JPH07241191A - Novel peptidase - Google Patents

Abstract

PURPOSE:To provide a novel peptidase specifically acting only on a peptide comprising a D-amino acid and having an extremely characteristic advantage. CONSTITUTION:A novel peptidase has the following properties: (a) The action: the peptidase hydrolyzes oligopeptidase comprising D-phenylalanine. (b) The substrate specificity: the peptidase acts specifically on oligopeptidase having or not having a protecting group comprising D-phenylalanine, and does not act on an oligomer comprising L-phenylalanine. (c) The pH stability: stable in the pH range of 5-12. (d) The optimal pH: 9-12. (e) The optimal temperature: approx. 50 deg.C. (f) The mol.wt.: the peptidase exhibits a sub-unit, having a mol.wt. of approximately 38,000 by a high performance liquid chromatography gel filtration method and having a mol.wt. of approximately 39,0000 by a SDS- polyacrylamide gel electrophoresis method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptidase that specifically acts on a D-amino acid derivative and a method for producing the same.

[0002]

BACKGROUND ART There have been many reports on microbial-derived peptidases. For example, Robinson et al., J
individual of Biological Chem
istry, 202, 1 (1953) can be illustrated. All of these peptidases are peptidases that act on peptides consisting of L-amino acids. However, Japanese Patent Publication No. 61-68 reports a peptidase derived from actinomycetes belonging to the genus Streptomyces that acts on oligopeptides containing D-amino acids.
-It does not have the action of specifically hydrolyzing an oligopeptide consisting of phenylalanine.

[0003]

DISCLOSURE OF THE INVENTION The present inventors extensively screened a strain having a peptidase activity that specifically acts on a peptide consisting of D-amino acids, and found that Bacillus cereus specifically acts on a peptide consisting of D-phenylalanine. It has been found to produce a new peptidase that does. The present invention has been made based on such findings. The present invention provides the following novel peptidase, a method for producing the same, and a novel microorganism having the ability to produce the peptidase.

[0004] Peptidase having the following properties (a) Action Hydrolyze oligopeptide consisting of D-phenylalanine (b) Substrate specificity D-phenylalanine specific or non-protective oligopeptide Acts on L-
Does not act on oligopeptides consisting of phenylalanine (c) pH stability Stable at 5-12 (d) Optimum pH 9-12 (e) Optimum temperature around 50 ° C (f) Molecular weight In high performance liquid chromatography gel filtration method About 3
A subunit having a molecular weight of 8,000 and having a molecular weight of about 39,000 by SDS-polyacrylamide gel electrophoresis is shown.

Bacillus cereus, a microorganism capable of producing a peptidase that specifically hydrolyzes an oligopeptide composed of D-phenylalanine, is cultured, and a peptidase that specifically hydrolyzes an oligopeptide composed of D-phenylalanine from a culture product is cultured. A method for producing peptidase by a microorganism, which comprises collecting

Bacillus cereus DF4-B, a microorganism having a peptidase-producing ability that acts specifically on peptides consisting of D-phenylalanine and its derivatives. This novel peptidase has a very characteristic advantage in that it specifically acts only on peptides consisting of D-amino acids.

The present invention will be described in detail below. A typical example of the microorganism for producing the novel peptidase according to the present invention is Bacillus cereus. A new strain belonging to this species is Bacillus cereus DF4-B isolated by the present inventors. This bacterium is FERM P.
Deposited as 14143. The new strain was isolated from the soil of Sagamihara City, Kanagawa Prefecture, using the medium having the composition shown in Table 1 below.

Table 1 Peptone 1.0% Yeast extract 0.5 Sodium chloride 1.0 (D-Phe) ₄ 0.2 Tap water pH 7.0

A culture medium having the composition shown in Table 1 was placed in a test tube (φ18 mm).
5 ml each, and sterilize at 120 ° C for 15 minutes,
A small amount of soil samples collected from various places was added to each of the culture media, and the mixture was shake-cultured at 30 ° C for 1 week. A plate medium was prepared by adding 1.5% agar to the medium having the composition shown in Table 1. Using this plate medium, a part of each culture solution obtained above was streaked with a platinum loop and incubated at 30 ° C. for several days. Initially in the medium, the substrate (D-Phe) ₄ is insoluble in water and thus becomes cloudy, but in each medium (D-Phe) 4
A colony forming a clear zone by the decomposition of ₄ was collected, and the strain was picked up on a slant medium having the same medium composition as above to isolate the strain.

