JPS5840473B2 - Novel proline acylase and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel proline acylase that differs in various properties from known aminoacylases, and a method for producing the same.

The proline acylase of the present invention is an enzyme that catalyzes a reaction to generate L-proline by decomposing N-acyl-L-proline in which the amino group of L-proline is acylated,
Generally, an enzyme used in a method for efficiently producing optically active L-proline by asymmetrically hydrolyzing the racemate into N-acyl D-proline and L-proline using N-acyl-DL-proline as a substrate. It is.

L-proline is a type of amino acid that has biological activity, and is expected to be used as a pharmaceutical raw material and reagent.

Conventionally, aminoacylase, which acts on N-acyl-amino acids in which the amino group of L-amino acids is acylated, has been found in the animal kingdom in pig kidneys [Methods in Enzymology].
thods in Enzyrnology) Part 2
Vol., p. 109, 1955], but in the microbial world, Lactobacillus arabinosus
) (Park, R, W, and Fax, S,
W Journal of Biological Chemistry (
Journal of Biological
Chemistry) Volume 235, Page 3193, 19
1960], Corynebacterium
erium) (Okuda et al. Special Publication No. 13989/1989), Aspergillus (Aspergi 1lus)
) and Rhizopus family [
Chibata et al. Nibretin of the Agricultural Chemical Society of Japan (Eu1letn)
of the Agricultural Ch.
chemical 5ociety of Japan
) Volume 21, No. 5, pp. 304-307, 1957; 5olodovnikova, N, I, and
Mo1odova, G.A.

l5sed, In5t, Biosin, Be1ko
vykhVeshchestv, Volume 1, No. 145~
151, 1972], etc. are known.
Moreover, the action of these aminoacylases on N-acyl-proline is very weak compared to other amino acids, showing only an activity of 1'/40,000 species.

In addition, as an enzyme that acts on a specific N-acyl-amino acid, for example, aspartoacylase that acts on N-acyl-aspartic acid, C Journal.
of Biological Chemistry (Journal
ofB iological Chem 1str
y) Vol. 194, p. 455, 1952], Acyl-lysine deacylase (Acylysine deacylase) that acts on N-acyl-lysine CW
, P., Pa1k et al.: Archives of Biochemistry and Biophysics (Archives of Biochemistry and Biophysics)
es of Biochemistry and
Biophysics, Vol. 6'9, pp. 56-66, 1957].

Therefore, in view of the current situation, the present inventors have determined that N-acyl-L
- As a result of extensive research into enzymes that specifically act on proline, we discovered that Alcaligenes
) was found to produce an enzyme with extremely high substrate specificity for N-acyl-L-proline and an unprecedented high proline acylase activity, and based on this knowledge, The invention has been completed.

That is, in the present invention, the amino group of proline is glycine, R
It is a novel proline acylase that specifically acts on N-acyl-L-proline bonded to a group or R-glycine (R is an amino group-protecting substance) or a carboxyl group of a fatty acid to produce L-proline. .

The present invention also involves culturing a strain belonging to alkaline earth metals and having the ability to produce the above-mentioned proline acylase in a medium,
This is a novel method for producing proline acylase, which is characterized by collecting the above-mentioned proline acylase from a culture.

The present invention will be explained in detail below.

First, the physicochemical properties of the purified enzyme (proline acylase) of the present invention will be described below.

(2) Action This enzyme decomposes N-acyl-L-proline to produce an acid with an N-acyl group corresponding to L-proline.

■ Substrate specificity If the amino group of proline is glycine, R group or R-glycine (glycine is the carboxy terminal) (R is an amino group protecting substance), or the carboxyl group of a fatty acid, preferably with a carbon number of C2 to CI6. It acts specifically on N-acyl-L-proline bonded to the carboxyl group of fatty acids to produce L-proline, and has almost no effect on other N-acyl-amino acids.

When the N-acyl group is glycine or R-glycine, it acts on the glycyl-L-proline moiety that is the bonding site with the C-terminus, liberating L-proline and exhibiting carboxypeptidase-like activity.

