JPS5995886A - L-tryptophan aminopeptidase and its preparation - Google Patents

Abstract

NEW MATERIAL:L-Tryptophan aminopeptidase. USE:A catalyst for the preparation of L-tryptophan, a reagent for determining the amino acid sequence of protein and peptide, etc. PROCESS:A bacterial strain belonging to Trichosporon genus and capable of producing L-tryptophan aminopeptidase, e.g. Trichosporon cutaneum IFO0173, etc. is cultured in a medium to accumulate L-tryptophan amino-peptidase in the bacterial cell, and the cells are collected by a centrifugal separator, etc. after the completion of cultivation. The collected cells are suspended in a buffer solution, etc., ground, centrifuged to separate the supernatant liquid, and added with ammonium sulfate. The produced precipitate is collected, and purified by ion exchange chromatography, adsorption chromatography with hydroxyapatite, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme, more particularly Z, -)
L that has the property of acting specifically on liftophanamide
- Relating to tryptophan amine peptidase.

L-) Lysotophan is an important compound as an essential amino acid, and is often used in amino acid infusions, animal feed additives, and sleep-inducing agents.
(l) Butophane derivatives, such as DL-tryptophan amide, are produced by optical resolution.

This time, in the present invention, a novel amine peptidase has been discovered from a certain type of bacterial cell of the genus Trichosporon that specifically acts on the amide bond of L-tryptophan amide to release L-)liptophan. It has been found to be extremely useful for the production of L-)liptophan by asymmetric hydrolysis of amide derivatives.

Conventionally, an aminopeptidase that specifically acts on the amide bond (or peptide bond) of 1L-trithophanamide has not been known, and in this specification, the enzyme will be referred to as kL-)liptophan aminopeptidase.

However, protamylase is known as an enzyme that hydrolyzes L-1, liptophan amide to produce L-) liptophan (Brbl 1. Agγ).

Ch, eyn, Soc, Japan, Vol, 2
1, AI, p 62-66.1957), this is thought to be an enzyme mixture derived from the pancreas, and it is unclear whether it is an enzyme that has specificity for L-tryptophanamide. However, because it is of animal origin, its production is not as easy as that of enzymes of microbial origin.

It has been reported that tryptophan aminopeptidase activity is observed in malnourished mice (J, C11
n, Inveat, 67 (1), p51-9.1
981), the active substance has not been separated and purified.
It is unclear whether this activity is due to the action of trinotophane aminopeptigase.

Leucine aminopeptidase (EC3,4,11,1)
However, if the activity towards L-leucylamide is 100, the activity towards L-) liptophanamide is only 24, and the activity towards hydrolyzing L-tryptophanamide is only 24. Very low (The Enzymes).

2tLd ed,, Vol, 4. p, 49,196 (J
).

Thus, no aminopeptidase having specificity for monotrigtophanamide has been known, and the enzyme of the present invention can be said to be a completely new enzyme.

The chemical properties of the enzyme provided by the present invention are as follows.

(1) Action Z: -acts on peptides having L-tryptophan at the amide and amine ends, and hydrolyzes the amide bond to release L-)Liptophan.

(2) Substrate specificity The substrate specificity of this enzyme is summarized in Tables 1 and 2 below.

Table 1 shows the specificity of this enzyme for various amino acid amides, and the relative activity of each substrate was determined as follows.

2S enzyme solution 0.01g, 8mM MnC
12 solution 0.05m/! 0.05 m/+e of each 0.12 M substrate solution was added to Lis-HCl buffer (pH 7,5) 009-, and reacted at 37°C for 10 minutes. Next, the reaction was stopped by heating at 90''C for 5 minutes, and 5 rrl of distilled water was added.

The generated ammonia is treated with dalcmate dehydrodanase (
The activity against each substrate was determined by an enzymatic method (Clinical Test Methods Revised 28th Edition, V!-47) using EC1,4,1,4), and the activity against L-tryptophanamide was set as 100.

