JPS6156997B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes a peptidase-producing bacterium belonging to the genus Flavobacterium, and the peptidase produced within the bacterium,
An endo-type enzyme that specifically breaks down peptide bonds between proline and other amino acids: peptidase and its production method. Many proteases and peptidases that decompose proteins or peptides have been found in animals, plants, microorganisms, etc. and have been isolated and purified, but they act specifically on peptide bonds between proline and other amino acids in proteins or peptides. As for the endo-type peptidase that degrades the protein, Walter found it in the kidney of lamb (R.Walter:
Biochem.Biophys.Acta, 422 , 138, 1976), but only separated. Such peptidases are of course useful for structural analysis of proteins, but they can also be used to degrade proteins or peptides to reduce toxicity, prepare useful active substances, etc.
Furthermore, it has many applications, such as chemically modifying decomposition products to induce useful active substances, and is expected to be used in the future. The present inventors focused on such peptidases and, as a result of intensive research, discovered a bacterium in the genus Flavobacterium that produces such a unique peptidase, and destroyed the bacterial cells to separate and extract the peptidase.
succeeded in refining it. That is, an object of the present invention is to utilize a peptidase-producing bacterium belonging to the genus Flavobacterium that produces a peptidase that specifically acts on and decomposes peptide bonds involving proline to produce peptidases and enzymes having the specific degrading action as described above. The purpose is to provide a manufacturing method for the same. That is, the peptidase of the present invention is characterized by having the enzymatic chemical properties described below. The peptidase of the present invention can be produced by culturing a peptidase-producing bacterium belonging to the genus Flavobacterium and collecting the peptidase accumulated within the bacterium. For example, a precipitate produced by adding ammonium sulfate to the intracellular fluid of a peptidase-producing bacterium belonging to the genus Flavobacterium is dissolved, and the precipitate is subjected to chromatography using a cation exchanger to produce a peptidase-producing bacterium containing peptidase. It can be produced by separating a fraction, then loading the fraction onto a hydroxyapatite column, and purifying it by gel filtration. Examples of peptidase-producing bacteria belonging to the genus Flavobacterium used in the present invention include Flavobacterium and Meningosepticum IFO12535.
(Flavobacterium meningosepticum
IFO12535), Flavobacterium TY−78−74
(Flavobacterium TY-78-74) Microtechnological Institute Deposit No. 4849, etc. Flavobacterium TY-78-74 was isolated from soil collected by the present inventors in Unebiyamayamarei, Nara Prefecture, and has the mycological properties described below. Now, the method for producing peptidase of the present invention will be explained in more detail. Peptidase-producing bacteria belonging to the genus Flavobacterium are usually aerated and cultured using a broth liquid medium. The most appropriate culture temperature is around 30 to 40°C, and the culture time is usually about 15 to 20 hours. The cells obtained by culturing in this manner are destroyed by a conventional method such as using glass beads or ultrasonic waves, and the cell walls or their fragments are removed to remove the peptidase. Obtain intracellular fluid. Ammonium sulfate is added to this intracellular solution to perform salting out.The preferred processing conditions are to first add ammonium sulfate to a saturation of 0.65, separate the supernatant, and then add to this supernatant 0.9 It is best to add ammonium sulfate to saturation and separate the precipitate. According to such treatment, first, nucleic acids can be removed, and enzymes can be separated more efficiently. Note that, if necessary, before adding ammonium sulfate, protamine sulfate may be added and centrifugation may be performed. Examples of the cation exchanger used in the present invention include cation exchange cellulose such as carboxymethyl (CM)-cellulose and phospho(P)-cellulose, and CM-Sephadex (trade name, manufactured by Pharmacia Fine Chemicals, Sweden). There are cation exchange cephadexes such as (name). Enzyme fraction separation using this cation exchanger is performed as follows. onto a cation exchanger column that has been buffered in advance with a buffer in the pH range of 5.8 to 7.0.
A precipitate containing the enzyme is loaded, and the enzyme fraction is separated using the same buffer as an eluent while linearly increasing the sodium chloride concentration from 0M to 0.1M.
