JP4374945B2 - Novel serine protease - Google Patents

Description

  The present invention relates to a novel serine protease and a method for producing the serine protease.

  Serine protease is an endopeptidase in which three amino acids, histidine, aspartic acid, and serine, are involved in the expression of catalytic activity, and are generally inactivated by diisopropylfluorophosphate (DFP), which chemically modifies the serine residue of the active group. is there. Typical animal-derived serine proteases include trypsin from bovine and porcine pancreas, and in microorganisms, trypsin-like serine proteases from Streptomyces griseus, Streptomyces fradiae, Streptomyces erythreus have been reported, Their properties have been reported (see Table 6). These conventionally known enzymes cleave arginine (Arg) -X bond and lysine (Lys) -X bond with respect to peptide substrates, and bonds other than those bonds, such as tyrosine (Tyr) -X The existence of an enzyme that also cleaves bonds and methionine (Met) -X bonds is not known.

  In order to solve the above problems, the present invention provides a serine protease that hydrolyzes arginine-X bond, lysine-X bond, tyrosine-X bond, methionine-X bond, and glutamic acid-X bond (where X represents an amino acid). The issue is to provide

  As a result of intensive studies on the microorganisms discovered by the present inventors for the first time, the present inventors have succeeded in obtaining a novel serine protease by culturing the microorganisms, thereby completing the present invention.

The present invention is an invention of a novel serine protease having the following properties a to d.
a. pH: optimum pH; 7.5-11.0, stable pH range; 4.5-10.5
b. Temperature: Optimal temperature; 70 ° C, temperature stability range 10 ° C to 70 ° C
c. Molecular weight: about 29,000 (SDS-PAGE method)
d. A sequence of 16 residues from the N-terminal amino acid:
Ile Ile Gly Gly Ile Asp Val Ala Asn Gly Lys Phe Pro Phe Met Val
The present invention also includes a prosthesis that is biologically equivalent to the above novel serine protease. That is, a biologically equivalent protease is a part of a trypsin-like protease which is the novel serine protease of the present invention or one or more amino acids deleted or added from the protease, and / or Alternatively, it refers to a protease in which one or more amino acids in the protease are replaced with other amino acids and has the biological properties of the protease of the present invention.

The present invention also relates to a method for producing a serine protease characterized by culturing a microorganism having the following properties a to l and not requiring methionine and glutamic acid for growth and collecting the microorganism from the culture: It is.
a. Form: Neisseria gonorrhoeae b. Size: 0.7 ~ 0.8 × 2.0 ~ 3.0μm
c. Gram stainability: negative d. Spore: negative e. Motility: negative f. Attitude toward oxygen: aerobic g. Oxidase: positive h. Catalase: positive i. OF test: negative j. Set color: no characteristic pigment or pale yellowish k. Quinone series: Q-8
l. GC content of intracellular DNA: 70 mol%
The microorganism is newly isolated and identified from the soil by the present inventors. The present inventors have identified Lysobacter sp IB-9374 (hereinafter abbreviated as Rhizobacter IB-9374). And is entrusted to the Institute of Biotechnology, Institute of Industrial Technology (1-3 East, 1-3 Tsukuba City, Ibaraki Prefecture), which is the international depositary body of the Budapest Treaty (accession number FERM P-16928).

  The serine protease of the present invention is capable of hydrolyzing arginine-X bond, lysine-X bond, tyrosine-X bond, methionine-X bond and glutamate-X bond (where X represents an amino acid). . A trypsin-like serine protease having such a hydrolysis function has not been known so far.

  In addition, the enzyme of the present invention has 150 to 1000 times higher enzyme activity than known trypsin-like activity, and also cleaves tyrosine-X bond, which is the cleavage point of pepsin, so that casein, α-lactalbumin, β -It is an effective enzyme which has the effect which a known trypsin-like serine protease does not have, such as being able to reduce these allergen activities by using for food proteins, such as lactoglobulin.

  The serine protease of the present invention is collected by culturing Rhizobacter sp. IB-9374, and the culturing can be performed by a conventional method. That is, the culture temperature is usually 20 to 35 ° C., preferably 25 to 35 ° C., and the pH of the medium is usually 6 to 8, preferably 6.5 to 7.5. Further, the culture is preferably performed under aerobic conditions by means such as shaking or aeration stirring, and the culture time is usually 150 to 250 hours.