Table 2 shows the substrate specificity of this enzyme for bezotides. The numerical values in the table are 1% of the reaction solution obtained by reacting in the same manner as described above (however, using 0.05ml of 20rnM each group solution) and adding 5ml of distilled water after stopping the reaction.
570? of free amino acids produced using the ninhydrin method per ml? Z? Increase in absorbance at lZ (△0
D) Show Q. Therefore, when ΔOD is zero, there is no effect at all, and the larger ΔOD is, the stronger the effect is.

This enzyme acts on each glaze amino acid amide, but does not act on N-acetyltributophane or hippuric acid, indicating that it is an aminopeptidase.

In particular, L-tryptophan amide and the amino terminus
-) It can be called tryptophan aminopeptidase because it acts most strongly on the peptide J-gutophane. It also works relatively well on amino acid amides with hydrophobic groups and peptides whose amino terminal amino acids have hydrophobic groups.

Since the activity towards L-leucine amide is 26, this enzyme is clearly different from leucine aminopeptidase.

The esterase activity of this enzyme was investigated in Table 3 (1d) below, and the activity against L-)liptophanamide was increased by 100
The relative activity was determined as

Table 3 (Note) The substrate temperature during the reaction is 30mM.

As shown in Table 3, this enzyme only slightly hydrolyzes L-)IJ butophane ester.

(3) Titer determination method The titer determination method consists of a two-step reaction. The first step is the hydrolysis reaction of L-tryptophanamide, and 0
24M DI,-tryptophanamide solution (pH 7,
The reaction was carried out in the same manner as in the measurement of relative activity in the previous section (2), except that 0) was used as the substrate.

The second step is the determination of L-butophane produced using tryptophanase (EC4,1,99,1).
Ehγ1ich by decomposing tryptophan into indole
Test rack - Add H and measure absorbance at 570 nm.

The reaction conditions for this second step are as follows: tryptophanase solution 0.1-o, 0512M potassium phosphate buffer containing 2mM 2-mercaptoethanol (p 1
17.8) Add 0.1 yd and 6mM pyridoxal-5'-phosphorus 01πlf and heat at 37°C for 10 minutes. To 0.3 ml of this enzyme solution was added the reaction solution from the first stage (
5m7 of distilled water after the reaction has stopped! )0.2m1.
(Add r,' and incubate at 37℃ for 10 minutes. Ehr
The reaction is terminated by adding 1 tnl of lich reagent (Kyoto University Faculty of Agriculture, Agricultural Chemistry Department, Cotton, New Revised Edition Agricultural Chemistry Laboratory, Vol. 2, p. 876), and after standing for 20 minutes, the absorbance at 5 TOnm is measured. L -+- which I created at the same time! L-butophane produced from the shielding line of J-butophane is emitted as γm.

The tryptophanase used here is Y, Morin.

and E, E, Sne l l t7) Method (k
iethods in, Bnzymo-1ogy, V
ol,”) (■I, A, p, 439-446
It was prepared from a cell-free extract of the E. coli K-12 strain by heat treatment and ammonium sulfate fractionation according to the method of E. coli K-12.

The enzyme unit of this enzyme is the amount of enzyme that releases 1 μmol of L-butophane per minute under the above reaction conditions.

(4) Optimal pH and stable pH range The relationship between pH and enzyme activity of this enzyme is shown in the graph of FIG. As is clear from FIG. 1, this enzyme acts in neutral to slightly alkaline conditions, and its optimum pH is 9.5. The graph in FIG. 2 shows the relationship between the pH of an aqueous solution of this enzyme and the residual activity after standing at 10° C. for 20 hours. The stable pH range is pH 7.5 to 8.5 as shown in FIG.

(5) Suitable temperature range for action Figure 3 shows the enzyme activity when the reaction was carried out outside the thread in the same manner as in the titer measurement, but at different temperatures.

3rd [xB crow 1] The suitable temperature for the role is 40 ~
At 45°C, the effective temperature is 30-55°C.

