JPH09173062A - D-amino acid oligopeptide synthase and microorganism capable of producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject new enzyme useful for synthesis, etc., of a D-amino acid oligopeptide useful as a D-amino acid-containing antibiotic substance or a peptide hormone, etc., by culturing a new actinomyces having the ability to produce a D-amino acid oligopeptide synthase. SOLUTION: This new D-amino acid oligopeptide synthase is capable of catalyzing a synthetic reaction for an oligopeptide by using a D-amino acid as a substrate, has about 7.0 optimum pH, about 40-45 deg.C range of the optimum action temperatures, 6.5 isoelectric point and about 60kDa molecular weight when measured by an acrylamide get electrophoresis. The synthase is useful for synthesis, etc., of a D-amino acid oligopeptide useful as a D-amino acid- containing antibiotic substance, a peptide hormone, etc. The enzyme is obtained by culturing an actinomyces, having the ability to produce an enzyme capable of catalyzing a synthetic reaction of the D-amino acid oligopeptide and belonging to the genus Saccharothrix [e.g. Saccharothrix sp. (FERM P-5341)] by using a D-amino acid as a substrate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an enzyme which catalyzes an oligopeptide synthesis reaction using a D-amino acid as a substrate, an actinomycete which produces this enzyme, and a method for synthesizing a D-amino acid oligopeptide using this enzyme or actinomycete. .

[0002]

2. Description of the Related Art Up to now, an enzyme which catalyzes an oligopeptide synthesis reaction using a D-amino acid as a substrate and a microorganism capable of producing such an enzyme have not been known.

[0003]

The present invention is useful for the synthesis of D-amino acid oligopeptides that can be used for D-amino acid-containing antibiotics and peptide hormones, which are expected to be widely used in the pharmaceutical field. The object of the present invention is to provide an enzyme for synthesizing a D-amino acid oligopeptide, a microorganism producing the enzyme, and a method for synthesizing a D-amino acid oligopeptide using the enzyme or the microorganism. So far, an enzyme that catalyzes an oligopeptide synthesis reaction using a D-amino acid as a substrate and a microorganism that produces the enzyme have not been known.

[0004]

The present inventors have found that Saccharo
The present invention was completed by isolating an enzyme that synthesizes a D-amino acid oligopeptide from a newly isolated actinomycete belonging to the genus thrix .

The enzyme of the present invention is an enzyme which catalyzes an oligopeptide synthesis reaction using D-amino acid as a substrate (hereinafter referred to as the present enzyme). This achieves the above objective.

The present enzyme has the following characteristics. (1) Optimum pH: about 7.0. (2) Optimum temperature range of action: about 40 ° C to 45 ° C. (3) Isoelectric point: 6.5. (4) Thermal stability: Stable up to about 55 ° C when kept at pH 7.0 for 30 minutes. (5) Molecular weight by 10% SDS polyacrylamide gel electrophoresis: about 60 KDa.

The method for synthesizing a D-amino acid oligopeptide includes the step of reacting the D-amino acid with the present enzyme which catalyzes the oligopeptide synthesis reaction using the D-amino acid as a substrate. This achieves the above objective.

In this method for synthesizing a D-amino acid oligopeptide, the D-amino acid is an ester of the D-amino acid. More preferably, it is a hydrophobic D-amino acid ester.

The enzyme is produced by the actinomycete of the present invention (hereinafter referred to as the actinomycete). A particularly preferred actinomycete is Saccharothrix deposited on December 13, 1995 at the Institute of Biotechnology, Institute of Biotechnology (abbreviated as Life Research Institute).
sp. (Life Science Research Institute No. 15341; FERMP-15341). This achieves the above objective.

The method for synthesizing a D-amino acid oligopeptide also includes a step of contacting the D-amino acid with the present actinomycete which produces an enzyme for synthesizing the D-amino acid oligopeptide. This achieves the above objective.

In this method for synthesizing a D-amino acid oligopeptide, the D-amino acid is preferably an ester of the D-amino acid. More preferably, the D-amino acid is preferably a hydrophobic amino acid, and more preferably esterified.

The enzyme is an inhibitor, diazoacetyl-DL.
-Norleucine methyl ester, 1,2-epoxy-3
It is inhibited by-(p-nitrophenoxy) propane and diisopropylfluorophosphate.

The D-amino acid oligopeptide synthase having the above characteristics and the actinomycete producing the same have not been known so far.

Hereinafter, the present invention will be described in detail.

The enzyme of the present invention is, for example, Saccharothrix.
Actinomycete, Saccharothrix sp.
Issue: FERM P-15341). This microorganism is
The present inventors have isolated a new microorganism as a result of screening the bacteria producing the enzyme which catalyzes the oligopeptide synthesis reaction using D-amino acid ester as a substrate from the soil. The properties of Saccharothrix sp. Are described below.

(1) Morphology and culture Table 1 shows the mycological properties of the microorganism of the present invention.

[0017]

[Table 1]

(Yeast / malt agar medium) 4 after the start of culture
At 8 hours, both the front and back of the colony were opaque cream yellow, the colony was round,
It grows to a diameter of 3 mm and is free of aerial hyphae. No spore formation is observed. These characteristics indicate that the microorganism of the present invention belongs to actinomycetes. The more detailed mycological properties of the actinomycetes of the present invention are as follows.

It contains meso-diaminopimelic acid,
It does not contain mycolic acid and forms mycelium divided into Bacillus-like elements. In addition, the detected sugar is
Galactose, mannose, and rhamnose. All these characteristics indicate that the actinomycete belongs to the genus Saccharothrix .

The growth range of this actinomycete is broth medium (1
% Polypeptone, 1% bonito meat extract, 0.5% NaCl), the temperature is about 25 to 32 ° C., the pH is about 7 to 8, and the suitable growth temperature is 27 to 30 ° C. A particularly preferred growth condition is 27 ° C.
The pH is about 7.0.

Next, the conditions for producing the enzyme of the present invention from the present actinomycete will be described. The actinomycete can produce the enzyme by liquid culture. The medium for growing the present actinomycete is not particularly limited, and an ordinary liquid medium is used. Glucose may be used as the carbon source. Polypeptone or bonito meat extract may be used as the nitrogen source. These medium components may have a concentration that does not inhibit the growth of the actinomycete, the carbon source is 0.5 to 5% by weight, preferably 1 to 3% by weight, the nitrogen source is 1 to 3% by weight, preferably 1 to
2% by weight.

The pH of the medium is adjusted to 7.0 and sterilized before use. The culture temperature may be any temperature at which actinomycetes can grow, and is 25 to 32 ° C, preferably 27 to 30 ° C in practice. When the present actinomycete is liquid-cultured, aeration culture or shaking culture is preferable. The culture time varies depending on various culture conditions, but in the case of aeration or shaking culture, it is usually preferably 3 to 4 days.

(2) Separation and purification of the present enzyme from the culture broth To separate and purify the present enzyme which catalyzes the oligopeptide synthesis reaction using the D-amino acid of the present invention as a substrate from the culture broth of the present actinomycete, known purification Methods such as dialysis, salting out, various column chromatography, isoelectric focusing, gel filtration, and electrophoresis can be used alone or in combination.

After culturing, insoluble matters such as bacterial cells are removed by centrifugation, filtration with a filter paper or filter cloth, etc., and a culture solution containing the present enzyme (crude enzyme solution) is recovered. The enzyme can be purified from the obtained crude enzyme solution by a commonly used enzyme purification method such as concentration, ammonium sulfate fractionation (salting out), dialysis, and various chromatographies. For example, the above crude enzyme solution is subjected to salting out, ion exchange chromatography, isoelectric focusing,
By subjecting it to gel filtration in order, a fraction containing the purified highly active enzyme of the present invention can be obtained.

(3) Measurement of the activity of the present enzyme The activity of the present enzyme in the present invention was determined by using D-phenylalanine methyl ester (D-PheOMe) as a substrate and 1 μmol of D-Phe-D-Phe- per hour. OM
The amount of enzyme that produces e was defined as 1 unit (U; unit). Specifically, in the present specification, 0.5 M of 0.8 M D-PheOMe dissolved in 0.1 M phosphate buffer of pH 7.0 was used.
Add 1.5 ml of sample solution and incubate at 30 ℃ for 30 minutes. Then stop solution (0.75N HCl in 0.2M phosphate buffer, pH 2.
The reaction was stopped by adding 2 ml of 1), diluted 10 times with 50% acetonitrile, and the product was analyzed by high performance liquid chromatography (HPLC). The measurement wavelength was 220 nm. HPL
C uses a Chemcosolve 5-ODS-UH column and is 0.0
Elution was performed with a concentration gradient of 30% to 80% acetonitrile containing 5% trifluoroacetic acid. At this time, the flow rate is 0.8 ml / min,
The column temperature was 40 ° C.

The present invention is described in more detail in the following examples, but these do not limit the present invention in any way.

[0027]

【Example】

Example 1 (culture of Saccharothrix sp.) 100 mL of broth medium (1
% Polypeptone, 1% Skipjack Meat Extract, 0.5% NaCl pH
In a 500 mL shake flask (30) containing 7), 27 ℃,
Culture was carried out with shaking for 4 days (120 rpm, reciprocal shaking).

Example 2 (Purification of this enzyme) The supernatant obtained by centrifuging the culture solution obtained in Example 1 above (at 6,000 rpm for 30 minutes) was used as a crude enzyme solution. Ammonium sulfate was added to this crude enzyme solution to 80% saturation, and ammonium sulfate salting out was performed. The resulting precipitate was dissolved in 10 mM phosphate buffer and dialyzed against the same buffer to obtain an enzyme solution.

The above solution was added to 10 mM phosphate buffer (pH 7.0)
It was tested on DEAE-Toyopearl 650M (manufactured by Tosoh Corporation) equilibrated with. After washing the column with the same buffer, the present enzyme adsorbed on the column was eluted by a concentration gradient with the same buffer (pH 7.0) containing 1M NaCl.

Subsequently, the above-mentioned active fraction was subjected to isoelectric focusing. Electrophoresis was carried out at 500 V for 20 hours and subsequently at 700 V for 20 hours using Pharmalite (pH 5-8, manufactured by Pharmacia). This active fraction was then tested on Sephadex G-100 (Pharmacia) equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl. This enzyme was eluted with the same buffer and isolated and purified.

Table 2 shows the protein amount, transfer activity, specific activity, recovery rate, and degree of purification of this enzyme in each of the above purification steps.
Shown in

[0032]

[Table 2]

Example 3 (Molecular weight of this enzyme) 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) method (Laemmli, UK, Nature, 227, 6)
80 (1970)) and the molecular weight of this enzyme was measured.

The purified present enzyme obtained in Example 2 and 6 kinds of proteins known as molecular weight markers (molecular weights of about 97,400, 66,300, 42,400, 30,000, 20,100 and 14,400) were simultaneously electrophoresed. After completion of the electrophoresis, the product was stained with Coomassie Brilliant Blue, and the molecular weight of the present enzyme was measured from the relative migration distance between the present enzyme and each molecular weight marker.

By this SDS-PAGE, this enzyme showed a single band. The molecular weight of this enzyme on SDS-PAGE is about 60,0.
00.

Example 4 (Adequate temperature range of action and thermostability of the present enzyme) (1) Optimum temperature range of action The activity of the purified present enzyme obtained in the above Example 2 was measured by various methods in the above activity measurement. Temperature conditions (20, 25, 30,
The results measured at 35, 40, 50 ° C) are shown in Fig. 1. The horizontal axis in the figure represents the reaction temperature (° C.), and the vertical axis represents the relative activity (%) when the maximum activity is 100. The optimum temperature for this enzyme is 4
It is 0 to 45 ° C.

(2) Thermostability The purified present enzyme obtained in the above Example 2 was treated with various concentrations under different temperature conditions (20, 30, 35, 4) in 10 mM phosphate buffer (pH 7.0).
After incubating for 30 minutes under 0, 45, 50, 55, 60 ° C), the activity was measured at 30 ° C as described above. The results are shown in FIG. In the figure, the horizontal axis represents the treatment temperature (° C), and the vertical axis represents the relative residual activity (%) when the maximum activity value is 100. The enzyme retains 100% or more of the enzyme activity up to the treatment temperature of 55 ℃ and is stable.

Example 5 (Optimum pH and stable pH range of this enzyme) (1) Optimum pH Depending on the pH range, 0.2M acetate buffer, 0.2M phosphate buffer or 0.1M borate buffer was used. Use a pH of 3.0 to 10 (3.0, 4.
Buffer solution adjusted to 0, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0), the purified present enzyme obtained in Example 2 above,
Mix 8M D-Phe-OMe 2: 1: 1 at 30 ° C.
After 30 minutes of reaction, the activity of this enzyme was measured. Fig. 3 shows the results.
Shown in 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. Optimum pH of this enzyme
Is about 7.0.

(2) Stable pH range The pH of the enzyme solution obtained in Example 2 was adjusted according to the pH range.
2.0 to 14.0 (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 0.2M phosphate buffer, 0.2M phosphate buffer, 0.1M borate buffer)
7.0, 7.5, 8.0, 9.0, 10.3, 13.0), 15 at 25 ℃
Incubated for hours. Next, adjust the pH of each treatment solution to 7.0.
After readjustment to 1, the residual activity of this enzyme was measured. FIG. 4 shows the results. The horizontal axis in the figure represents the treated pH, and the vertical axis represents the relative residual activity (%) when the maximum activity value is 100. The stable pH range of this enzyme is 5.0 to 10.0.

Example 6 (Isoelectric point of the present enzyme) The isoelectric point of the present enzyme was measured by the isoelectric focusing method. As a result of the measurement, the isoelectric point of this enzyme was about 6.
5

Example 7 (Substrate specificity of the present enzyme) The substrate specificity of the present enzyme purified in the above Example 2 was examined. As substrate, various 0.2M
Using D-amino acid methyl ester,
The reaction mixture is allowed to act at ℃, pH 7.0 and the reaction mixture is subjected to thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).
Was analyzed. Specifically, 20 μl of 0.13 M phosphate buffer (pH 7.0) containing 4 μmol of the above substrate was reacted at 30 ° C. for 30 minutes, and then the reaction stop solution (0.75 N HC in 0.2 M phosphate buffer was used).
l, pH 2.1), and then add TLC and H
Analyzed by PLC. Table 3 shows the results.

[0042]

[Table 3]

The present enzyme showed particularly high specificity for hydrophobic D-amino acid methyl esters such as D-phenylalanine methyl ester and D-tryptophan methyl ester.

Example 8 (Inhibitor of the present enzyme) The inhibitor of the present enzyme purified in the above Example 2 was examined. As an inhibitor, diazoacetyl-D
L-norleucine methyl ester, 1,2-epoxy-
3- (p-nitrophenoxy) propane, diisopropylfluorophosphoric acid, phenylmethanesulfonyl fluoride, 4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride, dithiothreitol, ethylenediaminetetraacetic acid salt, trans-epoxysuccinyl −
Using L-leucylamide (4-guanidino) butane, p-chloromercuribenzoate, phosphoramidone,
The inhibitory activity was examined by measuring the residual activity after mixing these inhibitors with this enzyme and leaving it at 30 ° C. for 30 minutes. The inhibitory activity was represented by setting the residual activity after standing at 30 ° C. for 30 minutes when no inhibitor was added as 100. 1 mM of CuCl ₂ was allowed to coexist with diazoacetyl-DL-norleucine methyl ester. 1,2-epoxy-3- (p-
The nitrophenoxy) propane was left overnight at 20 ° C. Table 4 shows the results.

[0045]

[Table 4]

The enzyme is diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3- (p-
Strongly inhibited by nitrophenoxy) propane and diisopropylfluorophosphate.

Example 9 (Hydrolytic activity of the present enzyme) The hydrolytic activity of the present enzyme purified in the above Example 2 was examined. As a substrate, L-phenylalanyl-L-phenylalanine, L-phenylalanyl-D-phenylalanine, D-phenylalanyl-L-phenylalanine, D-phenylalanyl-D-
Phenylalanine, D-phenylalanyl-D-proline, carbobenzoxy-D-phenylalanyl-L-phenylalanyl-glycine, carbobenzoxy-D-phenylalanyl-L-valine, acetyl-D-phenylalanine Nyl-L-tyrosine, D-phenylalanyl-L-
Valine-p-nitroanilide, D-phenylalanine-
Using p-nitroanilide, glycyl-D-phenylalanine-β-naphthylamide, these substrates were treated with 10 m.
In M phosphate buffer (pH 7.0) at a substrate concentration of 5 mM,
It was mixed with this enzyme and reacted at 30 ° C. for 1 hour. After the reaction, the reaction solution and the reaction stop solution (0.75N HCl in 0.2M phosphate buffer, pH 2.
The reaction was quenched by mixing 1) 1: 1 and the product was analyzed by TLC and amino acid analyzer. D-Phenylalanine-p-nitroanilide was added and the increase in absorbance at 405 nm was measured. Table 5 shows the results.

[0048]

[Table 5]

This enzyme did not hydrolyze any substrate.

Example 10 (Synthesis of oligopeptide by this enzyme) 0.5 ml of 0.8 M D-PheOMe dissolved in 0.1 M phosphate buffer of pH 7.0
Add 5 ml of sample solution and react at 30 ° C. for 0 and 1 hour. Then, stop solution (0.75N HC in 0.2M phosphate buffer)
2 ml of pH 2.1) is added to stop the reaction. The product was analyzed by HPLC as above. Results are shown in FIG.
(A) is the analysis result of the reaction solution after 0 hour, and (B) is the analysis result of the reaction solution after 1 hour by high performance liquid chromatography.
The horizontal axis in the figure represents the retention time.

1 to 7 depending on the retention time in the above HPLC
In order to estimate the substance corresponding to the peak of 1, the di, tri, tetra-L-Phe and their methyl esters were analyzed under the same conditions as the standard, and the retention times were compared. Then, the peak fractions of 1 to 7 were collected by HPLC,
The molecular weight was measured by mass spectrum. Furthermore, the amino acid composition and stereoisomerism of the hydrolyzate of each peak were measured. Table 6 shows the results.

[0052]

[Table 6]

It was confirmed that all the products were D-Phe oligopeptides.

[0054]

INDUSTRIAL APPLICABILITY According to the present invention, an enzyme that synthesizes a D-amino acid oligopeptide and an actinomycete that produces this enzyme are provided. This enzyme catalyzes a peptide synthesis reaction using D-amino acid as a substrate. Therefore, the present enzyme is useful for the synthesis of D-amino acid oligopeptides that can be used for D-amino acid-containing antibiotics and peptide hormones, which are expected to be widely used in the pharmaceutical field. Since the present enzyme is produced by culturing the present actinomycete, the present enzyme can be provided inexpensively in a large amount and can be widely used for pharmaceutical or medical applications.

[Brief description of the drawings]

FIG. 1 shows the relative activity when this enzyme activity is measured in the range of 20 to 50 ° C.

FIG. 2 shows the relative residual activity of this enzyme measured after incubation for 30 minutes at 20 to 60 ° C.

FIG. 3 shows the relative activity when the present enzyme activity is measured in the pH range of 2.5 to 10.