Any buffer can be used as long as it has a pH of 5.8 to 7.0 and a concentration of 20 to 50 mM. Examples include Tris-hydrochloride buffer, phosphate buffer, acetate buffer, and the like. In addition, for purification using hydroxyapatite, the enzyme fraction is loaded onto a hydroxyapatite column that has been buffered in advance with a buffer of pH 6.5 to 7.5, and after washing with a buffer of 10 mM or less, the same buffer is used as an eluent. While increasing the concentration to 0.5M, pour from the top of the column and elute to obtain the enzyme fraction. Any buffer can be used as long as it has a pH in the range of 6.5 to 7.5. Furthermore, purification by gel filtration is carried out by loading the enzyme fraction onto a column packed with a gel filtering agent, such as Sephadex. The column should be pre-adjusted to pH
6.5 to 7.5 and a buffer solution with a concentration of 20 to 100 mM, and elute using the same buffer as an eluent. By the way, Flavobacterium TY-, which was cited as an example of peptidase-producing bacteria used in the present invention,
76-74 was isolated from collected soil that was suspended in water, filtered, and then cultured by applying the filtrate to a broth agar medium (PH7.2). Its mycological properties are as follows, and Bergey's manual of
According to Determinative Bacteriology, 8th ed., it is presumed to be Flavobacterium ferrugineum. Microscopic findings (cultured on broth agar medium, 30℃, 48 hours) Cell shape and size: Slightly elongated, 0.4-0.6 x 1.2-4 microns;
It is a partially curved rod. Presence or absence of cell pleomorphism: Exist alone or in pairs, with no pleomorphic properties. Presence or absence of motility Almost no motility Presence or absence of spores Not formed Gram staining Negative Growth status in each medium Broth agar plate culture When cultured at 30℃ for 48 hours, round colonies with a diameter of 3 to 4 mm were formed, with a smooth surface. Form a village. Colony color is pale yellow. Juicy agar slant culture Grows well in a wide spread shape when cultured at 30℃ for 48 hours. The color of the colony is pale yellow with a dull luster. Meat juice liquid culture When cultured statically at 30°C for 24 hours, a thin film is formed on the upper layer and a precipitate is observed on the bottom. Meat juice gelatin puncture culture When statically cultured at 20°C for 1 to 30 days, the gelatin grows well on the surface and upper layer, and gelatin liquefaction is observed. Litmus milk After culturing at 30℃ for 5 days, alkalinization and peptonization are observed. Potato medium Grows slightly at 30℃ for 48 hours. Physiological properties 1 Production of indole - 2 Production of cytochrome oxidase + 3 Production of catalase + 4 O-F test Oxidation 5 Simon's citrate - 6 Reduction of nitrate - 7 Production of lysine decarboxylase - 8 Production of ornithine decarboxylase - 9 Arginine Production of dehydrolase - 10 Production of DNA degrading enzyme - 11 Production of acylamidase + 12 Presence or absence of acid production from the following sugars (1) Glucose + (2) Maltose + (3) Xylose + (4) Mannitol - Also, this The enzymatic chemical properties of the peptidase of the invention are as follows. Action and substrate specificity It acts specifically on and decomposes specific proline-related peptide bonds. a Things to be decomposed (↓ indicates the bond to be decomposed)

【table】 b.Things that cannot be decomposed

[Table] In the above formula, N is a carbobenzoxy group, 2-NNap
is β-naphthylamide, ONp
is p-nitrophenyl ester, Pht is phthalyl group, For is formyl group
respectively. Optimal action PH 7.0 Stable PH range 5.0-9.0 Optimum action temperature 40°C Thermostability 42°C or less Inhibitory action Not inhibited by pancreatic and limabine, trypsin, inhibitors, ovomucoid, pepstatin, etc. Molecular weight: approximately 78,000 Isoelectric point: PH9.1 The above peptidase of the present invention is different from the similar peptidase that Walter discovered in lamb kidney.
First of all, in addition to having different origins, Walter's enzyme is a different enzyme, with an optimal pH of 7.8 and an optimal temperature of 47°C, and different enzymatic chemical properties. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by these descriptions unless the gist thereof is exceeded. Example 1 Flavobacterium TY-78-74 was inoculated into a liquid medium (PH7.0) containing 1% broth, 1% polypeptone, and 0.5% sodium chloride, and was incubated at 8/min.
The mixture was kept at 30°C for 16 hours while aerating air, and then centrifuged to obtain 50 g of bacterial cells. Glass beads were added to 50 g of the bacterial cells, treated for 10 minutes using a Vibrogen Cell Mill (manufactured by Edmund Boehler, Germany), and then centrifuged to obtain 800 ml of supernatant.
Protamine sulfate was added to the obtained supernatant at a concentration of 0.2% by weight, centrifuged, and then ammonium sulfate was added to perform salting out. That is, ammonium sulfate was added to give a saturation of 0.65, the supernatant was separated, and then ammonium sulfate was added to the supernatant to give a saturation of 0.90. The resulting precipitate was collected. After desalting this precipitate,
It was loaded onto a CM-cellulose (Brown, USA) column (3.5 x 30 cm) buffered with 20mM Tris-HCl buffer (PH6.2), and the sodium chloride concentration in the same buffer was adjusted from 0M to 0.1M. Pour the eluent from the top of the column while increasing linearly to
Elution was performed at a rate of 60 ml per hour. 300~400ml
Enzyme fractions were obtained in 100 ml of the eluate range. This enzyme fraction had an activity of 20 units per mg of protein, as calculated from the absorbance at 280 nm. Next, 40 ml of the obtained enzyme fraction was loaded onto a hydroxyapatite column (2 x 30 cm) that had been buffered in advance using 10 mM phosphate buffer (PH7.0).
Pour the same buffer solution from the top of the column as an eluent while linearly increasing the concentration from 10mM to 0.5M.
Elution was performed at a flow rate of ml/hr. 500-600ml eluate
The enzyme fraction was obtained in 100ml. The enzyme activity of the fraction is
It was 95 units/mg protein. Next, the fraction was concentrated to about 10 ml by ultrafiltration method, and then Cephadex G-150 (Sweden, Pharmacia The sample was loaded onto a column (2 x 120 cm) manufactured by Co., Ltd., and the same buffer solution was poured as an eluent from the top of the column and eluted at a flow rate of 30 ml/hr. After desalting 105 ml of the eluate from 600 to 705 ml, lyophilization was performed to obtain 1.5 mg of white powder. The powder exhibited an activity of 130 units/mg and its enzymatic chemical properties were the same as described above. Example 2 Flavobacterium meningosepticum IFO12535, which was provided by Institute of Fermentation (IFO, Osaka), was used. Flavobacterium meningosepticum IFO12535 was inoculated into a liquid medium (PH7.0) containing 1% broth, 1% polypeptone, and 0.5% sodium chloride, and 8 min.
After keeping the mixture at 30°C for 16 hours while aerating air, 80 g of bacterial cells were collected by centrifugation. Glass beads were added to 50 g of this bacterial cell, and using a Vibrogen cell mill (manufactured by Edmund Boehler, Germany),
After treatment for 10 minutes, 800 ml of supernatant was obtained by centrifugation. The resulting supernatant was treated with protamine sulfate at a concentration of
After adding to 0.2% by weight and centrifuging,
Ammonium sulfate was added to perform salting out in the same manner as in Example 1, and the resulting precipitate was collected. After desalting this precipitate, it was loaded onto a CM-cellulose (manufactured by Brown, USA) column (3.5 x 30 cm) that had been buffered in advance using 20mM Tris-HCl buffer (PH6.2). While increasing the sodium chloride concentration in the solution linearly from 0M to 0.1M, the eluent was poured from the top of the column and eluted at a rate of 60ml per hour. Enzyme fractions were obtained in 180 ml of eluate ranging from 300 to 480 ml. This enzyme fraction had an activity of 33.8 units per mg of protein, as calculated from the absorbance at 280 nm. Next, 180 ml of the obtained enzyme fraction was loaded onto a hydroxyapatite column (2 x 30 cm) that had been buffered in advance using 10 mM phosphate buffer (PH7.0), and the concentration of the same buffer was adjusted from 10 mM to Pour as eluent from the top of the column while increasing linearly to 0.5M.
Elution was performed at a flow rate of 35 ml/hr. The enzyme fraction was obtained in 80 ml of eluate from 230 to 310 ml. The enzyme activity of the fraction is
It was 110 units/mg protein. Next, the fraction was concentrated to about 50 ml by ultrafiltration, and then buffered using 20 mM Tris-HCl buffer (PH 7.0) containing 100 mM sodium chloride. Co., Ltd. column (2
It was eluted at a flow rate of After desalting 25 ml of the 210-235 ml eluate, it was freeze-dried to obtain about 6 mg of white powder. The powder exhibited an activity of 130 units/mg protein and its enzymatic chemical properties were the same as described above. In addition, the enzyme activity was determined by 0.2mM Z-Gly-Pro-2.
−0.1M phosphate buffer (PH
6.9) Add 0.25 ml of enzyme solution to 1.25 ml, keep at 35°C, react the resulting β-naphthylamine with fast garnet GBC, and measure the resulting diazo pigment. At this time, the amount of enzyme that released 1 μmole of β-naphthylamine per minute was defined as 1 unit.

Claims (1)

[Claims] 1. Enzyme chemical properties: Action and substrate specificity a It specifically acts on and decomposes the following proline-related peptide bonds (↓ indicates the bond to be decomposed). [Table] b The following proline-related peptide bonds are not broken down. [Table] [Table] (In the above formula, Z is a carbobenzoxy group, 2-
NNap is β-naphthylamide, ONp is p-nitrophenyl ester, Rht is phthalyl group,
For represents a formyl group. ) Optimum PH for action: 7.9 Stable PH range: 5.0-9.0 Optimal temperature for action: 40°C Thermostability: 42°C or less Inhibitory action: Not inhibited by pancreatic and rimavine trypsin inhibitors, ovomucoid, and pepstatin. An endo-type peptidase characterized by having a molecular weight of about 78,000 and an isoelectric point of PH9.1. 2. It is characterized by culturing peptidase-producing bacteria belonging to the genus Flavobacterium and collecting the peptidase accumulated in the bacterial cells. The following enzyme chemical properties: Action and substrate specificity a. Specificity for the following proline-related peptide bonds. acts on and decomposes (↓ indicates the bond to be decomposed). [Table] b The following proline-related peptide bonds are not broken down. [Table] (In the above formula, Z is a carbobenzoxy group, 2-
NNap is β-naphthylamide, ONp is p-nitrophenyl ester, Pht is phthalyl group,
For represents a formyl group. ) Optimum PH for action: 7.0 Stable PH range: 5.0-9.0 Optimal temperature for action: 40°C Thermostability: 42°C or less Inhibitory action: Not inhibited by pancreatic and rimavine trypsin inhibitors, ovomucoid, and pepstatin. A method for producing an endo-type peptidase with a molecular weight of approximately 78,000 and an isoelectric point of PH9.1.