Note that R is, for example, a peptide, a carbobenzoxyl group, t
ert -7” thyloxycarbonyl group, tert
- means an amino group-protecting substance which is an amyloxycarbonyl group, a benzoyl group, or a bond between a peptide and the above-mentioned substance other than the peptide.

■ Optimum pH and stable pH range As shown in Figure 1, the stable pH range is 6 to 8.5, and the range of pH 3.0 to 8.0 is
McIlvaine) buffer, pH 5.0-8.
2 to 0.05M phosphate buffer, and pH 8.0~
The optimum pH when measured with 0.0!5M boric acid buffer up to 12.0 is 7.5-8.0 as shown in Figure 2.
It is 0.

■Method for measuring titer-1 0.5M phosphate buffer (pns, o) 0.1ml
and the substrate 0.2M N-acetyl-L-proline 0.25
ral and 0.9 ml of distilled water. Enzyme solution 0 after
．． Add 1 rul and react at 30°C for 10 to 30 minutes, then add 0.1 ml of 1M hydrochloric acid to stop the enzyme reaction.

The reaction stop solution is centrifuged at 250 Or, p, m, for 15 minutes, and 0.5 ml of the supernatant is passed through an amino acid analyzer (Hitachi model 034 amino acid force analyzer) to quantify the amount of proline produced.

The enzyme activity to produce 1 micromole of L-proline from the substrate in 1 minute was defined as 1 unit.

Method-2 0.5M phosphate buffer (pH 8.0) 0.1ml and 0.
．． Dilute 0.25 ml of 2M N-acetyl-L-proline to 0.9 ml with distilled water, add 0.1 ml of the enzyme solution, react at 30°C for 10 to 30 minutes, and then heat to 00°C.
The enzyme reaction is stopped by keeping it in a hot water bath for 2 minutes.

The amount of proline liberated from the substrate in this reaction solution was determined by Ye
mm-Cocking's ninhydrin colorimetric method Ros
Modified method by en (H, Rosel: Arch, Bi
ochem, Boiphys, Vol. 67, p. 10, 1957), and the enzyme activity that produces 1 micromole of L-proline in 1 minute is defined as 1 unit.
And so.

Note that unless otherwise specified, the titer was measured by this method.

In addition, the specific activity was measured by the method described above, and the method of Lineweaver and Burke (M,
D 1xonand E, C, Webb: Enz
yme 1, page 69, 1962, Longmansl
Green and Co.

The activity (Unit) calculated according to t, ta, is expressed in enzyme units per 1 inch of enzyme protein, and the protein was quantified using the copper-holein method (Lowry et al.) using bovine serum albumin as the standard protein. ,0.H6,R
Osenbrough, N. J.: Journal of Biological Chemistry
l of Biological Chemistry
y) Vol. 193, pp. 265-275, 1951].

■ Range of suitable temperature for action As shown in FIG. 3, it is within the range of 20 to 50'C.

■ Inactivation conditions such as pH 1 temperature As shown in Figure 4, the residual activity is 1 at 40℃ for 10 minutes.
00%, but it is completely inactivated in 10 minutes at 50℃※※.

At pH 4 or 10, it is completely inactivated at 40°C in 20 minutes, but
It is inactivated by 68% at pH 5 and 90% at pH 9.

■ Inhibition, activation, and stabilization Various metal ions, metal chelating agents, and other drugs that are normally thought to be involved in the stability of enzyme proteins were added to the reaction solution at a final concentration as shown in Table 1. Table 1 summarizes the effects on the enzyme activity when added.

The relative activity in Table 1 is expressed as the ratio of the activity when the drug is added to the activity when the drug is not added.

■ Purification method The crude enzyme solution of the proline acylase of the present invention was adsorbed onto a DEAE-cellulose column equilibrated with 0.01M phosphate buffer (pH 8,0), and after adsorption, it was thoroughly washed with the same buffer. The active fraction is collected by elution with the same buffer containing 1M potassium chloride.