(6) Condition for inactivation by heat Regarding inactivation by heat, the residual activity was measured after treating the enzyme aqueous solution for 10 minutes at each temperature. The results are shown in Figure 4. Fourth
As is clear from the figure, the addition of sucrose increases the thermal stability. The residual activity after heat treatment at 60'C for 10 minutes is 5
With a coefficient of 0, this enzyme is a relatively thermostable enzyme.

(7) Inhibition and activation The effects of each rod inhibitor and Kinmin Io were examined.

The effect of the tag agent was on the activity after treatment at 4°C for 30 minutes.
It was measured according to the titer measurement method described in IJ, and the influence of metal ions was measured by J-
The activity of MnC1 was measured and expressed as relative activity to MnC1. The results are shown in Table 4 below.

Table 4 This enzyme was frequently inhibited by α, αL dipyrisole and N-methylmaleimide. Among the metal ions, M gSO, and Z? 1. SO4 has no activation effect at all (CoCL).

MnCl2, which showed an activity of 25%, has the best activation effect.

(8) Purification method This will be explained later.

(9) Molecular weight: Approximately 270,000 as determined by the gel filtration method using Sephadex G-2oo. According to dodecyl sulfate polyacrylamide gel electrophoresis, this enzyme has a molecular size of 1j68,00
It is presumed to consist of four subunits: 0.

0ω Disc City 1 Air Swimming ML B, J, I) avis (7) p II 9.5
tvr le CAnn, N, Acad.

Sci., 121.404 (1964)), and according to the disk's tunic swimming proboscis, when the migration distance of the promofunol pull processed as a marker is 100, the migration distance is 21. A single band was observed.

Electrofocusing performed according to the method shown in En Isoelectric Point Japanese Biochemistry Extra Line, Biochemistry Experiment Course, Volume 1 Chemistry of White Matter L 7), 270-2T6 (published by Tokyo Kagaku Doujin on March 30, 1978) As a result, the isoelectric pH was 4.7.

0z Crystal Structure This enzyme is a regular hexagonal crystalline substance as shown in FIG.

t1 powder Km value of the enzyme of the present invention, -)Lyptophanamide and Mn
The apparent Km value for Cl2 is 5.6mM, respectively.
and 0.2w, M.

The enzyme of the present invention is produced by culturing a bacterial cell belonging to the genus Trichosporon and having the ability to produce L-tricytophan aminopeptidase in a medium, producing and accumulating L-tryptophan aminopeptidase in the bacterial cell, and then producing L-tryptophan aminopeptidase from the bacterial cell. It can be produced by preparing peptidase in minutes.

The strain producing the enzyme of the present invention is not particularly limited as long as it produces the enzyme of the present invention, but suitable strains include, for example, Trichosporon cutaneum I F 0
0173, Trichosporon bapley (Tri-ch
osporon beigelii) I F
O0598, Trichosporon fermentus (T.
richosporon F'errn, entas)
I F O1199, Trichosporon Capitatum
IFQ1197 etc. can be used.

Nutrient sources for the culture medium for culturing such bacterial strains are widely used, and nitrogen sources include, for example, peptone, meat extract, yeast, soy flour, cornstarch liquor, urea, Examples of carbon sources include starch, dextrin, sucrose, glucose, maltose, molasses, glycerin,
Examples include ethanol, mannitol, and sorbitol.

Examples of 4-hole salt-free salts include magnesium, calcium, potassium, sodium, phosphorus, iron, manganese,
Salts such as molybdenum are used as appropriate.

In particular, it is preferable to use corn stable liquor as the nitrogen source and glycerin as the carbon source.

Generally, liquid culture is suitable as the culture format, and industrially, it is advantageous to carry out deep aeration agitation culture.

In addition, as culture conditions, the temperature is generally in the range of 25 to 37°C, and the initial pH of the medium is generally 6.5.
-8.0, preferably 7.0-7.5. Cultivation can usually be carried out under such conditions for about 30 to 150 days, thereby making it possible to obtain the desired amount of the porridge of the present invention.