FIG. 4 shows the relative residual activity of this enzyme measured after incubation for 15 hours at 25 ° C. under conditions of pH 3 to 13.0.

FIG. 5 shows the results of high performance liquid chromatography analysis of the products after oligopeptide synthesis reaction with the present enzyme using D-PheOMe as a substrate.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 1/20 C12R 1:01) (C12P 21/02 C12R 1:01) (72) Inventor Tetsuo Muro Kyoto 8 Unilife Uji A Building No. 310, No. 32, Gokasho Nishigawara, Uji City, Fukushima Prefecture (72) Inventor Tetsuo Sugihara 6-87 Senjo, Itami City, Hyogo Prefecture (72) Inventor Takenishi Shigeyuki, Nara City, Nara City 1 Chome 6 Heijo No. 1 Housing Complex 19-302 (72) Inventor Yuji Shimada 4-231 Kushiya-cho Higashi, Sakai City, Osaka Prefecture (72) Toshihiro Nagao Ichiro Higashi 1-Chome, Ikuno-ku, Osaka City, Osaka Prefecture (72) (72) Inventor Yoshio Tominaga 2-7-2 Utashima, Nishiyodogawa-ku, Osaka-shi, Osaka

Claims (19)

[Claims] 1. An enzyme which catalyzes an oligopeptide synthesis reaction using a D-amino acid as a substrate. 2. The enzyme according to claim 1, wherein the enzyme is D-amino acid transferase. 3. The enzyme according to claim 1, wherein the optimum pH of the enzyme is about 7.0, and the optimum temperature range of action is about 40 ° C. to 45 ° C. 4. The enzyme according to claim 1, wherein the enzyme has an isoelectric point of 6.5. 5. The enzyme according to claim 1, which is stable up to about 55 ° C. when the enzyme is kept at pH 7.0 for 30 minutes. 6. The enzyme has a molecular weight of about 60 KDa as measured by acrylamide gel electrophoresis.
An enzyme according to claim 1. 7. A method for synthesizing a D-amino acid oligopeptide, which comprises a step of reacting an enzyme that catalyzes an oligopeptide synthesis reaction with a D-amino acid as a substrate and a D-amino acid. 8. The method for synthesizing a D-amino acid oligopeptide according to claim 7, wherein the D-amino acid is an ester of a D-amino acid. 9. The method for synthesizing a D-amino acid oligopeptide according to claim 7, wherein the D-amino acid is a hydrophobic amino acid. 10. The method for synthesizing an oligopeptide of a D-amino acid according to claim 8, wherein the D-amino acid is a D-amino acid methyl ester. 11. An actinomycete which produces an enzyme that catalyzes a D-amino acid oligopeptide synthesis reaction using a D-amino acid as a substrate. 12. The actinomycete is Saccharothrix sp. (Science and Life Science Institute No. 15341; FERM P-15341).
Actinomycetes described in. 13. The actinomycete according to claim 11 or 12, wherein the enzyme is a D-amino acid transferase. 14. A method for synthesizing a D-amino acid oligopeptide, which comprises a step of contacting a D-amino acid with an actinomycete that produces an enzyme that synthesizes the D-amino acid oligopeptide. 15. The method according to claim 14, wherein the actinomycete belongs to the genus Saccharothrix . 16. The method for synthesizing a D-amino acid oligopeptide according to claim 14 or 15, wherein the D-amino acid is an ester of a D-amino acid. 17. The method for synthesizing a D-amino acid oligopeptide according to claim 14, 15, or 16, wherein the D-amino acid is a hydrophobic amino acid. 18. The method for synthesizing a D-amino acid oligopeptide according to claim 16, wherein the D-amino acid is a D-amino acid methyl ester. 19. The method according to claim 15, wherein the actinomycete is Saccharothrix sp. (Life Science Research Institute No. 15341; FERM P-15341).
The method for synthesizing a D-amino acid oligopeptide according to 1.