From this active fraction, the protein fraction precipitated at 25-80% saturation with ammonium sulfate was mixed with a small amount of 0.1 M potassium chloride.
．． OIM! Sephadex G1 dissolved in J acid buffer (pH 8,0) and equilibrated in advance with a buffer of the same composition.
00, the proline acylase active fraction was collected by elution with a buffer of the same composition.

Next, this effective category was divided into 0.0. OI
It is adsorbed onto a QAE-Sephadex column equilibrated with MIJ acid buffer, and after adsorption, it is eluted with a potassium chloride concentration gradient from 01 to 1.0M to collect the active fraction.

This active fraction was ultrafiltered through collodion bag (Sartorius Membrane Filter Co., West Germany) and concentrated.
A purified enzyme preparation of the proline acylase of the present invention is obtained by applying the column to a Biogel A-15m column equilibrated with a 0.01M phosphate buffer containing 1M potassium chloride and eluting with a buffer having the same composition.

■ Molecular weight The molecular weight according to the gel method is about 600,000 to 700,000, but S
In DS disk electrophoresis, it is about 55,000.

[Phase] The result of measurement using isoelectric point ampholine (pH 3 to 10) is pH 5.0.

0 Elemental analysis value C51.69%, H6.73%, N16.69% @ Amino acid analysis value (1 of each amino acid in 100♂ enzyme protein
Number) Aspartic acid 8.57, Threonine 4.90, Serine 2.70, Glutamic acid 11.35, Proline 5.88
, glycine 5.14, alanine 7.96, 6.
61, methionine 3.60, inoleucine 4.07, leucine 6.73, tyrosine 6.40, phenylalanine 5.29, lysine 3.02, histidine 2.55, arginine 9.92, tryptophan 3.07.1/2 -Cystine 0.37° Looking at the specific activity of the enzyme of the present invention for each substrate, it is as shown in Table 2. It is a proline acylase that has substrate specificity completely different from conventional aminoacylases and has extremely high Since the enzyme of the present invention exhibits this activity, it is different from any known aminoacylases and is recognized as a novel enzyme.

Next, specific means for producing the present enzyme will be described below.

First, the raw materials for production may be of any origin, but for example, those of microbial origin, especially Alcaligenes (Alcaligenes).
It is particularly advantageous to use a strain that belongs to the genus P. igenes and has the ability to produce the above-mentioned proline acylase in producing the present enzyme.

A specific example of a strain belonging to alkaline earth metals is Alcaligenes A-131, and variants or mutant strains of this strain can also be used.

The above-described Alcaligenes A-131 is a strain newly isolated by the present inventors from humic soil, and its mycological properties are as shown below.

The mycological properties were generally based on the methods described in the Manual of Microbiological Methods (published by McGraw-Hill Book Company, 1959).

(a) Morphological microscopic observation (cultivated on broth agar medium at 30°C for 12 hours) ■Cell shape and size: 0.5-0.75X0.7
A short bacillus with a nearly spherical shape of 5 to 1.5 microns.

■ Cell pleomorphism: Not observed.

■ Motility: Has periflagella and is motile.

■ Presence or absence of spores: Not formed.

■ Durham staining: Negative.

■ Anti-acidity: Negative.

(b) Growth status in each medium ■ Circular colonies with a diameter of 0.5 to 1WL11L after cultured in broth liquid culture at 30°C for 48 hours.

Surface flattened, slightly head-shaped. It has a gold rim, a slightly yellowish tinge, and is transparent.

■ Meat juice agar slant culture Growth is good, grows in a spread pattern, and does not form colonies. The color is yellowish.

■ Meat juice liquid culture Grows well and uniformly and does not produce sediment.

■ Meat juice gelatin puncture culture Although it grows slightly, it does not liquefy at 30°C for 148 hours.

■ Lidmus Milk no change.

(c) Physiological properties ■ Nitrate reduction: negative.

■ Denitrification reaction: Negative.

■ MR test: Negative.

■ VP test: Positive.

■ Indole production: Negative.

■ Hydrogen sulfide formation: Negative.

■ Starch hydrolysis: negative.

■ Use of citric acid: Koser's medium, Chris
It is not used as a medium for Tensen.

■ Use of inorganic nitrogen sources: Both nitrates and ammonium salts are used in small amounts.