Recovery of the enzyme of the present invention from bacterial cells in which the enzyme of the present invention has been optically accumulated in this way can be carried out in accordance with the method used for the separation and purification of intracellular enzymes. For example, microbial cells collected by centrifugation may be mixed with an appropriate amount of Tris-HCl buffer (
pH 7, s), Dyno Mill, French Press,
Grind in a mortar etc.

Next, centrifugation is performed to remove unbroken bacterial cells and to isolate the crude pancreatic fluid. To obtain the target enzyme from this crude enzyme solution, first, the crude enzyme solution is heat-treated at 50 to 55°C for 5 to 10 minutes, and the resulting precipitate is removed by centrifugation. At this time, for enzyme stabilization, about 10 parts of glycerol or sucrose may be added as needed to perform heat treatment.

The resulting enzyme solution is then purified by subjecting it to ammonium sulfate salting out, DEAE cellulose ion exchange chromatography, hydroxyapatite adsorption chromatography, electrofocusing, and the like.

The Z,-)lysotophan aminopeptidase provided by the present invention has high amide hydrolysis ability and can be used as a catalyst for producing L-tryptophan (see, for example, Japanese Patent Application No. 55-84618). It can also be used as a reagent for amino acid sequencing of proteins and peptides.

It may also be used as an anti-inflammatory agent in the field of food processing or as a medicine.

Next, the present invention will be further explained with reference to Examples.

Example 1 5 parts glycerol, 5% corn steep liquor,
1 part of 11N/- potassium phosphate buffer (pH 7,0),
Mineral rlli combination (q (Mg504-78202
%, FeS04°Tl1200.5 fb, CaC
1,0,2%, IvfnCl, -48200,02%
, N aAio O4・211,00.01%, N
aC10,01 section) Iq the 1% composition to pH 7.0?
A Trichosporon cutaneum I in the conditioned medium 201
1.51 of Oki culture solution of FOO173 was inoculated and grown at 20Orpm, loN/mi in a 501 volume gloss fermenter.
Incubate for 30 hours at 0°C under 1 m of air. After culturing,
Collect the solids using a Sharpless centrifugal preparator, and
-Cold 50mM) Lys containing mercaptoethanol-
Hydrochloric acid buffer (pH 7,5) 10j! The microbial cells are suspended in water and ground using Inomil. This operation and the following operations are all performed at 4°C or lower.

Add 10% glycerol to the supernatant that was centrifuged at 1F, heat-treated at 55°C for 5 minutes, and remove the resulting precipitate by centrifugation. Add ammonium sulfate 5.3K9 to this supernatant, remove the resulting precipitate by centrifugation, and then add ammonium sulfate 1.86K2 to the strainer. The resulting precipitate is collected by centrifugation and dialyzed against the aforementioned buffer for 20 hours.

After dialysis, a DEAE cellulose column (φ36 x 3
After adsorbing the dialysate (0) and washing thoroughly with the same buffer containing 0.075 MKCl, it is bathed with the same buffer 911 containing 0.075 MKCl, and then with the same buffer 911 containing 0.075 MKCl.

0, I Ammonium sulfate 69 in the active category obtained in MMC1 category
The precipitate formed by adding 0.07 was collected and added to 5mM phosphorus-rich potassium buffer (p
20 hours of dialysis against H7,5). After dialysis, the dialysate is adsorbed onto a hydroxyapatite column (φ3.6 x 6 ca) that has been subjected to XIL equilibration with the same buffer and thoroughly washed with the same buffer. o. Elution was performed with 200 ml of IM potassium phosphate buffer, followed by 0.2 M potassium phosphate buffer (both containing 0.05% 2-mercaptoethanol), and the sample was obtained in a 0.2 MIJ potassium phosphate buffer section. Add active class VC ammonium sulfate 921i'. Collect the resulting precipitate and mix with 5mM) Lithium salt containing 0.05% 2-merzyl ethanol.
(pH 7.5) for 20 hours. After centrifuging to remove insoluble matter, 6 ml of the supernatant was poured into a B Prod.
p II 4 using a Ucterg 110-volume column
Electrofocusing is performed in the range of ~6. The results are shown in Figure 5. Active category (Fluxi'eln 39-5
0) An enzyme preparation showing a single peak in disk electrophoresis was obtained by collecting i. In addition, equal'r+3: the point is pH 4
, q. The yield and specific activity of each refinement stage are as follows.