Next, 5 ml of the medium having the composition shown in Table 1 was dispensed into the test tube and sterilized in the same manner as above. Each of the strains collected above was shake-cultured in this medium at 30 ° C. for 16 hours, and a strain in which the (D-Phe) ₄ content in the supernatant was remarkably reduced was selected. The novel strain obtained as described above has the mycological properties shown in Table 2.

Table 2 Observation items a) Morphology 1 Cell type Bacillus 2 Polymorphism presence / absence-3 Motility presence / absence + flagella epithelial state Periflagellum 4 spores presence / absence +5 Gram staining +6 acid resistance-

B) Growth state in each medium 1 Meat broth agar plate culture (30 ° C., 3 days) a) Colony shape (diameter) 1.3 cm b) Colony shape Coarse circular c) Colony surface shape Smooth d) Colony Elevated state of the plane E) Colony peripheral corrugation F) Color tone of the colony Beet) Transparency of the colony Opacity) No luster of the colony) Soluble pigment formation −

2 Meat broth agar slope culture (30 ° C, 3 days) a) Good growth Good growth b) Colony no gloss 3 Meat broth liquid culture (30 ° C, 1 day) b) No surface growth b) Total turbidity C) Precipitating powdery d) No gas generation 4 Meat juice gelatin (30 ° C, 10 days) Gelatin liquefaction + 5 Litmus milk (30 ° C, 3 days) Coagulation

C) Physiological properties 1 Reduction of nitrate + 2 Denitrification + 2 MR + 4 VP − pH> 75 Indole formation − 6 Hydrogen sulfide formation −

7 Starch hydrolysis +8 Utilization of citric acid a) Koser-Ro) Christensen + C) Bergey's Manual +9 Utilization of nitrate-10 Pigment formation a) King A medium + Ro) King B medium + 11 Urease-12 oxidase + 13 catalase + 14 Growth range a) pH 6.0 to 9.0 b) Temperature 10 to 40 ° C 15 Attitude toward oxygen Aerobic 16 OF test (glucose) Fermentative

17 Generation of Acid and Gas from Sugars Acid gas 1 L-arabinose-2-D-xylose-3-D-glucose + -4D-mannose-5D-fructose + -6D-galactose --- 7 Maltose + -8 Sucrose--9 Lactose--10 Trehalose + -11 D-Sorbit--12 D-Mannitol--13 Glycerin + -14 Starch + -15 Raffinose ---

D) Other properties Formation of dioxyacetone-Deamination reaction of phenylalanine ± Salt tolerance 2% + 5% + 7%-Egg-yolk reaction + Lysozyme resistance + Starch hydrolysis + Utilization of propionic acid ＋ Casein degradability ＋ Tyrosine degradability ＋ Gelatin degradability ＋ Sabouraud dextrose Growth in medium ＋

Based on the above-mentioned mycological properties, Bergey '
s Manual of Systematic Ba
cterology 9th ed. , Vol.
1. , Strain p1122, the strains were identified as follows. This bacterium is an aerobic, Gram-positive bacillus with sporulation and motility, and grows in meat extracts and peptones. In addition, it was identified as a bacterium belonging to the genus Bacillus because it produces an acid from D-glucose with a positive catalase reaction. Furthermore, it was identified as Bacillus cereus because it was positive for Egg-yolk reaction, resistant to lysozyme, and hydrolyzes casein, gelatin, and starch.

It is also possible to mutate the bacterium to obtain a strain with higher productivity. In addition, the gene involved in the production of peptidase existing in the cells of this strain is excised and inserted into an appropriate vector, for example, a plasmid, and this vector is used to produce an appropriate host, for example, Escherichia coli (Escherichia coli). )
It is also possible to artificially create the peptidase-producing strain of the present invention by transforming a heterologous host such as or yeast or a homologous host such as Bacillus.