[Phase] Pigment formation: Negative.

0 Urease: Slightly present.

@ Oxidase: Positive.

◎ Catalase: Positive.

■ Growth range: pH 6-11. Temperature 10-40℃8 [Phase] Attitude towards oxygen: Aerobic.

@0-F test: Negative.

0 Generation of acids and gases from sugars: L-arabinose, D-xylose, D~glucose,
D-mannose, D-fructose, D-galactose,
No acid or gas production was observed with any of the sugars: maltose, sucrose, lactose, tre-lose, D-sorbitol, D-mannite, inosit, glycerin, and starch.

The taxonomic properties of the above-mentioned strains capable of producing proline acylase are described in the 8th edition of "Virge Ace Manual of Determinative Bacteriology" (1974).
In contrast to the classification of ``Alcaligenes faecalis'', Alcaligenes aquamarinus, Alcaligenes europhus, and Alcaligenes baradoxas, the above-mentioned Vergeace manual states that alkaline earth metals include Alcaligenes faecalis, Alcaligenes aquamarinus, Alcaligenes eurohus, and Alcaligenes baradoxas. Only four species have been described, and they differ from the four bacterial species mentioned above due to their isolation source and a few properties.

That is, Alcaligenes A-131, which has the above-mentioned ability to produce proline acylase, is a short bacillus that is negative in Durham staining.
It is extremely aerobic, shows no polymorphism, does not produce acid or gas from glucose, is oxidase positive, has periflagella, and is classified as an alkaline earth metal due to its motile properties.・Faecalis (A 1cal
It is considered to be closely related to I. genesfaecalis), but it has physiological properties such as not using acetic acid, propionic acid, or butyric acid as a single carbon source or energy source, unchanged lidmus milk, and producing this enzyme. There are differences.

In addition, Alcaligenes A-131 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 4437 (FERM-P).
4437), and was also deposited in the American Tight Culture Collection (American Tight Culture Collection).
pe Culture Co11ection) AT
It has been deposited as CCA, 31415.

Next, the production of proline acylase using the above bacteria capable of producing proline acylase will be described.

Examples of the culture medium used for culturing the above-mentioned bacteria capable of producing proline acylase include those used for culturing microorganisms belonging to alkaline earth metals.

The nitrogen source for the culture medium may be any available nitrogen compound, such as one or more organic compounds such as yeast extract, hefton, meat extract, gelatin, corn steep liquor, amino acids, soybean, wheat rip extract, ammonium sulfate, ammonium nitrate, etc. Alternatively, an inorganic nitrogen source is used.

Add one or more appropriate inorganic salts such as manganese, phosphoric acid, potassium, magnesium, and calcium to the above nitrogen source, and if necessary, add carbon sources necessary for bacterial growth or enzyme production, such as sugars, various organic substances, and inorganic substances. , vitamins, and the like are preferably used as a medium.

Although the bacteria may be cultured by solid culture, it is usually preferable to employ a liquid culture method, and it is preferable to culture the bacteria aerobically by shaking culture, stirring culture, aeration culture, or the like.

Industrially, it is preferable to use a liquid medium and perform submerged culture with aeration and agitation.

The culture temperature is 25 to 35°C, preferably around 30°C.

The optimum pH during culture is 7.0. The culture time varies depending on the culture format, but is 14 to 48 hours.

For example, when culturing aerobically using shaking culture or aerated agitation culture, this enzyme is efficiently produced and accumulated within the bacterial cells within 48 hours, but if necessary, the culture may be continued for an additional 24 hours to allow autolysis. This allows the enzyme to be excreted from the bacterial body.

General enzyme extraction and purification methods can be used to extract and purify the present enzyme from the culture thus obtained.

For example, after separating bacterial cells from a culture by an appropriate method, the bacterial cells are ground in the presence of an abrasion agent, using a lytic enzyme such as lysozyme, applying ultrasonic energy, or applying osmotic shock. After the enzyme is destroyed by a known method such as the method of 100 ml, or shaken or left in the presence of a surfactant such as soteium cholic acid, Triton X100, toluene, etc., the enzyme is excreted from the bacterial cell. The solution is treated with an appropriate operation such as irradiation or centrifugation to remove solid matter to obtain a bacterial cell extract, or extracted with water, buffer, or an appropriate solvent, and this is used directly as a crude extract. Obtained as an enzyme solution.

