or cells of shiitake mushroom (Lentinus edodes) and a method for producing the same. Conventionally, specific aminopeptidases include:
aminopeptidase B that specifically decomposes peptides whose amino terminus is arginine or lysine;
Acyl amino acid liberating enzyme derived from mammalian organs that specifically degrades peptides whose amino terminus is an acylamino acid, pyrrolidonyl peptidase that specifically degrades peptides whose amino terminus is pyroglutamic acid, and pyrrolidonyl peptidase that specifically degrades peptides whose amino terminus is an aromatic amino acid. Phenylaminopeptidase that specifically decomposes a certain peptide, and various aminopeptidases derived from animals or microorganisms that decompose peptides whose amino terminus is any amino acid are known, although there are some differences in their actions. As a result of intensive research on aminopeptidase present in the fruiting body of shiitake mushrooms, the present inventors found that
The present inventors have discovered a proline iminopeptidase that specifically decomposes peptides whose amino terminals are amino acids having a pyrrolidine ring, such as proline or hydroxyproline, and have completed the present invention. The purpose of the present invention is to provide an aminopeptidase that specifically acts on peptides having proline at the amino terminal, which has not been seen before, and this purpose is achieved by the proline iminopeptidase of the present invention. was completed. The proline iminopeptidase of the present invention has the enzymatic chemical properties shown below. 1 Molecular weight: 190,000 (gel filtration method, tetramer subunit 49,000) 2 Stable PH (PH range where the residual activity is 100% when kept at 4°C for 2 hours): 6-10 3 Optimal pH for action: 6-10 8 4 Optimum temperature for action: 37°C 5 Isoelectric point: 4.9 6 Activator: Not particularly required. 7 Inhibitors: Zn ²⁺ (10mM) 94% inhibition Cu ²⁺ (10mM) 99% inhibition Hg ²⁺ (0.1mM) 94% inhibition Also inhibited by p-chloromerkyuribenzoate. Zn ²⁺ , Cu ²⁺ , Hg ²⁺ by EDTA
Recovers inhibition of activity caused by 8 Substrate specificity: Only peptides and synthetic substrates that have proline or hydroxyproline at the amino end are well hydrolyzed, and synthetic substrates and peptides that have other amino acids at the amino end are not degraded at all. In addition, as a prolyl peptide, MSH Releasing Inhibition Factor (MSH Releasing
Inhibition Factor), Substance P Pentapeptide
It also degrades natural oligopeptides such as One of the points that the proline iminopeptidase of the present invention differs from other enzymes with similar specificity is that it does not particularly require an activator. Such proline iminopeptidases, like enzymes derived from bacteria, ascomycetes, or higher animals, are not activated by Mg ²⁺ , and their activity does not decrease even when metal ions are removed by dialysis or other treatments, and their activity does not decrease with the addition of EDTA. No decrease in activity was observed even after treatment. Furthermore, activation by cysteine and other SH protecting agents, such as enzymes of Escherichia coli and Bacillus brevis, is not observed. Next, regarding the substrate specificity of the proline iminopeptidase of the present invention, 0.5% of the substrate was added to 0.1N Tris-maleate buffer (PH6.1).
Dissolved in mM, added enzyme solution, reacted at 37°C for 10 minutes, and L-proline-β-naphthylamide (L-
The degradation rate, optimal pH, and Km value of each substrate are shown when the degradation rate of Pro-β-NA) is taken as 100%.

【table】

[Table] The method for producing proline iminopeptidase of the present invention involves extracting the shiitake mushroom fruiting body or bacterial cells using a near-neutral salt solution containing ethylenediaminetetraacetic acid (EDTA), and purifying the resulting extract. It is characterized by: Below, the manufacturing method of the present invention will be explained in more detail. The shiitake mushrooms used in the present invention include shiitake mushroom cells that are self-cultured at 20 to 25°C using a normal medium (PH5 to 6) containing yeast extract, malt extract, and glucose, as well as commercially available shiitake mushrooms. Bacterial cells may also be used. To the shredded shiitake mushroom cells or fruiting bodies, first, a solution of salt and EDTA with pH around neutrality is added, treated with a blender, and immediately centrifuged to remove insoluble matter, and an extract solution is obtained. get. Examples of the salt used for extraction include Tris-HCl. Preferably, the salt concentration of such a solution is in the range of 0.05-0.5M. Concentrations below 0.05M may result in insufficient extraction and reduced yield;
At concentrations exceeding 0.5M, there is no significant difference in the extraction effect, so there is no need to use a particularly high concentration. Although it is possible to extract with water alone, the yield is low and it is not preferable from the viewpoint of enzyme stability. Further, the EDTA concentration of the solution used for extraction is suitably around 5 mM in view of the effect on yield and cost. The reason for adding EDTA is that it forms a complex with the metal in the container, which prevents the enzyme from being oxidized and inactivated by heavy metals, and prevents the extract from being colored by polyphenol oxidase. These processes and subsequent processes are described in 4.
It is preferable to carry out at a low temperature of ℃ or less. Ammonium sulfate is added to the extract thus obtained to make it 40% saturated, and the resulting precipitate is removed, and further ammonium sulfate is added to make it 60% saturated, and the resulting precipitate is collected. The resulting precipitate is dissolved, ammonium sulfate is removed by conventional means such as dialysis, and anion exchange cellulose, such as diethylaminoethyl cellulose (hereinafter abbreviated as DEAE cellulose) is added.
Load the column and add eluent. The eluent is
It consists of a buffer solution with a pH near neutrality and a neutral salt solution, and the neutral salt concentration in the eluent is added while increasing linearly from the low concentration side. After removing the neutral salt from the proline iminopeptidase fraction obtained by elution, repeat the same procedure.
Treatment is performed using DEAE Sepharose. The buffer used in the above operation preferably has a pH near neutrality and a concentration of 50 mM or less. The neutral salt to be added is not particularly limited, but includes, for example, sodium chloride, potassium chloride, etc., and its concentration is linearly increased from 0 to 0.7M. The proline iminopeptidase fraction obtained by ion exchange chromatography is then treated with hydroorbitic chromatography for further purification. As the filler used in this case, phenylcephalose is preferable. The eluent consists of the 50 mM neutral buffer and a neutral salt solution, and is added while decreasing the neutral salt concentration in the eluent linearly from the higher concentration side. When the neutral salt concentration is high, contaminant proteins with low hydrophobicity are eluted, and at low concentrations, the target proline iminopeptidase is eluted. The type of neutral salt is not particularly specified, but a common one such as ammonium sulfate is sufficient, and its concentration is varied linearly from around 10% to 0%. The active fraction obtained by hydro-orbit chromatography is further purified by gel filtration. The solvent used in this case may be any buffer as long as it has a pH around neutrality, but the concentration is preferably about 50 mM. As a gelling agent, one with an exclusion molecular weight of about 150,000 is suitable, such as Cephadex G-150.
It is preferable to use The active fraction obtained by gel filtration is further purified by hydroxyapatite column chromatography, and the active fractions are collected to obtain proline iminopeptidase. The solvent in this case is
Use a phosphate buffer solution with a pH around neutrality centered around 10mM, and adjust the buffer concentration from 10mM to 400mM.
Add in linear increments up to mM. As a result, pure proline peptidase, which is 2500 times more purified than the crude extract, is obtained with a high active yield of 13.5%. The proline iminopeptidase of the present invention does not degrade any peptides other than those whose amino terminus is proline or hydroxyproline. Also, prolyl β-naphthylamide or hydroxyprolyl β-
It effectively degrades not only synthetic substrates such as naphthylamides, but also prolyl dipeptides and hydroxyprolyl dipeptides, and has the ability to effectively degrade prolyl dipeptides and hydroxyprolyl dipeptides.
-Leu-Gly- _NH2 )) and Substance P Pentapeptide (Pro-Lys-Pro
It also acts well on natural oligopeptides such as -Glu-Glu)). To date, there have been no reports of proline iminopeptidases that have been confirmed to exhibit high specificity for amino-terminal proline and hydroxyproline and also act well on natural oligopeptides. Additionally, Mg ²⁺
Proline iminopeptidases, which do not require metal ions such as, and do not require SH protectants, are completely new. Furthermore, the proline iminopeptidase of the present invention is not only useful for structural analysis of proteins, but also useful for modifying proteins and peptides.
It also has the potential to increase the stability of protein and peptide drugs, foods, etc. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto unless it exceeds the gist thereof. Example 500g of commercially available shiitake fruiting bodies, which had been cleaned with soil clods and wood chips, were removed and washed with water, and shredded with 3 times the amount of 5mM EDTA
-Crushed with 20mM Tris-HCl buffer (PH7.4). Insoluble materials such as cell walls were removed by centrifugation at 8000 rpm for 30 minutes, and the supernatant was collected. Add solid ammonium sulfate to the clear brown supernatant to 40%
The resulting precipitate was removed by centrifugation at 8000 rpm for 30 minutes, and solid ammonium sulfate was gradually added to the supernatant to achieve 60% saturation. After standing at 4°C overnight, the precipitate was collected by centrifugation, and a small amount of 20mM
It was dissolved in Tris-HCl buffer (PH7.4) and dialyzed against the same buffer to remove ammonium sulfate. Enzyme concentrated by ammonium sulfate fractionation was adsorbed onto a DEAE cellulose column equilibrated with 20mM Tris-HCl buffer (PH7.4), first washed with buffer to elute non-adsorbed proteins, and then Add KCl to the buffer solution and adjust its concentration to
Elution was performed in linear increments up to 0.7M. The fraction with activity towards proline β-naphthylamide was eluted at approximately 0.3M-KCl. Collect the fractions showing activity and incubate them with Diaflow-DM-10 (Diaflow).
-DM-10) Concentrate with membrane and 20mM Tris
- Dialysis was performed against HCl buffer (PH7.4).
This enzyme solution was then mixed with DEAE Sepharose-
It was adsorbed onto a column of CL-6B (20mM Tris-HCl buffer, PH7.4), and a linear concentration gradient of KCl (0→
0.7M). Collect the enzyme active fraction from the eluate and dilute to 50mM
Dialysis was performed against a Tris-HCl buffer (PH7.4)-12% ammonium sulfate solution. Next, this enzyme solution was placed on a phenylsepharose column (2.2 x 13.5 cm) equilibrated with a solution with the same composition as above, and a linear concentration gradient (12% to 0%) of a buffer solution not containing ammonium sulfate was applied. Elution was performed and the eluted active fractions were collected. This active fraction containing 0.1M KCl
Gel filtration was performed by the ascending method using a Sephadex G-150 column equilibrated with 20 mM Tris-HCl buffer (PH 7.4). Pro-β-NA
Fractions having activity were collected, dialyzed against 10 mM Na-phosphate buffer (PH6.0), equilibrated with the same buffer, and adsorbed onto a hydroxyapatite column. When eluted with a linear concentration gradient (10mM→400mM) of Na-phosphate buffer, the active fraction of proline iminopeptidase was eluted as a sharp peak. Through the above operations, the proline iminopeptidase of the shiitake fruiting body was purified approximately 2500 times, and the yield was 13.5%. The purity of the purified enzyme was examined by 5% polyacrylamide gel electrophoresis at each PH, and Coomassie Brilliant Blue R-250 (Coomassie
Brilliant Blue R-250) showed a single band as shown in the figure, and the activity staining band using proline β-naphthylamide as a substrate and the protein staining band completely matched.

[Brief explanation of drawings]

The figure shows the results obtained when 50 μg of purified shiitake fruiting body proline iminopeptidase enzyme was stained with Coomassie blue and subjected to 5% polyamide gel electrophoresis.
The electrophoretic pattern at PH is shown.

Claims (1)

[Scope of Claim] A proline iminopeptidase having the following enzymatic chemical properties. Molecular weight: 190000 (gel filtration method, tetramer) Stable pH (activity residual rate when kept at 4℃ for 2 hours)
100% PH range): 6-10 Optimum pH for action: 6-8 Optimum temperature for action: 37°C Isoelectric point: 4.9 Activator: Not particularly required. Inhibitors: Zn ²⁺ (10mM) 94% inhibition Cu ²⁺ (10mM) 99% inhibition Hg ²⁺ (0.1mM) 94% inhibition Also inhibited by p-chloromerkyuribenzoate. Zn ²⁺ , Cu ²⁺ , Hg ²⁺ by EDTA
Recovers inhibition of activity caused by Substrate specificity: Only peptides and synthetic substrates that have proline or hydroxyproline at the amino group end are well hydrolyzed, and synthetic substrates and peptides that have other amino acids at the amino end are not degraded at all. In addition, prolyl peptides include MSH Releasing Inhibition Factor (MSH Releasing Inhibition Factor).
It also easily degrades natural oligopeptides such as Biochemistry (Bition Factor) and Substance P Pentapeptide. 2. Extraction is carried out from the shiitake mushroom fruiting body or fungal cells using a near-neutral salt solution containing ethylenediaminetetraacetic acid, and the resulting extract is purified, and the prolyl iminopropylene has the following enzymatic chemical properties. Method for producing peptidase. Molecular weight: 190000 (gel filtration method, tetramer) Stable pH (activity residual rate when kept at 4℃ for 2 hours)
100% PH range): 6-10 Optimum pH for action: 6-8 Optimum temperature for action: 37°C Isoelectric point: 4.9 Activator: Not particularly required. Inhibitors: Zn ²⁺ (10mM) 94% inhibition Cu ²⁺ (10mM) 99% inhibition Hg ²⁺ (0.1mM) 94% inhibition Also inhibited by p-chloromerkyuribenzoate. Zn ²⁺ , Cu ²⁺ , Hg ²⁺ by EDTA
Recovers inhibition of activity caused by Substrate specificity: Only peptides and synthetic substrates that have proline or hydroxyproline at the amino group end are well hydrolyzed, and synthetic substrates and peptides that have other amino acids at the amino end are not degraded at all. In addition, prolyl peptides include MSH Releasing Inhibition Factor (MSH Releasing Inhibition Factor).
It also easily degrades natural oligopeptides such as Biochemistry (Bition Factor) and Substance P Pentapeptide.