The strain of the present invention can be preserved according to a conventional method, for example, on an agar slant or by a freeze-drying method. As the agar slant medium, a medium commonly used for the preservation of Bacillus bacteria, for example, the above-mentioned medium for separating the bacteria can be used. Also, freeze-drying can be performed according to a conventional method.
When the peptidase of the present invention is to be produced by culturing the above-mentioned microorganism, any medium can be used as the basal nutrient medium as long as the microorganism can be increased. This medium contains, as a nitrogen source, one or more kinds of yeast extract, peptone, meat extract and the like. If necessary, D-glucose, starch, glycerin and the like can be added to this medium as a carbon source. This medium contains inorganic salts such as sodium chloride,
It is preferable to add dipotassium phosphate, magnesium sulfate and the like.

The culture may be carried out in either a solid medium or a liquid medium, but in order to obtain a large amount of the target enzyme, the liquid medium is used and cultured under aerobic conditions by shaking culture, aeration / agitation culture or the like. Is preferably performed. The culturing temperature may be any temperature within the temperature range in which the bacteria grow and peptidase is produced, but it is preferably 20 to 40 ° C.
The pH is in the range of 5-10, preferably 6-9. The culture time may be selected such that the enzyme activity is expressed, but it is preferably 6 to 72 hours.

Next, the peptidase of the present invention is collected from the obtained culture broth and cells, and the usual enzyme purification method can be used as the purification method. Since this enzyme is mainly present in the culture medium, the bacterial cells are removed by centrifugation, etc. to obtain a crude enzyme solution, which is then concentrated by ultrafiltration, etc., salting out, organic solvent precipitation, adsorption chromatography, ion exchange. Chromatography, hydrophobic chromatography, gel filtration chromatography and the like can be performed to obtain a uniform enzyme preparation.

Regarding the enzyme in the bacterial cells, the bacterial cells are collected by centrifugation or the like, the solid is crushed by a mechanical method such as ultrasonic treatment or dynomill, and the solid matter such as cell debris is removed by centrifugation or the like. The same can be done as described above.

Method for measuring the titer of enzyme In the present invention, the titer was measured by the following method. Tris-HCl buffer (pH 9.0) 50 μmol, (DP
he) ₄ 1 μmol and an appropriate amount of sample 0.5 m
The resulting mixture (D-Phe) ₂ was subjected to high performance liquid chromatography (COSMOSIL 5C18-MS 4.6 × 150).
mm, eluent 35% methanol-phosphoric acid (pH 3.
0), flow rate 1 ml / min, detection wavelength 254 nm)
And the (D-Phe) in the reaction solution from the calibration curve.
_Two quantities were determined. 1 μmol (D-Phe) per minute
One unit was the amount of enzyme that produced ₄ to 2 μmol of (D-Phe) ₂ .

Properties of Enzyme The peptidase of the present invention has the following properties. (A) Action Hydrolyzes a peptide composed of D-phenylalanine. (B) Substrate specificity L-, which acts specifically on an oligopeptide having no protective group or having a protective group consisting of D-phenylalanine,
It does not act on oligopeptides consisting of phenylalanine. (C) Optimum pH 9 to 12 is optimum. (D) pH stability It is stable at 5 to 12 when the residual activity is measured after incubation at 40 ° C for 3 hours in a buffer solution (0.1 M) of each pH.

(E) Optimal temperature The activity is maximum at around 50 ° C. (F) Molecular weight Approximately 3 in high performance liquid chromatography gel filtration method.
A subunit having a molecular weight of 8,000 and having a molecular weight of about 39,000 by SDS-polyacrylamide gel electrophoresis is shown.

Next, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

【Example】

Example 1 Purification of Peptidase from Bacillus cereus DF4-B Meat extract 1.0%, peptone 1.0%, sodium chloride 0.5% were contained in a medium of 140 liter prepared to pH 7.2, 120 ° C. After heat sterilization for 15 minutes, Bacillus cereus DF4-B (Microtechnology Research Institute, FERM P-14)
143) was inoculated and cultured aerobically at 30 ° C. for 48 hours.