Further, a crude enzyme powder can also be obtained by carrying out a conventional enzyme recovery method, that is, a freeze-drying method, an alcohol precipitation method, an acetone precipitation method, etc., which are appropriately selected and carried out on the extract.

To obtain a further purified enzyme preparation from the above crude enzyme solution or crude enzyme powder, for example, a gel 1-pass method using Sephadex or biogel, an adsorption/elution method using an ion exchanger, an electrophoresis method using a polyacrylamide gel, etc. ,
Purification can be achieved by appropriately selecting and combining adsorption and elution methods using hydroxyapatite, sedimentation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation methods using molecular sieve membranes or hollow fiber membranes, etc. A sample of this enzyme can be obtained.

The present enzyme can also be obtained by treating the culture p solution obtained by extending the culture time to allow autolysis to occur.

Next, by using an N-acyl-L proline degrading enzyme having such specificity, that is, the proline acylase of the present invention, the amino group of L-proline is converted to N
-Glycyl-L-proline, N-acyl-L-prodine in which the amino group of L-proline is bonded to an R group or R-glycine, such as carbobenzoxy-L-proline, carbobenzoxy-glycyl-L-7"loline , L-leucyl-glycyl-L-7''loline, carbobenzoxy-chrycyl-L-floryl-L-leucyl-glycyl-L-
Florin, tert-7” tyloxycarbonyl-L-
7'lolin, tert-amyloxycarbonyl-L-
Proline, pensoyl-L-7'loline, or N-acyl-proline in which the amino group of L-proline is bonded to the carboxyl group of a fatty acid, preferably a C2-C16 fatty acid, such as N-acetyl- L-7"
Loline, N-phthyryl-L-proline, etc., generally act on their racemic forms to decompose N-acyl-L-proline and efficiently obtain L-proline.

In this case, the optimal conditions for the proline acylase used are adopted as the operating conditions, but in the case of the above-mentioned proline acylase obtained from a microorganism that has the ability to produce the above-mentioned proline acylase belonging to alkaline earth metals, the action pH is is in the range of 6 to 8.5, and the operating temperature is appropriately in the range of 20 to 50°C.

The action time is appropriately determined depending on the substrate concentration and enzyme concentration.

After completion of this reaction, L-florin can be recovered from the reaction solution by a conventional method.

The L-proline thus obtained can be expected to have excellent effects as a raw material for pharmaceuticals, a biochemical reagent, etc.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Lactose 10f, polypeptone 10? , yeast extract 31, phosphoric acid 1 potassium 0.25? , 2 g of disodium phosphate, 0.25 f of magnesium sulfate, and 0.0 IP of ferrous sulfate were dissolved in 11 parts of distilled water, and the culture medium adjusted to pH 7.2 was added to 200 m
Dispense 1 portion into each 500 ml shake flask, and add 120
After sterilization for 10 minutes at ℃, Alcaligenes A 131 (
FERM PA, 4437 r inoculated and incubated at 30°C for 4 hours.
Shaking culture was performed at 8 o'clock time width 5CrrL1120r, pom.

After the culture is completed, 10,100 O of the culture solution is centrifuged, and then the supernatant is separated.

After suspending the thus obtained bacterial cells in 0.01M phosphate buffer (pH 8.0) 200-, ultrasonic treatment (
(manufactured by Kaiyo Denki Co., Ltd., 20KC for 15 minutes), the solid portion was removed by centrifugation, and the cell extract was extracted at 180 mA!
I got it.

Using this cell extract as an enzyme solution, the proline acylase activity was measured by the method 1 described above, and the result was 0.8 units/
It was rILl.

Example 2 Gelatin IOP, yeast extract 31, phosphoric acid 1 potassium 0.2
5P, disodium phosphate 21, magnesium sulfate 0.
25f and 0.01f of ferrous sulfate were dissolved in 10,100O of distilled water, and the medium was adjusted to pH 7.2. 200ml of the medium was dispensed into each 500ml shaking flask, and incubated at 120°C for 10
After sterilization for minutes, this was added to Alcaligenes A-13i (FE
RM-PA, 443't) and cultured at 30°C for 48 hours as described in Example 1.

After the culture was completed, 10,100 O of the culture solution was centrifuged, and the supernatant was separated.

The bacterial cells thus obtained were suspended in 200 ml of 0.01 M phosphate buffer (pH 8,0), and Triton X-100 (polyoxyethylene alkylphenol ether, Rohm and Haas Co.) was added to the suspension.
was added to a final concentration of 5%, stirred well to ensure uniformity, and then left at room temperature overnight.

Next, centrifuge to remove solids and extract 190ml of cell extract.
I got 1.

When the proline acylase activity of this preparation was measured by method 1, it was 2.4 units/TILl.

Example 3 1617rrLl of the crude enzyme solution obtained in Example 2 was diluted to 0.
It was adsorbed onto a DEAE-cellulose column pre-equilibrated with 01M phosphate buffer (pH 8,0).

After thoroughly washing this column with the same buffer, it was eluted with the same buffer containing 1M potassium chloride to obtain active fraction 10100O.

Ammonium sulfate is added to this proline acylase active fraction to precipitate the protein fraction at 25-80% saturation with ammonium sulfate. OIM'J
After dissolving in 100 ml of acid buffer (pH 8,0) and centrifuging to remove unbathable substances, the supernatant was adsorbed onto Sephadex G-100 that had been equilibrated with a buffer of the same composition.
Chromatography was performed using a buffer solution of the same composition to collect the proline acylase activity fraction.

This active fraction was then treated with 0.01M potassium chloride.
QAE- equilibrated with M phosphate buffer (pH 8,0)
After adsorption onto a Sephadex column, chromatography was performed using an elution method in which the concentration of potassium chloride in the same buffer was continuously increased from 0.1 to 1.0M.

The proline acylase activity fraction thus obtained was ultrafiltered using a collodion bag (Sartorius Membrane Filter, West Germany), concentrated to 2 mg, and then 0.1
0.01M phosphate buffer containing M potassium chloride (pH 8.0
Biogel (Biogel) equilibrated with )
The proline acylase of the present invention was obtained by applying chromatography to an A-15m column and using a buffer solution having the same composition.

Table 3 shows the total activity, specific activity, and yield in each of the above purification steps.

In Table 3, * indicates the value obtained by the titer measurement method according to Method-1, and ** indicates the value obtained by the titer measurement method according to Method-2.

Example 4 3.14P of N-acetyl-DL-proline was dissolved in water and adjusted to 8.0 with NaOH to give a total volume of 10 ml.

Next, 60 units of the enzyme obtained by the QAE-Sephadex treatment in Example 3 were added to this solution, and the mixture was heated at 37°C.
The reaction was carried out for 24 hours.

Next, after adding hydrochloric acid to adjust the pH to 1.5, it was adsorbed on a Towex 50WX8 (H+) column, and 100TL of water was added.
After washing with 5% NH4OH and eluting with 5% NH4OH, the active fraction was evaporated to dryness in a rotary enovolator.

Next, this evaporated dry product was dissolved in a small amount of methanol and heated at 5°C.
Acetone was added thereto for crystallization.

Recrystallization was performed from methanol-acetone to obtain 0.91 L-proline.

The yield was 78% (based on L-proline in DL proline).

The physical and chemical properties of this substance are as follows.

[α] Do 84.5° (C-1, H2O) Optical purity
99.4% Elemental analysis value: HN as C3H9NO2 Theoretical value (%) 52.16 7.88 12.17 Actual value (%) 52.25 7.91 12.13 In the same manner, the substances shown in Table 4 were used as raw materials. L-proline was obtained in the yield shown in Table 4.

