JPH0775540B2 - Method for producing proline dipeptidase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for producing a proline dipeptidase of the present invention. More particularly, it relates to a novel method for producing proline dipeptidase by a novel strain. Proline dipeptidase (EC.3.4.13.9) decomposes proline-containing dipeptides that are hardly decomposed by proteases and other peptidases.
It is also a very valuable enzyme for use in the production of amino acid-containing substances such as diet food.

Conventionally, as a method for producing proline dipeptidase (EC.3.4.13.9), pigs (Biochimica et Biophysica Acta), 327 , 457
-470 (1973), cattle (Journal of Biochemitry), 94 , 1889-1896
(1983)), Monkey (Indian Journal of Biochemistry and Biophysics (Indian)
Journal of Biochemistry and Biophysics), 11 , 7-1
1 (1974)) and other methods such as germinated soybean (Pharmaceutical Journal, 97 (1), 111-115 (1977)) and other plants. In addition, as a method for producing a proline dipeptidase of microbial origin, Streptococcus (Agricultural and Biological Chemistry) is used.
ural and Biological Chemistry), 48 (12), 3035-30
40, (1984)), Arthrobacter genus (Acta Chemica Sca)
ndinavica), 25 , 847-858 (1971)), genus Escherichia (Acta Biotechnologyka 2 (A
cta Biotechnologica2), 2 , 161-170 (1982), Neurospora genus (Biochimica et Biophysica Act)
a), 370 , 530-540 (1974)) have been reported.

The present inventors have conducted a search for a microorganism capable of producing the above-mentioned proline dipeptidase in high yield, and as a result, glycyl-
The present invention has been completed by finding that a microorganism belonging to the genus Pseudomonas or the genus Trichosporone that can assimilate L-proline as a carbon source and a nitrogen source produces proline dipeptidase.

That is, the present invention is a method for producing proline dipeptidase, which comprises culturing a proline dipeptidase-producing bacterium belonging to the genus Pseudomonas or the genus Trichosporon in a medium, and separating and collecting proline dipeptidase from the obtained culture. . Hereinafter, the present invention will be described in detail.

The strain used in the present invention may be any strain as long as it is a strain belonging to the genus Pseudomonas or the genus Trichosporone and having the ability to produce proline dipeptidase,
A variant or mutant strain of these bacteria may also be used. Pseudomonas fluorescens IFO12 is a specific example of a strain belonging to the genus Pseudomonas and capable of producing proline dipeptidase.
055 is mentioned. In addition, as a specific example of a strain belonging to the genus Trichosporon and capable of producing proline dipeptidase, Trichosporon sp.
p.) 107-1.

The Pseudomonas fluorescens IFO12055 is a publicly known strain and can be obtained from the Fermentation Research Institute. The Trichosporon SP 107-1 strain is a strain newly obtained from soil in Himeji City, Hyogo Prefecture, and its mycological properties are as follows. The Yeast-a Taxonomic Study 3rd ed Krger-v was used to examine the mycological properties.
The method and medium composition described in an Rij (1984) Elsevier) were used.

Mycological properties of Trichosporon SP 107-1 (a) Growth state in each medium Yeast extract / malt extract liquid culture After culturing at 30 ° C. for 3 days, the cells are cylindrical cells and form budding cells and mycelia. The size of the cell is (7-11)
× (5-6) μ Yeast extract / malt extract agar culture Cultivated at 30 ° C for 3 to 20 days, good growth, yeast-like in the early stage of culture, but immediately colony surface became powdery and aerial hyphae were observed. Become a state. The colony is yellowish white, matte, and has a convex cone shape. Does not grow at 37 ° C.

Slant culture on potato-glucose agar medium With mycelia, no pseudohyphae, vegetative cells only divide and no budding cells. The tip of the hypha divides to form a meiosis.

(B) Ascospore formation None was observed by any of the yeast extract / malt extract agar medium, the Gorodkova improved medium, and the Mc Clary method.

(C) Formation of injection spores Not observed on yeast extract / malt extract agar medium.

(D) Physiological properties 1) Optimal growth conditions pH 5.5 Temperature 25 ° C 2) Growth range Does not grow at 37 ° C 3) Potassium nitrate does not grow as a single nitrogen source.