After culturing, cells were removed by a centrifuge to obtain 130 liters of culture supernatant. This was concentrated to 9.9 liters by ultrafiltration using a hollow fiber cartridge H10P10-20 to obtain a crude enzyme solution. Solid ammonium sulfate (3,130 g) was added to the crude enzyme solution to give 5
It was saturated with 0% ammonium sulfate. After stirring for 1 hour, −
The supernatant (11 liters) obtained by centrifugation at 15,000 × g for 40 minutes was further added with solid ammonium sulfate (2,
354 g) was added to make 80% ammonium sulfate saturation.

The enzyme-active precipitate obtained by centrifugation at 19,000 × g for 40 minutes was dissolved in a small amount of 0.01 M phosphate buffer (pH 7.0), and further 0.1 mM was added.
0.01M phosphate buffer containing EDTA (pH 7.
It dialyzed by 0). This enzyme solution is 0.1 mM in advance
0.01M phosphate buffer containing EDTA (pH 7.0)
Passed through a column of DEAE-Toyopearl 650 M equilibrated with 0.1 mM EDTA and 0.1 M NaC
Elution was carried out with 0.1 M phosphate buffer (pH 7.0) containing 1 liter.

The active fractions were collected and dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA.
The sample was again passed through a column of DEAE-Toyopearl 650M which had been equilibrated with the same buffer solution in advance to obtain 0.1 mM ED.
Elute the enzyme with a linear concentration gradient from 0.01 M phosphate buffer (pH 7.0) containing TA to 0.1 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 0.1 M NaCl. did.

The active fractions were collected, ammonium sulfate was added to 30% saturation, and then 0.1 mM was prepared in advance.
Pass through a column of Butyl Toyopearl 650 M equilibrated with 0.01 M phosphate buffer (pH 7.0) containing EDTA and 30% saturated ammonium sulfate to give 0.1 m
The enzyme was eluted with a linear gradient of 0.01 M phosphate buffer (pH 7.0) containing 30% to 0% saturated ammonium sulfate containing M EDTA.

The active fractions were collected, ammonium sulfate was added to 30% saturation, and then 0.1 mM was prepared in advance.
Re-pass through a column of Butyl Toyopearl 650M equilibrated with 0.01 M phosphate buffer (pH 7.0) containing EDTA and 30% saturated ammonium sulfate,
0.01M Phosphate buffer (pH 7.0) containing 30% to 0% saturated ammonium sulfate containing 1 mM EDTA
The enzyme was eluted with a linear concentration gradient of.

The active fractions were collected, dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and concentrated by ultrafiltration. This is 0.1 mM ED
It was attached to a column of Mono Q HR5 / 5 initialized with 0.02 M phosphate buffer (pH 7.0) containing TA,
0.02 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA to 0.1 mM EDTA and 0.2
0.02M phosphate buffer containing M NaCl (pH 7.
Elution was performed with a linear concentration gradient of 0) at a flow rate of 1.0 ml / min.

The active fractions were collected, concentrated by ultrafiltration, and then ammonium sulfate was added so that the concentration became 30%. This was mixed with 0.1 M phosphate buffer (pH 7.%) containing 0.1 mM EDTA and 30% saturated ammonium sulfate.
0) Phenyl Superose HR initialized
Attach to a 5/5 column and include 0.1 mM EDTA 3
The enzyme was eluted with a linear concentration gradient of 0.1 M phosphate buffer (pH 7.0) containing 0% to 0% saturated ammonium sulfate at a flow rate of 0.5 ml / min.

Thus, the peptidase was purified about 300 times in a yield of about 3%. Table 3 shows the specific activity and recovery rate in this purification process. This enzyme preparation was homogeneous on SDS-polyacrylamide gel electrophoresis.

[0036]

* 1 unit is 1 μmol of (DP
he) The amount of enzyme that yields ₄ to 2 μmol of (D-Phe) ₂ .