  The medium composition of Rhizobacter sp. IB-9374 may be either a natural medium or a synthetic medium as long as the microorganism can grow. The medium may be a solid or liquid medium. These media include, for example, sugars such as glucose, maltose and mannose as carbon sources, organic acids such as citric acid and malic acid, alcohols such as ethanol and glycerol, peptone as a nitrogen source, meat extract, In addition to general natural nitrogen sources such as yeast extract, protein hydrolyzate, and amino acids, various inorganic and organic ammonium salts are included. In addition, inorganic salts such as potassium, magnesium, iron, and calcium salts are required. Depending on the case, it is added appropriately.

The Rhizobacter genus IB-9374 is a microorganism having the following properties a to l and does not require methionine and glutamic acid for growth.
a. Form: Neisseria gonorrhoeae b. Size: 0.7 ~ 0.8 × 2.0 ~ 3.0μm
c. Gram stainability: negative d. Spore: negative e. Motility: negative f. Attitude toward oxygen: aerobic g. Oxidase: positive h. Catalase: positive i. OF test: negative j. Set color: no characteristic pigment or pale yellowish k. Quinone series: Q-8
l. GC content of intracellular DNA: 70 mol%
A specific method for obtaining the serine protease of the present invention using Rhizobacter IB-9374 is as follows.

  That is, Rhizobacter sp. IB-9374 was cultured, and when the amidase activity in the culture solution became high, the culture solution was filtered. The obtained culture filtrate was subjected to (1) ammonium sulfate fraction (0 to 50%), (2) Isoelectric point precipitation treatment, (3) Fractionation with 10-100 mM, preferably 30-50 mM ammonium sulfate, (4) Precipitation treatment with 50-90%, preferably 70-80% ethanol, (5) Column chromatography treatment such as gel filtration column chromatography (6) Precipitation treatment with 50 to 90%, preferably 70 to 80% ethanol (7) Buffer substitution, (8) Heat treatment, (9) Focal electrophoresis, etc. It is obtained by attaching. These processes are preferably performed sequentially in the order of (1) to (9).

The enzymatic chemistry of the novel serine protease of the present invention is described below.
a. pH: optimum pH; 7.5-11.0, stable pH range; 4.5-10.5
b. Temperature: Optimal temperature; 70 ° C, temperature stability range 10 ° C to 70 ° C
c. Molecular weight: about 29,000 (SDS-PAGE method)
d. A sequence of 16 residues from the N-terminal amino acid:
Ile Ile Gly Gly Ile Asp Val Ala Asn Gly Lys Phe Pro Phe Met Val
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Purification of Serine Prosthesis from Culture Solution of Xanthomonas Rhizobacter IB-9374 Substrate and Reagent In this experiment, the following substrate and reagent were used.

Substrate
Nα-Benzoyl-L-arginine-p-nitroanilide hydrobromide
(Hereinafter abbreviated as Bz-Arg-pNA) Peptide Research Institute Reagents Tris base; Wako Pure Chemical Industries, Ltd., Toyopearl HW-55F; Tosoh Corporation,
N-cyclohexyl-3-aminopropanesulfonic acid (CAPS); Nacalai Tesque,
Brij 35; Nacalai Tesque,
Pharmalite; Amersham Pharmacia,
LMW Electrophoresis Calibration Kit E; Amersham Pharmacia, Ethanol (99.5%); Wako Pure Chemical Industries, Ltd.
All other reagents used were special grades.

2. Experimental method
(1) Cultivation of Rhizobacter IB-9374 As a result of examining the composition of various media, the hydrolysis activity (amidase activity) for Bz-Arg-pNA in 1 ml of Rhizobacter IB-9374 culture solution is the highest (0.102 unit) The culture was carried out in the medium composition shown in Table 1 below.

  The culture solution was adjusted to pH 7.2, dispensed 100 ml each into three 500 ml Sakaguchi flasks that had been sterilized by heating, and then sterilized at 121 ° C for 20 minutes and shake cultured at 30 ° C for 24 hours. A preculture was used. Dispense 20 ml (5%) from this preculture into 7 1,000 ml Sakaguchi flasks (2,800 ml) containing 400 ml of freshly prepared culture, and then incubate at 30 ° C. The cells were cultured for 228 hours so that the amidase activity (substrate; Bz-Arg-pNA) was maximized. The results are shown in FIG. FIG. 1 also shows the change in UV absorption of trypsin-like enzyme.

  After culturing, the cells were removed at 31,000 xg at 4 ° C using a continuous cooling centrifuge (Hitachi; HIMAC SCR20B type, continuous centrifuge rotor; RPRC18-3), and the supernatant was used as crude enzyme solution (2,565 ml). )

  In addition, the amidase activity shown in FIG. 1 was measured as follows. Further, unless otherwise specified below, amidase activity was measured according to the following method.