4) Decompose fat.

5) Urease negative 6) Do not liquefy gelatin.

7) Does not produce carotenoids.

8) Does not generate organic acid.

9) Does not produce starch-like substances.

10) Does not decompose arbutin.

11) Grows well in vitamin-deficient medium of squid ham.

12) No vitamin requirement.

(E) Fermentability of each carbon source and assimilation ability Fermentability of sugar 1) D-glucose-2) sucrose-3) lactose-4) D-galactose-5) maltose-6) raffinose-of a single carbon compound Assimilation ability 1) D-glucose + 2) D-xylose + 3) D-mannitol + 4) D-fructose + 5) D-galactose + 6) saccharose ± 7) L-arabinose-8) raffinose-9) Inositol-10) D-ribose-11) Soluble starch-12) Arbutin-13) α-Ketogluconate potassium-14) D-sorbit-15) Trehalose ± 16) Maltose ± 17) Lactose-above mycological and physiological Due to various properties, this strain forms (1) a true mycelium, and the mycelium later divides into segmented spores.
(2) It also grows by budding. (3) The cells are cylindrical. (4) Ejected spores, ascospores, Teliospores and Basidiospor
e) does not form. (5) Does not form carotenoid primary pigment. (6) Does not grow like sarcinus-like aggregates. Based on the above results, it was determined that the bacteria belong to the genus Trichosporone.

Furthermore, when this bacterium is compared with the bacteria belonging to the known Trichosporon genus, Trichosporon brassicae (T.brassicae), acreatum (T.aculeatum), and capitatum (T.capitat)
um), T. erinse and T. erinse.
sericeum). However, Brassica flies are positive for arbutin degrading in urease test and do not grow in vitamin-deficient medium. Acreatum has a positive urease test and arbutin degradability, and does not grow in a vitamin-deficient medium. Capitatum grows at 37 ° C and does not grow on vitamin-deficient medium. Erinse is a weak and slow fermenter of glucose, is positive for arbutin degrading and does not grow in vitamin deficient medium. Cericeum tested positive for urease and does not grow in vitamin deficient medium. Therefore,
The above species and this bacterium are different. From the above points, this bacterium was identified as a new strain belonging to the genus Trichosporon.

In addition, the above-mentioned Trichosporon SP 107-1 can be obtained from the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, by the Micromachine Research Institute No. 9489 (FERM P
−9489) has been deposited.

Next, as the medium used for producing proline dipeptidase using the above-mentioned strain used in the present invention, a medium containing a carbon source, a nitrogen source, and an inorganic salt can be appropriately used. A medium in which vitamins are further added to the medium can also be used. As the carbon source, for example, glycerin, sodium acetate and any other usable strains of the above-mentioned strains can be used,
It is desirable to add it at a rate of 0.5 to 2% (wt / vol) per medium. As the nitrogen source, an inorganic nitrogen source such as ammonium sulfate or ammonium chloride, or a natural nitrogen source such as peptone or yeast extract can be used.
It is desirable to add in the ratio of. As the inorganic salt, metal salts such as magnesium, calcium, magazine and ferric iron can be used. Magnesium is 0.05-0.1
%, Calcium, magazine, ferric iron is preferably added in the proportion of 0.001 to 0.003%. In addition, glycyl-L-proline, polypeptone, etc. were used as inducers in the medium at 0.5%.
When added at a rate of ~ 1%, the production amount of proline dipeptidase is increased.

Culturing is usually preferably carried out for 18 to 40 hours under shaking or aeration and stirring. In this case, the pH of the medium is about 5-8,
The culture temperature is preferably 20 to 40 ° C. After completion of the culture, the produced proline dipeptidase is mainly contained in the cells, so in order to collect the proline dipeptidase from the culture solution, the culture solution is filtered or centrifuged to collect viable cells, The cells can be disrupted by a known method such as French press, ultrasonic wave, alumina, etc. to obtain a cell-free extract having proline dipeptidase activity, which can be used as an enzyme preparation. Among the strains used in the present invention, the enzymatic properties of proline dipeptidase produced by Trichosporon sp. 107-1 and Pseudomonas fluorescens IFO 12055 will be described.

* Properties of proline dipeptidase derived from Trichosporon sp. 107-7 Action: Amino acid and L by acting on aminoacyl-L-proline
-Produces proline.

Substrate specificity; most preferably acts on L-methionyl-L-proline,
Glycyl-L-proline, L-leucyl-L-proline, L
-Ceryl-L-proline, L-valyl-L-proline, L-
Histidyl-L-proline, L-isoleucyl-L-proline, L-lysyl-L-proline, L-phenylalanyl-
It also acts on L-proline, L-proline-L-proline and the like.

Activator: Remarkably activated by Mn ²⁺ ions, and also activated by Co ²⁺ ions. Also, the M ²⁺ ion concentration is
1-2 mM is preferred.

Inhibitor: N -ethylmaleimide (hereinafter abbreviated as NEM), almost completely inhibited by iodoacetamide, ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), ethylene glycol bis (β-
It is also inhibited by aminoethyl) tetraacetic acid (hereinafter abbreviated as EGTA).

★ Properties of Pseudomonas fluorescens IFO12055-derived proline dipeptidase Action: Amino acid and L by acting on aminoacyl-L-proline
-Produces proline.

Activator; activated by Mn ²⁺ ions.

Inhibitor: Completely inhibited by EDTA and EGTA, but not by NEM and iodoacetamide.

In addition, the activity of proline dipeptidase is
Hydrochloric acid buffer pH 8.0 50 μmol, glycyl-L-proline 10 μ
mol, 500 μl of reaction solution containing 5 μmol of manganese chloride
The reaction was performed for 30 and 30 minutes, and the produced glycine was quantified by an amino acid analyzer. However, 1 U was the amount of enzyme that catalyzes the production of 1 μmol of glycine per minute.

According to the present invention, a novel process for producing proline dipeptidase can be found, which is industrially significant. Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

(Example) Example 1 Production of proline dipeptidase by Trichosporon sp. 107-1 (1) (Effect of nitrogen source on enzyme productivity) Glycerin 1% and glycyl-L-proline 0.5 in medium A
%, And poly (peptone), yeast extract, or ammonium sulfate as a nitrogen source, 0.5% of Trichosporon SP-107-1 was cultivated for 20 hours at 30 ° C in 5 ml of pH 5.0 medium, and the cells were separated by centrifugation. Recovered. The collected cells were suspended in 1 ml of a buffer solution having a concentration of 50 mM Tris-hydrochloric acid buffer pH 7.5, 1 mM EDTA, 0.02% 2-mercaptoethanol, and the cells were disrupted by ultrasonic waves. The crude enzyme solution obtained by removing cell debris and unbroken cells by centrifugation was diluted with the same buffer solution 2
It dialyzed twice for the time. The proline dipeptidase activity of this cell-free extract was measured by the above method, and the results shown in Table 1 were obtained. The specific activity is the enzyme activity per 1 mg of protein (U / mg), and the protein determination of the crude enzyme solution was carried out by the Lowry method (Journal of Biological Chemistory), 193 , 265,
(1951)). The total activity was defined as the activity per medium.

Example 2 Production of proline dipeptidase by Trichosporon sp. 107-1 (2) (Effect of carbon source on enzyme productivity) Polypeptone 0.5%, glycyl-L-proline was added to medium A.
0.5% and glycerin or sodium acetate were added at a ratio of 1%. Trichosporon SP in 5 ml of pH 5.0 medium
107-1 was cultured and the proline dipeptidase activity was measured in the same manner as in Example 1. The results shown in Table 2 were obtained.

Example 3 Production of Proline Dipeptidase by Trichosporon SP 107-1 (3) (Effect of Glycyl-L-Proline on Enzyme Productivity) Carbon source-free medium of Example 2, sodium acetate-containing medium and respective glycyl- Trichosporon sp. 107-1 was cultured in an L-proline-free medium, and the proline dipeptidase activity was measured in the same manner as in Example 1. The results are shown in Table 3.

Example 4 Production of proline dipeptidase using Pseudomonas fluorescens IFO12055 (Effect of glycyl-L-proline on enzyme productivity) Medium A containing 1% glycerol and 0.5% ammonium sulfate, 0.5% glycyl-L-proline Pseudomonas fluorescens IFO12055 was cultivated in 5 ml each of the pH 7.5 medium and the glycyl-L-proline-free pH 7.5 medium, and the proline dipeptidase activity was measured in the same manner as in Example 1. The results are shown in Table 4.

Example 5 Effect of enzyme inhibitor on proline dipeptidase activity derived from Trichosporon sp. 107-1 Trichosporone sp. 107-1 was cultured in a medium without addition of carbon source and glycyl-L-proline of Example 3,
The enzyme solution obtained in the same manner as in Example 1 was treated with an inhibitor at 50 mM.
After dialyzing in Tris-hydrochloric acid buffer pH 8.0 for 15 hours, the reaction was carried out in reaction solution B500μ containing the same inhibitor as the dialysate, and proline dipeptidase activity was measured. The results are shown in Table 5, when the relative activity was expressed as 100 when the enzyme inhibitor was not added.

Example 6 Effect of metal ion on activity of proline dipeptidase derived from Trichosporon sp. 107-1 The enzyme solution obtained in Example 5 was reacted in 500 ml of reaction solution B added with a concentration of 1 mM of metal ion, and the activity was measured. The results in Table 6 were obtained when the relative activity was expressed by setting the case where no metal ion was added as 100.

Example 7 Effect of Mn ²⁺ ion concentration on proline dipeptidase activity derived from Trichosporon sp. 107-1 The enzyme obtained in Example 5 was reacted in 500 ml of a reaction solution B to which various concentrations of Mn ²⁺ ions were added, and the activity was measured. did. When the Mn ²⁺ ion was added at a concentration of 1 mM as 100, the results were shown in Table 7 as a relative activity.

Example 8 Substrate specificity of proline dipeptidase derived from Trichosporon sp. 107-1 Aminoacyl-L-proline, which is a substrate, was added to reaction solution C in an amount of 10%.
The enzyme solution was added at a concentration of mM and the activity was measured using the enzyme solution obtained in Example 5. The results are shown in Table 8 when the activity against glycyl-L-proline is set to 100 and expressed as a relative activity.

Example 9 Effect of Enzyme Inhibitor on Proline Dipeptidase Activity Derived from Pseudomonas fluorescens IFO12055 Pseudomonas fluorescens IFO12055 was cultured in broth medium and the activity was measured in the same manner as in Example 5 and expressed as relative activity. The results are shown in the table.

Example 10 Effect of metal ion on Pseudomonas fluorescein IFO12055-derived proline dipeptidase activity The activity was measured in the same manner as in Example 6 using the enzyme solution obtained in Example 9. However, when metal ion is not added
When expressed as a relative activity of 100, the results are shown in Table 10.

Example 11 Effect of Mn ²⁺ ion concentration on Pseudomonas fluorescens IFO12055-derived proline dipeptidase activity The activity was measured in the same manner as in Example 7 using the enzyme solution obtained in Example 9, and Mn ²⁺ ion was measured. Table 1 shows the results when the relative activity was expressed as 100 when 1 was added at a concentration of 1 mM.

Medium A Yeast extract 0.02 (% W / V) KH 2 PO 4 0.1 Na 2 HPO 4 · 12H 2 O 0.1 MgSO 4 · 7H 2 O 0.05 CaCl 2 1 × 10 -3 FeCl 2 1 × 10 -3 MnCl 2 1 × 10 ^-3 Pyridoxy hydrochloride 2 × 10 ^-4 Nicotinic acid 2 × 10 ^-4 Biotin 4 × 10 ^-6 Reaction solution A Tris-hydrochloric acid buffer solution 100 mM Glycyl-L-proline 20 mM Manganese chloride 10 mM Reaction solution B Tris-hydrochloric acid buffer solution 100 mM Glycyl-L-proline 20 mM Reaction solution C Tris-HCl buffer 100 mM Manganese chloride 1 mM

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Claims (1)

[Claims] 1. A method for producing proline dipeptidase, which comprises culturing a proline dipeptidase-producing bacterium belonging to the genus Pseudomonas or the genus Trichosporon, and separating and collecting proline dipeptidase from the obtained culture.