The properties of the peptidase thus purified are shown below. (1) Specific activity of purified enzyme: 126 (2) Substrate specificity: It acts specifically on (D-Phe) ₄ and does not act on (L-Phe) ₄ . In addition, (D-
Phe) ₆ , (D-Phe) ₃ , (D-Phe) ₂ , N
-T-butoxycarbonyl- (D-Phe) ₄ , Nt
It also acts on -butoxycarbonyl- (D-Phe) ₃ and Nt-butoxycarbonyl- (D-Phe) ₂ . (3) Optimum pH: pH 9-12 (Fig. 1) (4) pH stability: Stable at pH 5-12 (40 ° C, 3
Time treatment) (Fig. 2) (5) Optimum temperature: around 50 ° C (Fig. 3)

(6) Temperature stability: stable up to 40 ° C. (p
H7.0, treatment for 60 minutes) (Fig. 4) (7) Effect of metal ion and inhibitor: This enzyme
Fe is activated by the addition of a divalent metal such as Mg ²⁺ ,
It is inhibited by ²⁺ and Cu ²⁺ . (8) Molecular weight: high performance liquid chromatography (TSK
It is calculated to be about 38,000 by gel G-3000SW). (9) Molecular weight of subunit: Calculated to be about 39,000 by SDS-polyacrylamide gel electrophoresis. (10) Homogeneity: SDS-polyacrylamide gel electrophoresis (10.0%, pH 7.2) gives a single band as shown in FIG.

Example 2 Bacillus cereus DF4-B
(D-Phe) ₄ Decomposition Reaction by Partially Purified Enzyme of Using peptidase purified to step 7 shown in Table 3 as follows. That is, Tris-hydrochloric acid buffer solution (pH 9.0) 50 μmol, magnesium sulfate 1
μmol, DMSO 10 μl, (D-Phe) ₄ 1 μm
mol, peptidase 0.4 unit containing a reaction solution 0.
5 ml were reacted at 30 ° C. and at each time HClO ₄
After the reaction was stopped at 1, the substrate (D-Phe) ₄ and the produced (D-Phe) ₂ , D-Phe were quantified using high performance liquid chromatography. The result was as shown in FIG.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between pH and reaction rate of peptidase produced by Bacillus cereus DF4-B strain.

FIG. 2 is a graph showing pH stability of peptidase produced by Bacillus cereus DF4-B strain.

FIG. 3 is a graph showing the temperature and reaction rate of peptidase produced by Bacillus cereus DF4-B strain.

FIG. 4 is a graph showing the temperature stability of peptidase produced by Bacillus cereus DF4-B strain.

FIG. 5 is a photograph of SDS-polyacrylamide gel electrophoresis (10.0%, pH 7.2) showing the homogeneity of peptidase produced by Bacillus cereus DF4-B strain.

FIG. 6 is a graph showing the time course of (D-Phe) ₄ degradation reaction by peptidase produced by Bacillus cereus DF4-B strain.

Claims (3)

[Claims] 1. A peptidase having the following properties: (a) Action Hydrolyze an oligopeptide consisting of D-phenylalanine (b) Substrate specificity D-phenylalanine-specific oligopeptide having no protecting group or having a protecting group Action, L-
Does not act on oligopeptides consisting of phenylalanine (c) pH stability Stable at 5-12 (d) Optimum pH 9-12 (e) Optimum temperature around 50 ° C (f) Molecular weight In high performance liquid chromatography gel filtration method About 3
A subunit having a molecular weight of 8,000 and having a molecular weight of about 39,000 by SDS-polyacrylamide gel electrophoresis is shown. 2. A microorganism Bacillus cereus having a peptidase-producing ability that specifically hydrolyzes an oligopeptide composed of D-phenylalanine is cultured, and the oligopeptide composed of D-phenylalanine is specifically hydrolyzed from the culture product. A method for producing peptidase by a microorganism, which comprises collecting the peptidase. 3. A microorganism Bacillus cereus DF4-B having a peptidase-producing ability that acts specifically on a peptide consisting of D-phenylalanine and a derivative thereof.