  That is, a known method (for example, Hoppe-Seyler's Z. Physiol. Chem., 329, 278 (1962) measuring p-nitroaniline released by enzymatic action at 405 nm using Bz-Arg-pNA as a substrate. )) Was used. In addition, Bz-Arg-pNA was used by dissolving 50 mg in pure water to 25 ml and storing it frozen (4.6 mM).

Specifically, first, 0.15 ml of 4.6 mM Bz-Arg-pNA solution and 1.3 ml of 200 mM Tris-HCl buffer (pH 9.2) were each dispensed into a test tube (1.5 x 13 cm), and a constant temperature water bath at 30 ° C. After 5 minutes, 0.05 ml of the enzyme solution was added and allowed to react. After 15-20 minutes, 0.5 ml of 45% acetic acid solution was added to stop the reaction, and the absorbance at 405 nm was measured. As a control sample, a substrate aqueous solution 0.15 ml, 200 mM Tris-HCl buffer (pH 9.2) 1.3 ml, 45% acetic acid solution 0.5 ml and enzyme solution 0.05 ml were sequentially added and mixed quickly. The enzyme activity that liberates 1 μmol of p-nitroaniline (molecular extinction coefficient 9,620) per minute under the above conditions was defined as 1 unit, and the number of units (amidase activity) was determined from the absorbance value. The enzyme activity 1 (unit / ml) was calculated according to the following formula.
Enzyme unit (unit / ml) = OD 405 nm correction value / 9,620 × 1,000 × 2 / 0.05 × dilution ratio / reaction time (min)

2. Purification of enzyme from culture medium of Rhizobacter IB-9374 The enzyme was purified from Rhizobacter IB-9374 by the purification method shown in FIG. Each generation process will be described in detail below. All purification operations in each purification process were performed at 4 ° C.
(1) Ammonium sulfate fraction of culture filtrate
Ammonium sulfate was added to 2,565 ml (223.0 units) of the culture filtrate cultured for 228 hours so as to achieve 50% saturation (temperature 0 ° C.). After standing overnight at 4 ° C, the precipitate obtained by batch centrifugation (31,000 xg, 20 minutes, 4 ° C) is placed in 25 mM Tris-HCl buffer (pH 7.0) containing a minimum amount of 10% glycerol. Suspended.

  The obtained suspension enzyme solution was further separated by centrifugation (31,000 × g, 20 minutes, 4 ° C.). 25 mM glycine-sodium hydroxide buffer (pH 10.0, hereinafter abbreviated as A buffer) containing 10% glycerol in a mortar well cooled on ice to extract the enzyme protein present in the resulting precipitate The resulting extract was centrifuged again (31,000 × g, 20 minutes, 4 ° C.). The amidase activity for Bz-Arg-pNA of the extract was measured, and this operation was repeated until the activity was not confirmed, and 400 ml (184.0 units) of the enzyme solution was obtained.

(2) Isoelectric precipitation
400 ml (184.0 units) of the brown enzyme solution obtained in (1) was sufficiently dialyzed against 25 mM Na2HPO4-Citrate (citrate monohydrate) buffer (pH 4.0) containing 10% glycerol. Thereafter, the suspended enzyme solution produced during dialysis was centrifuged (31,000 × g, 20 minutes, 4 ° C.) to obtain a precipitate. The resulting precipitate was completely dissolved in A buffer (pH 10.0) and then sufficiently dialyzed against the same buffer at 4 ° C. to obtain 52 ml (181.0 units) of an enzyme solution.

(3) 0.86% (50 mM) ammonium sulfate fraction
Ammonium sulfate (Nacalai Tesque, Inc., molecular weight 132.14, special reagent for biochemical research) was added to 52 ml (181.0 units) of the enzyme solution obtained in (2) so that the final concentration was 50 mM (0.86% saturation). did. After standing overnight at 4 ° C, the precipitate obtained by centrifugation (31,000 xg, 20 minutes, 4 ° C) is completely dissolved in A buffer (pH 10.0). After sufficiently dialyzing, 25 ml (216.0 units) of the enzyme solution was obtained. The total activity increased by this step. This is because the enzyme activity temporarily decreased due to inhibition of enzyme activity by ammonium sulfate in this step was eliminated by dialysis, and the isoelectric point precipitation of (2). This is considered to be higher than the value at.