Reference Example 1 0.5 me (0.5 units) of the purified enzyme obtained in Example 1 was added to 0.257 of 8 mM Mnc 12i'f3 solution! ,
0.24MDL-tricytophanamide%'((ff,
(pH 7,0) 0.251 nl.

After adding and reacting at 37°C for 60 minutes, heating at 90°C for 5 minutes to stop the reaction, L - When the tributophane was determined, the yield was 96%.

[Brief explanation of drawings]

Figure 1 is a graph showing the effect of pH on the activity of the enzyme of the invention, and Figure 2 is a graph showing the effect of pH on the activity of the enzyme of the invention.
Figure 3 is a graph showing the effect of pH on the activity of the enzyme of the present invention, and Figure 4 is a graph showing the influence of mud Ft on the stability of the enzyme of the present invention. FIG. 5 is a graph showing the results of electrofocusing, and FIG. 6 is an enlarged photograph (600 times) of crystals of the enzyme of the present invention. In FIGS. 1 and 2, when the curve a is used with Tris-maleide buffer, the curve is glycine-NαO1i.
It is a graph when a buffer solution is used. In addition, in Fig. 4, curve 1B 6 is when no sucrose is added, and curve d is sucrose 10.
% addition. 1,000 figure pH 7 Kayabori ≧ Temperature (0C) 2nd figure pH Rod 4 figure? 1 Flea 7L ('C) Half 5 figure fraction Nasivar! 0.8rnL/Fuku2shitan) Figure 6 Procedural Amendment (#F,) January 24, 1980 Patent Office Repliance J) A, 1. Indication of the case...; l157 middle page 1 self, 1+1 bow', 203ti0
9.2, Name of the invention 7.
e' and -inji-no-] No 1 Law 3 Supplemental relationship between the beach matter and what^ Patent applicant (l-sho Yama ['Maze IJ-i'it\Yamabanto-cho 1-12 W'32 +r name (o2o) J-I 10; 4+, ;H + book S', ri + 1 (name) 4th representative person〒107 (1) January I 1i -2'4'-y 1
2 Self it 4 Ej υc [-E,
Co11K-12" Y r A', C0A
Weigh it as ``L 8-12''. (2) lr'' H14m, (7) 4th:j<1-N-methylmaleimide'' in the 8'il position from middle 2 i N-: x-tilma 1/imide'' and d'' Stop. (3) In the same No. 1510441-2, there is "I-1\I-methylma)/imide"=f r N-En°γ'luma 1/
Imido” and 1. ] Correct. (411ijJ4.15self No. 91) 11 Stops when a certain verb "-Kale L1 side" is passed. (5) Same car 15 m"'Fd- et al; 4!, 3 Ij Oc 1A71.7+, N, J and i" Ann, ha
ti, J and i” Correct. Same, ax6↓4me"jλ1 j,, it l-gromofunorp and wo"gromofukonorp-j and M
1J ig rm from 4, 4i J
, n n t Q, sJ is rfgrm, enter
sJ and MJ correct. (8) gcap'ita t in the third line from the bottom of page 17.
, qbmJ is corrected as f "capitatum J. (9) Times d422 self; In line J46, "aoCm) dialysis" is corrected to f30cm) dialysis."that's all

Claims (1)

[Claims] 1.L-)Liptophan aminopeptidase. 2. Cultivating a bacterial strain belonging to the genus Trichosporon and having L-tryptophan aminopeptidase physiological activity in a medium, producing and accumulating L-)IJ butophane aminopeptidase in the bacterial cells, and then separating and purifying L-tryptophan peptidase from the bacterial cells. A method for producing L-tryptophan amine peptidase, characterized by: