JPH0244511B2 - TAINETSUSEIARUKARIKINZOKUPUROTEAAZEOYOBISONOSEIZOHO - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an alkali metal protease obtained from edible fungi and a method for producing the same, and more specifically, to a method for producing the alkali metal protease obtained from edible fungi, and more particularly to Grifola frondosa (Fr. .
The present invention relates to an alkali metal protease extracted from the fruiting body of F. Gray or its cultured cells. Conventionally, metalloproteases that require zinc as an essential component in order to exhibit catalytic activity include microorganisms such as Bacillus subtilis and Bacillus subtilis.
Neutral metalloproteases in the culture medium of Bacillus subtilis), Bacillus thermoproteolytieus , etc., and two types obtained from the culture medium of Aspergillus aspergillus, Aspergillus oryzae , Aspergillus sojae , etc. There is a low-molecular neutral metalloprotease present in the fruiting body of Arimillaria mellea .
In animals, alkali metal proteases, which are components of snake venom in snake vipers, pit vipers, and rattlesnakes, and neutral metal proteases obtained from rabbit kidneys and bovine pituitary glands are known. There are many types of these metalloproteases with molecular weights ranging from 14,000 to 90,000, and some contain calcium atoms in addition to zinc atoms. Most neutral metalloproteases have an optimal pH
is in the range of 6 to 7, but the optimum pH of snake venom alkali metal protease is on the weakly alkaline side of 8 to 9. In addition, these neutral and alkali metal proteases have the specificity that the neutral metal proteases in the fruiting body of Aracea mushroom preferentially decompose the peptide bond, -X-CO-NH-Lys-, which has a lysine residue on the N-terminal side. In addition to peptide bonds where the N-terminal amino acid is leucine, phenylalanine, or valine, -N-CO-NH-Leu
-, -X-CO-NH-Phe-, -X-CO-NH-
It is known that it has the specificity of mainly decomposing Val- (X represents an amino acid residue). The purpose of the present invention is to develop novel proteases with different properties and specificities compared to neutral or alkali metal proteases derived from bacteria, fungi, snake venom, or mammalian organs that have been revealed through previous research. An object of the present invention is to provide a thermostable alkali metal protease. As a result of comprehensive research on proteolytic enzymes contained in the extract of maitake fruiting bodies, the present inventors found that
It has a molecular weight of 22,000, contains one atom of zinc per protein molecule, has an optimal pH of 9 to 10 for the decomposition of casein or azocol, and has a leucine, phenylalanine, valine, or lysine residue on the N-terminal side. We have discovered an alkali metal protease that exhibits endopeptidase activity that specifically hydrolyzes peptide bonds, and have completed the present invention. That is, the thermostable metalloprotease of the present invention has the following enzymatic chemical properties. Molecular weight: Approximately 22,000 (gel filtration method, SDS gel electrophoresis method) Approximately 21,300 (amino acid analysis) Stable PH range: 3 to 10 (PH range with 100% residual activity when kept at 4℃ for 24 hours) Temperature Stability: 60℃ (Temperature at which 100% residual activity occurs when heat treated at PH7.0 for 60 minutes) 70℃ (Temperature at which 70% residual activity occurs when heat treated at PH7.0 for 60 minutes) Optimum pH for action: 9-10 (Substrate: casein, azocol, albumin) Optimum temperature for action: 70℃ (Temperature when reacting for 10 minutes at PH9 using azocasein as a substrate) Isoelectric point: 7.5 Metals contained: 1 zinc per molecule of protein Atomic activator: Not particularly required. Inhibitor: ethylenediaminetetraacetate (EDTA), 1,10-phenanthroline, α,
α'-dipyridyl, dithiothreitol, β-mercaptoethanol, glutathione (reduced form). Inhibition by EDTA is inhibited by Zn ²⁺ , Mn ²⁺ or Co ²⁺
The activity is restored by adding . Substrate specificity: casein, azocasein, azocol, congolet elastin, albumin,
It specifically hydrolyzes the N-terminal peptide bonds of leucine, phenylalanine, valine, and lysine residues in proteins or peptides such as insulin and polylysine. Moreover, the manufacturing method is characterized by extracting the maitake fruiting body or its bacterial cells using a salt solution of pH 3 to 10, and purifying the obtained extract. The maitake mushrooms used in the present invention may be self-cultured maitake cells grown at 20 to 25°C in a normal medium (PH5 to 6) containing yeast extract, malt extract, and glucose, but commercially available You may also use the fruiting body. The method for producing the protease of the present invention will be described in detail below. First, shredded maitake fruit bodies or bacterial cells obtained by culturing are treated with a neutral salt solution in a blender, and then immediately centrifuged to obtain an extract. The pH range of the solution used for extraction may be from 3 to 10, and the salt concentration may be from 0.05M to 0.5M. These processes and subsequent processes are described in 4.
It is desirable to conduct the test at a low temperature below ℃. Add ammonium sulfate to the extract thus obtained, remove the precipitate formed at 30% saturation, add more ammonium sulfate, and collect the precipitate formed at 80% saturation. Next, the obtained precipitate is dissolved, and ammonium sulfate is removed using dialysis or a gel filtration agent column such as Cephadex G-25 (trade name), and the pH is adjusted to 5. ,
For example, by loading a carboxymethyl cellulose column and performing elution while linearly increasing the concentration of neutral salts in the PH5 eluent (buffer) used to buffer the column, alkali metal proteases may be present in the eluate. Obtain fractions. In this case, it is also effective to increase the concentration of the neutral salt stepwise and elute the enzyme intensively at a characteristic neutral salt concentration. After removing the neutral salt from this fraction, it is treated again in the same manner using SP-Sephadex (trade name). The pH of the eluent (buffer) should be weakly acidic, but its concentration should be 50
It is desirable that the amount is not more than mM. The neutral salt added and used for enzyme elution is not particularly limited, but sodium chloride or potassium chloride is suitable. During elution, the concentration of neutral salts varies from 0 to
Enhanced linearly up to 1.0M or 0.3M,
It will be increased step by step to 0.5M and then 0.7M. Next, the fraction obtained by ion exchange chromatography is subjected to gel filtration for further purification. As the gel filtration agent used in this purification step, Cephadex G-100 (trade name) or Cellulofine GC-200-m (trade name) is suitable. Gel filtration uses a neutral eluent with a concentration of 50mM or less, but it is desirable that a neutral salt of 0.5M or less is present. After gel filtration, the eluted alkali metal protease fractions are collected, desalted and concentrated to obtain a purified sample.
Through the above purification process, maitake alkali metal protease can be purified 230 times with a high yield of 60%. As mentioned above, the alkali metal protease of this invention exists in large quantities in the hyphae of maitake fruiting bodies.
It can be easily extracted, fractionated with ammonium sulfate, carboxymethylcellulose and
It is possible to purify the enzyme protein in a high yield to a pure enzyme protein by column chromatography using a cation exchanger such as SP-Sephadex and gel filtration using a gel filtration agent such as Sephadex G-100. The characteristics of maitake alkali metal protease and the differences in properties, specificity, etc. between it and conventionally reported zinc-containing metal proteases are as follows. 1 The molecular weight of maitake alkali metal protease is approximately 22,000, the neutral metal protease of Bacillus subtilis (molecular weight: 40,000), and the two extracellular neutral metal proteases of Kojikin (molecular weight: 17,000 and
42,000), which is clearly different from the neutral metalloprotease from the fruiting body of Araceae (molecular weight: 13,500) and the neutral metalloprotease derived from mammalian organs (molecular weight: 90,000). 2 Maitake alkali metal protease has an optimal pH of 9 to 10, a molecular weight of 23,000 to 24,000,
It is similar in molecular weight, optimum pH, and substrate specificity to metalloproteases originating from snake venom, which has an optimum pH of 8.5 to 9.5, but has poor pH stability (snake venom enzymes: PH5 to 10) and thermostability. (PH8.5, 60℃,
(completely inactivated by heat treatment for 5 minutes), there is a clear difference. 3. The extremely high thermostability of maitake alkali metal proteases has been shown to be beneficial for Bacillus subtilis, especially Bacillus.
thermoproteolytieus neutral metalloprotease,
It is comparable to Thermolysin, and is a remarkable property of this enzyme. The intracellular alkali metal protease of maitake takes advantage of its stable nature and limited specificity to act on proteins and peptides at various temperatures up to 70°C and over a wide range of pH from 5 to 10.
Not only is it useful for analyzing and modifying its structure, but it can also be used for modifying and processing physiologically active proteins or peptides, and for processing and increasing the stability of foods. Furthermore, by using an enzyme that has been made insolubilized by taking advantage of its stability, it is also possible to decompose proteins at various temperatures and pH levels. At this time, there is an advantage that the reaction can be stopped by adding EDTA or restarted by adding an appropriate amount of Zn ²⁺ or Mn ²⁺ . Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by these unless the gist of the invention is exceeded. Example Fresh, commercially available maitake fruiting bodies (production amount, 1500 g) washed with water were crushed in a blender with 3 times the weight of 20 mM Tris-HCl buffer (PH7.2), and alkali metal protease was extracted from the bacterial cells. Insoluble residues such as cells were removed by centrifugation at 13,000 xg for 30 minutes, and the supernatant was collected. Solid ammonium sulfate powder was gradually added to the supernatant (approximately 6) while stirring well to achieve 30% saturation. After being left at 4°C for 3 hours, the resulting precipitate was removed by centrifugation at 13,000 xg for 30 minutes. Next, solid ammonium sulfate was added to the supernatant to achieve 80% saturation, and the mixture was left at 4° C. for 20 hours. The precipitate was collected by centrifugation at 13,000 x g for 30 minutes, dissolved in a small amount of 20 mM acetate buffer (PH5.0), and passed through a Sephadex G-25 column (5 x 38 cm) to remove ammonium sulfate. did. Collect fractions with protease activity,
Carboxymethylcellulose (Watmann, CM) equilibrated with 20mM acetate buffer (PH5.0)
-32) column (3.5 x 25 cm). Elution of the protease was performed using a concentration gradient in which the concentration of potassium chloride dissolved in a total volume of 3 volumes of 20 mM acetate buffer (PH5.0) was linearly increased from 0 to 1.0M. The protease active fraction was eluted with 0.7M to 0.8M potassium chloride. The protease active fractions were collected and desalted by passing through a Sephadex G-25 column equilibrated with 20 mM acetate buffer (PH5.0). At this stage, the alkali metal protease was adsorbed onto a carboxymethylcellulose column, and first, 20ml of 20ml containing 500ml of 0.3M potassium chloride
Wash the column with M acetate buffer (PH5.0) to elute the protein, and then elute the protease using a linear concentration gradient of potassium chloride from 0.3M to 1.0, or use 20mM acetate buffer (PH5.0) to elute the protease. 0) is effective for separating and purifying maitake alkaline metal protease by increasing the concentration of potassium chloride in stepwise steps to 0.3M, 0.6M, and 0.8M to elute the enzyme. Next, the desalted protease fraction was adsorbed onto an SP-Sephadex column (2.0 x 13 cm) buffered with 20 mM acetate buffer (PH5.0), and the total volume was
Elution was carried out by gradient elution with linearly increasing concentrations of potassium chloride from 0 to 0.8M in 500ml of 20mM acetate buffer (PH5.0). To measure protease activity, collect the protease active fraction eluted with 0.4-0.5M potassium oxide and transfer to Diaflow membrane.
After ultraconcentration using DM-10, gel filtration was performed using a Sephadex G-100 column (2.4 x 100 cm) equilibrated with 20 mM Tris-HCl buffer (PH7.2). This gel filtration yielded an elution pattern for alkali metal protease in which the activity elution curve for azocasein and the protein elution curve completely matched. The active fractions were collected, concentrated using Diaflow membrane DM-10, and freeze-dried.
It was stored frozen at 80°C. In addition, the collected active fractions were divided into 10
It may be desalted using a Sephadex G-25 column equilibrated with mM ammonium bicarbonate and then freeze-dried. By the above purification method, the alkali metal protease of maitake fruiting body was purified 230 times from the crude extract, and the yield was 60%. The purity of the purified enzyme was determined by polyacrylamide gel electrophoresis (10% gel, PH4.3) and SDS polyacrylamide slab electrophoresis (Gradient Gel8~
Coomassie
A single protein-stained band could be detected with Brilliant Blue staining in all cases.

Claims (1)

[Scope of Claims] A thermostable alkali metal protease having the following enzymatic chemical properties. Molecular weight: Approximately 22,000 (gel filtration method, SDS gel electrophoresis method) Approximately 21,300 (arnoic acid analysis) Stable PH range: 3 to 10 (PH range with 100% residual activity when kept at 4°C for 24 hours) Temperature stability: 60℃ (Temperature at which the residual activity rate is 100% when heat treated at PH7.0 for 60 minutes) 70℃ (Temperature at which the residual activity rate is 70% when heat treated at PH7.0 for 60 minutes) Optimum pH for action :9-10 (Substrate: casein, azocol, albumin) Optimum temperature for action: 70℃ (Temperature when reacting for 10 minutes at PH9 using azocasein as a substrate) Isoelectric point: 7.5 Metal content: Zinc per molecule of protein Single-atom activator: Not particularly required. Inhibitor: ethylenediaminetetraacetate (EDTA), 1,10-phenanthroline, α,
α'-dipyridyl, dithiothreitol, β-mercaptoethanol, glutathione (reduced form). Inhibition by EDTA is inhibited by Zn ²⁺ , Mn ²⁺ or Co ²⁺
The activity is restored by adding . Substrate specificity: casein, azocasein, azocol, congolet elastin, albumin,
It specifically hydrolyzes the N-terminal peptide bonds of leucine, phenylalanine, valine, and lysine residues in proteins or peptides such as insulin and phorilysine. 2 From maitake fruiting body or its bacterial cells, PH3-10
A method for producing a heat-stable alkali metal protease having the following enzymatic chemical properties, the method comprising performing extraction using a salt solution of and purifying the obtained extract. Molecular weight: Approximately 22,000 (gel filtration method, SDS gel electrophoresis method) Approximately 21,300 (arnoic acid analysis) Stable PH range: 3 to 10 (PH range with 100% residual activity when kept at 4°C for 24 hours) Temperature stability: 60°C (temperature at which 100% residual activity occurs when heat treated at PH7.0 for 60 minutes) 70°C (temperature at which 70% residual activity occurs when heat treated at PH7.0 for 60 minutes) Optimum pH for action :9-10 (Substrate: casein, azocol, albumin) Optimum temperature for action: 70℃ (Temperature when reacting for 10 minutes at PH9 using azocasein as a substrate) Isoelectric point: 7.5 Metal content: Zinc per molecule of protein Single-atom activator: Not particularly required. Inhibitor: ethylenediaminetetraacetate (EDTA), 1,10-phenanthroline, α,
α'-dipyridyl, dithiothreitol, β-mercaptoethanol, glutathione (reduced form). Inhibition by EDTA is inhibited by Zn ²⁺ , Mn ²⁺ or Co ²⁺
The activity is restored by adding . Substrate specificity: casein, azocasein, azocol, congolet elastin, albumin,
It specifically hydrolyzes the N-terminal peptide bonds of leucine, phenylalanine, valine, and lysine residues in proteins or peptides such as insulin and phorilysine.