(4) 75% ethanol precipitation
Ethanol (Wako Pure Chemical Industries, Ltd., special grade) well cooled at 20 ° C was added to 25 ml (216.0 units) of the pale yellow enzyme solution obtained in (3) to a final concentration of 75%. . The mixture was allowed to stand at 4 ° C for 1 hour, and further separated by centrifugation (31,000 xg, 20 minutes, 4 ° C). The resulting precipitate was dissolved in 10 mM CAPS buffer (pH 10.5, hereinafter referred to as B buffer; abbreviated) containing 10% glycerol, and then sufficiently dialyzed at 4 ° C. against the same buffer. Thereafter, the solution was concentrated to 10 ml (193.0 units) using an ultrafiltration membrane (Amicon Millipore Ultrafiltration Membrane, PLCC Dia, 44.5 mm, 0.7 kg / cm ² ).

(5) Toyopearl HW-55 column chromatography
The enzyme solution 10 ml (193.0 units) concentrated in (4) was divided into 5 portions and applied to a Toyopearl HW-55 column. That is, the concentrated enzyme solution (193.0 units) was applied to a Toyopearl HW-55 column (1.5 × 20 cm) equilibrated with a B buffer solution (pH 10.5) containing 100 mM sodium chloride to separate impure proteins. At this time, only the impure protein was eluted, and the target enzyme protein was not eluted at all with the B buffer (pH 10.5) containing 200 mM sodium chloride and 0.5% Brij35, and remained on the top of the Toyopearl HW-55 column. It was in a state. Therefore, collect about 5 cm of resin from the top of the column, add an appropriate amount (about 5.0 ml) of B buffer (pH 10.5) containing 0.5% Brij35, and leave it at 30 ° C for 30 minutes. Was recovered. The same operation was repeated several times, and the enzyme solution whose activity was completely recovered was sufficiently dialyzed against the same buffer solution. Thereafter, the solution was concentrated to 16 ml (123.0 units) using an ultrafiltration membrane (Amicon Millipore Ultrafiltration Membrane, PLCC Dia, 44.5 mm, 0.7 kg / cm ² ).

(6) 75% ethanol precipitation
Ethanol (Wako Pure Chemical Industries, Ltd., special grade) well cooled at 20 ° C. was added to 16 ml (123.0 units) of the enzyme solution concentrated in (5) so that the final concentration was 75%. The mixture was allowed to stand at 4 ° C for 1 hour, and further separated by centrifugation (31,000 xg, 20 minutes, 4 ° C). The obtained precipitate was suspended in 20 mM Tris-HCl buffer (pH 7.0, hereinafter referred to as C buffer; abbreviated) containing 10% glycerol, and the suspension was kept at 4 ° C against the same buffer. Dialyzed thoroughly. Thereafter, the solution was concentrated to 16 ml (114.0 units) using an ultrafiltration membrane (Amicon Millipore Ultrafiltration Membrane, PLCC Dia, 44.5 mm, 0.7 kg / cm ² ).

(7) Buffer replacement
In 16 ml (114.0 units) of the enzyme solution concentrated in (6), the target enzyme protein does not dissolve in the C buffer (pH 7.0) and is visible in the buffer. The enzyme protein was further separated by centrifugation (31,000 × g, 20 minutes, 4 ° C.), and the resulting precipitate completely containing the amidase activity was completely dissolved in B buffer (pH 10.5). Then, dialyze thoroughly at 4 ° C against the same buffer, and use an ultrafiltration membrane (Amicon Millipore Ultrafiltration Membrane, PLCC Dia, 44.5 mm, 0.7 kg / cm ² ), 17 ml (104.0 units) Concentrated to.

(8) Heat treatment
The enzyme solution 17 ml (104.0 units) concentrated in (7) was heat-treated at 45 ° C. for 30 minutes. This operation was performed based on the temperature stability of the present enzyme, which was previously performed as a preliminary experiment. In other words, proteases other than the target enzyme protein considered to be present in the enzyme solution were denatured by heat treatment, deactivated and hydrolyzed by the enzyme. No inactivation of the target enzyme was observed by this operation (104.0 units / 17 ml).

(9) Focus electrophoresis
17 ml (104.0 units) of the enzyme solution heat-treated in (8) was subjected to focal electrophoresis. That is, amphoteric electrolyte carrier pharmalite (pH 2.5-5.0) is mixed with the enzyme solution, and 0.5% amphoteric electrolyte carrier pharmalite (pH 2.5-5.0) is contained in a 110 ml column (type LKB 8,100). A 0-50% sucrose density gradient was created. After electrophoresis at 600V for 48 hours at 4 ° C, elution from the column was performed. The pH, absorbance at 280 nm and enzyme activity (amidase activity) of each fraction were measured, and the peak pH of the activity category was measured to obtain the isoelectric point of the enzyme of the present invention. The result is shown in FIG.
Furthermore, after the obtained eluate was sufficiently dialyzed against B buffer solution, using an ultrafiltration membrane (Amicon Millipore Ultrafiltration Membrane, PLCC Dia, 44.5 mm, 0.7 kg / cm ² ), 12 Concentrated to ml (60.0 units).