[Brief explanation of drawings]

Figure 1 shows the p of the proline acylase of the present invention at 40°C.
FIG. 2 is a diagram showing the optimum pH of the proline acylase of the present invention, FIG. 3 is a diagram showing the operating temperature of the proline acylase of the present invention, and FIG. FIG. 2 is a diagram showing the thermostability of acylase.

Claims (1)

[Scope of Claims] 1. A novel proline acylase having the following physicochemical properties. (a) Action N-acyl-L-proline is decomposed to produce an acid with an N-acyl group corresponding to L-florin. (b) Substrate specificity An N-acyl loop in which the amino group of proline is bonded to glycine, an R group, or R-glycine (glycine is the carboxy terminal) (R is an amino group protecting substance), or a carboxyl group of a fatty acid. L-
It produces proline and has little effect on other N-acyl-amino acids. (c) Optimal pH and stable pH range The stable pH range is 6 to 8.5, and pH 3.0 to 8.5.
0.05 M phosphate buffer for pH 50 to 8.2, 0.05 M phosphate buffer for pH 50 to 8.2, and 0.05 M phosphate buffer for pH 8.0 to 1.
The optimum pH is 7.5 to 8.0 when measured with a 0.05M boric acid buffer up to 2.0. (d) The optimum temperature for action is within the range of 20 to 50°C. (e) Inactivation conditions such as pH1 temperature. At 40°C for 10 minutes, the residual activity was 100%, but 5
It is completely inactivated in 10 minutes at 0°C. At pH 4 or 10, it is completely inactivated at 40°C in 20 minutes, but
It is inactivated by 68% at pH 5 and 90% at pH 9. (f) Inhibition, activation, and stabilization The effects of various metal ions, metal chelating agents, and other drugs normally thought to be involved in the stability of enzyme proteins on the enzymatic activity of this enzyme are shown in Table 1 below. It is shown. (g) Molecular weight The molecular weight according to gel 1 filtration method is about 600,000 to 70,000, but S
In DS disk electrophoresis, it is about 55,000. (h) The result of 1'C measurement using isoelectric point ampholine (pH 3 to 10) is pH 5.0. 2. The novel proline acylase according to claim 1, which has a specific activity of 1 to 210. 3 Belongs to alkaline earth metals and has the following physical and chemical properties. (a) Action N-acyl-L-proline is decomposed to produce an acid with an N-acyl group corresponding to L-florin. (b) Substrate specificity An N-acyl loop in which the amino group of proline is bonded to glycine, an R group, or R-glycine (glycine is the carboxy terminal) (R is an amino group protecting substance), or a carboxyl group of a fatty acid. L-
It produces proline and has little effect on other N-acyl-amino acids. (e) Optimal pH and stable pH range The stable pH range is 6 to 8.5, and pH 3.0 to 8.5.
Up to 0, pine irvain buffer, pH 5.0-8.2
0.05M phosphate buffer and pH 8.0-1.
The optimum pH is 7.5 to 8.0 when measured with Q up to 2.0 and 05M boric acid buffer. (d) The optimum temperature for action is within the range of 20 to 50°C. (e) Inactivation conditions such as pH 1 temperature at 40°C for 10 minutes, the residual activity is 100%, but 5
It is completely inactivated in 10 minutes at 0°C. At pH 4 or 10, it is completely inactivated at 40°C in 20 minutes, but
It is inactivated by 68% at pH 5 and 90% at pH 9. (f) Inhibition, activation, and stabilization The effects of various metal ions, metal-killing agents, and other drugs normally thought to be involved in the stability of enzyme proteins on the enzymatic activity of this enzyme are shown in Table 1 below. As shown below. (g) Molecular weight The molecular weight according to the gel one-pass method is about 600,000 to 700,000, but S
In DS disk electrophoresis, it is about 55,000. (h) Isoelectric point The result of measurement using ampholine (pH 3 to 10) is pH 5.0. A novel method for producing proline acylase, which comprises culturing in a medium a strain capable of producing proline acylase having the following: and collecting the proline acylase from the culture. 4. The novel method for producing proline acylase according to claim 3, which comprises adding a surfactant to the bacterial cells obtained from the culture to release proline acylase.