(10) Activity yield and specific activity in each purification step Table shows the total protein amount converted from the volume, total activity, and absorbance measured values in each purification step, the total protein amount by Bradford method, specific activity, yield, and purity. It was shown in 2. The total activity is a value obtained by operating in the same manner as the method for measuring amidase activity described in (I), and the specific activity is a value obtained by dividing the total activity by the total protein amount by Bradford method. Yes, the yield is the ratio of the activity value of each purification step when the total activity of the culture filtrate is 100, and the degree of purification (purification ratio) is the ratio of each purification step when the specific activity of the culture filtrate is 1. The ratio of activity values is shown respectively.

  In the quantification of total protein by the Bradford method, bovine serum albumin (Protein Standard II, Bio-Rad) was used as a standard protein.

From these results, it was found that these purification operations resulted in an amidase activity yield of 27% and a specific activity of about 380 times. In addition, the isoelectric point precipitation of (2) can increase the specific activity by 15.5 times, and this operation was found to be very effective in removing contaminating proteins. From the results shown in FIG. 3, it was confirmed that the focal electrophoresis of (9) is an effective means for obtaining a purified enzyme preparation having an isoelectric point of pH 4.04.
It was also found that the isoelectric point of this enzyme (pH 4.04) was close to that of Streptomyces ericeus (pH 4.0) (see Fig. 3).
From the above results, it was confirmed that the enzyme derived from Rhizobacter IB-9374 is an acidic protein.

(11) Uniformity and molecular weight measurement of the purified enzyme obtained in (9) by SDS-PAGE The homogeneity and molecular weight of the purified enzyme preparation were measured using Laemmli's acrylamide with 12.5% separation gel and 2.5% concentrated gel acrylamide. It was measured by electrophoresis method. That is, the marker BPB was run at constant current of 10 mA in the concentrated gel, 20 mA constant current after entering the separation gel, and run for about 2 hours until the BPB line was 5 mm above the gel end. . After electrophoresis, staining was performed with a mixed solution of 0.1% CBB R-250 (w / v), 30% methanol (v / v), and 10% acetic acid (v / v). Decolorization was performed using methanol: acetic acid: water = 3: 1: 6 (v / v). LMW electrophoresis calibration kit E (phosphorylase b; 94,000, serum albumin; 67,000, ovalbumin; 43,000, carbonic anhydrase; 30,000, trypsin inhibitor; 20,100, α-lactalbumin; 14,400) Was used. In order to prevent self-digestion of the enzyme protein, this enzyme was previously inactivated by heat treatment for 3 minutes in boiling water at 95 ° C. The result is shown in FIG.

From this result, it was confirmed that the purified enzyme preparation was uniform by SDS-PAGE. The molecular weight of the enzyme was measured by SDS-PAGE, and was calculated to be about 29,000 (see FIG. 5). This value is determined from Streptomyces griseus-derived protease (MW 20,000, pI = 9.0), Streptomyces fradiae-derived protease (MW 16,500, pI = 9.0), Streptomyces erytheus-derived protease (MW 20,000, pI = 4.0) Compared with the molecular weight of bovine and porcine pancreatic trypsin (bovine; MW 24,000, pig; MW 23,300), it was confirmed to be considerably larger.
Based on the above, SDS-PAGE with 27% recovery of amidase activity against Bz-Arg-pNA and approximately 380-fold increase in specific activity after 9 steps from 2,565 ml of this bacterial culture It was found that a uniform enzyme can be obtained.

Experimental Example 1 Chemical properties of the enzyme of the present invention Reagents Enzyme (SDS-PAGE) homogenous enzyme sample obtained in Example 1 was stored at 4 ° C in 10 mM CAPS buffer (pH 10.5) containing 10% glycerol. I used something.
The following reagents were used.
EDTA ・ 3Na ・ 3H2O ； Dojin Research Institute Co., Ltd.
o-Phenanthroline; Nacalai Tesque
L-cysteine; Tokyo Chemical Industry Co., Ltd.
2-mercaptoethanol ： Wako Pure Chemical Industries, Ltd.
N-ethylmaleimide (NEM); Nacalai Tesque
Iodoacetic acid ： Nacalai Tesque
r-Chloromercury benzoic acid (PCMB); Nacalai Tesque
Diisopropyl fluorophosphate; Wako Pure Chemical Industries, Ltd.
Phenylmethanesulfonyl fluoride; Boehringer
N-tosyl-L-lysyl chloromethyl ketone (TLCK); Wako Pure Chemical Industries, Ltd.
N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK); Nacalai Tesque
Dithiothreitol (DTT); Nacalai Tesque
Tris base; Wako Pure Chemical Industries, Ltd.
Methanol; Wako Pure Chemical Industries, Ltd.
Acetic acid; Wako Pure Chemical Industries, Ltd.
Coomassie Brilliant Blue R-250 (CBB R-250); Nacalai Tesque
Pro Blot Membrane; Appli Biosystem Filter Paper; Advantech Leupeptin; Peptide Research Center Chimostatin; Peptide Research Center As a commercially available enzyme, TPCK-treated trypsin derived from bovine pancreas is manufactured by Worthington Biochemicals, derived from porcine pancreas A trypsin crystal manufactured by Wako Pure Chemical Industries, Ltd. was used.
The enzyme concentration of trypsin from bovine and porcine pancreas is determined using the molar extinction coefficient at 280 nm in the ultraviolet region, E _{1 cm} ^1% = 16.0 for bovine trypsin and _{1 cm} ^1% = 15.0 for porcine trypsin. It was.

2. experimental method
(1) Measurement of amidase activity using pNA substrate The amidase activity was measured according to 2 in Example 1 using Bz-Arg-pNA as a substrate. Measured according to the method described in.
The amidase activity was measured within an enzyme concentration of 0.08 μg, a substrate concentration of 0.46 mM or less, an absorbance of 0.4 or less, and a reaction time of 12 minutes or less.

(2) Optimum pH of this enzyme
We investigated the effect of pH on the amidase activity of this enzyme against Bz-Arg-pNA. That is, 115 mM various buffers [pH 2.0-4.5; glycine hydrochloride buffer (-○-), pH 3.0-6.0; citrate buffer (-x-), pH 4.5-8.0; potassium dihydrogen phosphate-phosphorus Dipotassium hydrogen phosphate buffer (-■-), pH 7.0-9.0; Tris-HCl buffer (-△-), pH 8.0-9.5; AMP-HCl buffer (-●-), pH 9.0-12.0; Glycine- Sodium hydroxide buffer solution (-□-)] After incubating a mixed solution of 0.1 ml of 4.6 mM Bz-Arg-pNA solution in 1.3 ml at 30 ° C for 5 minutes, CAPS buffer solution containing 0.05 μg of enzyme (pH 10.5) 0.05 ml was added and reacted. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured.
The result is shown in FIG. As is clear from this result, the optimum pH of the amidase activity of this enzyme for Bz-Arg-pNA was pH 7.5-11.0.

(3) Optimum temperature of this enzyme The effect of temperature on the amidase activity of this enzyme for Bz-Arg-pNA was examined. That is, after incubating a mixed solution in which 0.15 ml of 4.6 mM Bz-Arg-pNA solution was added to 1.3 ml of 200 mM Tris-HCl buffer (pH 9.2) at various temperatures (10-80 ° C) for 5 minutes, 0.05 μg of enzyme was added. 0.05 ml of CAPS buffer solution (pH 10.5) containing was added and reacted at various temperatures. After 2-20 minutes, the reaction was stopped by adding 45 ml of 45% acetic acid, the absorbance at 405 nm was measured, and the relative activity was calculated from the number of Units per ml. The result is shown in FIG.
From this result, it was found that this enzyme shows the maximum activity at 70 ° C.

(4) pH stability of the enzyme The stability of the enzyme at various pH was investigated.
That is, various buffer solutions of 100 mM containing 0.1 μg of enzyme [pH 2.0-4.5; glycine hydrochloride buffer (− ○ −), pH 3.0-6.0; citrate buffer (− × −), pH 4.5-8.0; phosphate Potassium dihydrogen-dipotassium hydrogen phosphate buffer (-■-), pH 7.0-9.0; Tris-HCl buffer, (-△-) pH 8.0-9.5; AMP-hydrochloric acid buffer (-●-), pH 9.0 -12.0; glycine-sodium hydroxide buffer (-□-)] 0.1 ml was allowed to stand at 4 ° C for 24 hours, and 0.05 ml of this enzyme solution (enzyme; 0.05 µg) was incubated at 30 ° C for 5 minutes in advance. The reaction was initiated by adding 1.3 ml of 200 mM Tris-HCl buffer (pH 9.2) and 0.15 ml of 4.6 mM Bz-Arg-pNA solution. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured. The result is shown in FIG.
From this result, it was found that this enzyme is stable in the range of pH 4.5-10.5.

(5) Temperature stability of the enzyme The stability of the enzyme at various temperatures was investigated.
That is, 0.1 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 0.1 μg of enzyme was allowed to stand at various temperatures (10-80 ° C.) for 30 minutes and then cooled sufficiently on ice. 0.05 ml of this enzyme solution (enzyme; 0.05 μg) was mixed with 1.3 ml of 200 mM Tris-HCl buffer (pH 9.2) and 0.15 ml of 4.6 mM Bz-Arg-pNA solution that had been incubated at 30 ° C for 5 minutes. To start the reaction. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured. The result is shown in FIG.
From this result, the enzyme is stable enough to have approximately the same activity as the maximum activity from 10 ° C to 50 ° C. It is about 90% of the maximum activity at 60 ° C and about 80% of the maximum activity at 70 ° C. It turned out to be stable. On the other hand, it was also found that at 80 ° C., it was completely deactivated.

(6) Effects of various enzyme denaturing agents on this enzyme The effects of urea, guanidine hydrochloride and sodium dodecyl sulfate (SDS) on the amidase activity of this enzyme were examined.
That is, the effect of urea, guanidine hydrochloride and SDS on this enzyme was determined by pretreating 0.2 ml of 40 mM Tris-HCl buffer (pH 8.0) containing various concentrations of denaturant and 0.05 μg of enzyme at 30 ° C for 30 minutes. Add 0.1 ml of this treatment solution (enzyme; 0.025 μg) to 1.25 ml of 200 mM Tris-HCl buffer (pH 9.2) containing various concentrations of urea, guanidine hydrochloride, and SDS that had been incubated at 30 ° C for 5 minutes in advance. 4.6 mM Bz-Arg-pNA solution was added to 0.15 ml of the mixed solution to start the reaction. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured.
Moreover, in order to compare the influence of this enzyme with respect to various protein denaturing agents, the influence was similarly measured using trypsin derived from bovine and porcine pancreas. Specifically, 0.2 ml of 40 mM Tris-HCl buffer (pH 8.0) containing various concentrations of denaturant and bovine-derived trypsin (20.0 μg) or porcine-derived trypsin (2.0 μg) was pretreated at 30 ° C. for 30 minutes. Residual activity was measured using 0.1 ml of the solution (bovine-derived trypsin; 10.0 μg, porcine-derived trypsin; 1.0 μg).

The results are shown in FIG. 10 (urea), FIG. 11 (guanidine hydrochloride), and FIG. 12 (SDS), respectively. The results of trypsin derived from bovine pancreas are shown as-●-, the results of trypsin derived from porcine pancreas are shown as -Δ-, and the results of this enzyme are shown as-◯-.
From this result, when the enzyme was pretreated with 1.0 mM urea, 0.1 M guanidine hydrochloride and 0.1% SDS for 30 minutes at 30 ° C in 40 mM Tris-HCl buffer (pH 8.0), the activity was 100%, 30% and 35% remained. In any case, the residual activity was higher than those of bovine and porcine pancreatic trypsin. In particular, the resistance of the enzyme to urea is more pronounced than that of bovine and porcine pancreatic trypsin, whereas in the presence of 5.0 M urea, bovine and porcine pancreatic trypsin is almost inactivated. With this enzyme, 50% residual activity was observed.

(7) Effects of various inhibitors of this enzyme The effects of metal chelating reagent, SH reagent and reducing agent on the amidase activity of this enzyme were examined.
Specifically, 0.2 ml of 40 mM Tris-HCl buffer (pH 8.0) containing 0.1-10 mM of various inhibitors and 0.05 μg of enzyme was pretreated at 30 ° C. for 30 minutes, and then 0.1 ml of this pretreatment solution (enzyme; 0.025 μg ) Is added to a mixture of 1.25 ml of 200 mM Tris-HCl buffer (pH 9.2) and 0.15 ml of 4.6 mM Bz-Arg-pNA solution containing the inhibitor at the specified concentration previously incubated at 30 ° C for 5 minutes. The reaction was started. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured. Table 3 shows the relative activities determined from the measured values.

  As is clear from this result, a metal chelating reagent (EDTA, o-phenanthroline), a reducing agent (cysteine, 2-mercaptoethanol (2-ME), dithiothreitol (DTT)) and SH reagent (N-ethylmaleimide ( NEM), MIA (monoiodoacetic acid), and r-chloromercuribenzoic acid (PCMB)) did not inhibit the activity of this enzyme, and thus were considered neither metal endopeptidase nor cysteine endopeptidase. In addition, diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) strongly inhibited the amidase activity of the enzyme, strongly suggesting that the enzyme is a serine endopeptidase. Furthermore, this enzyme is N-tosyl-L-lysyl chloromethyl ketone (TLCK) that reacts specifically with the histidine (His) residue present in the active center of trypsin and leupepsin, a trypsin inhibitor derived from actinomycetes. N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) reacts specifically to the His residue of chymotrypsin and not chymostatin, a chymotrypsin inhibitor from actinomycetes It was. These results suggest that this enzyme is a kind of trypsin-like serine endopeptidase.

(8) Influence of various metal salts of this enzyme The influence of metal salts on the amidase activity of this enzyme was examined.
Specifically, 0.2 ml of 40 mM Tris-HCl buffer (pH 8.0) containing various metal salts with a concentration of 1.0 mM and 0.05 μg of enzyme was pretreated at 30 ° C. for 30 minutes, and then 0.1 ml of this treatment solution (enzyme; 0.025 μg) Add to a mixed solution of 1.25 ml of 200 mM Tris-HCl buffer (pH 9.2) containing various metal salts at 1.0 mM concentration and 0.15 ml of 4.6 mM Bz-Arg-pNA solution that had been incubated at 30 ° C for 5 minutes in advance. The reaction was started. After 15 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured. Table 4 shows the relative activity determined from the measured values.

As is clear from this result, the amidase activity of this enzyme is 1.0 mM Co ²⁺ , Hg ²⁺ , Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , Ba ²⁺ , Cd ²⁺ , Ni ²⁺ , Sr ^{2 Although} not affected by ⁺ , Cu ²⁺ and Fe ²⁺ , it was weakly inhibited by 1.0 mM Zn ²⁺ .

(9) N-terminal amino acid sequence analysis of this enzyme
) The N-terminal amino acid sequence of this enzyme was measured up to 16 residues according to a known method [Akihiko Iwamatsu: Protein Experiment Note (bottom), P.83, published by Yodosha Co., Ltd. (2001)]. . That is, the electrophoretically uniformly purified enzyme was transferred from a gel after SDS-PAGE to a PVDF membrane by electroblotting (semi-dry) method and analyzed by a gas phase protein sequencer (Upride Biosystems Model 494). The results are shown in Table 5 in comparison with animal / microbe-derived trypsin-like serine protease.

  As is apparent from Table 5, the N-terminal amino acid of this enzyme was isoleucine (Ile). In addition to the N-terminal residue isoleucine (Ile), the third, fourth glycine (Gly), the tenth glycine (Gly), the thirteenth proline (Pro), 16 The residue valine (Val) was well conserved compared to any trypsin-like serine protease. Among these trypsin-like serine proteases, the N-terminal 16 residues have 56.3% identity at the amino acid level with Streptomyces griseus-derived trypsin. From the 12th residue phenylalanine (Phe), 16 residues The exact same sequence was confirmed up to the valine (Val). It also showed 31.3% identity with bovine trypsin, 50% with crayfish trypsin, 37.5% with porcine kallikrein, 43.8% with porcine thrombin B, and 43.8% with bovine Facter Xa. In particular, the enzyme and acrosin derived from goat showed 62.5% amino acid identity with the enzyme.

3. Examination of chemical properties of the enzyme of the present invention The above results were combined with other bovine-derived trypsin, protease derived from Streptomyces griseus, protease derived from Streptomyces erytheus and protease derived from Streptomyces fradiae. Table 6 shows.

The optimum pH of this enzyme had a wide range from neutral to alkaline compared to known trypsin-like proteases derived from microorganisms. Furthermore, the pH stability was close to that of Streptomyces fradiae (pH 4.0-9.0), but that of trypsin-like protease from other microorganisms (pH 2.0-5.0) and that of bovine pancreatic trypsin. It existed in a wider area on the alkaline side than (pH 2.0-3.0).
The temperature stability was 10 ° C. higher than that of bovine trypsin (40 ° C.), and 30 ° C. higher than that of bovine trypsin (40 ° C.).
The enzyme was inhibited by DFP, PMSF, TLCK and leupepsin. On the other hand, metal chelating reagents (EDTA, o-phenanthroline), reducing agents (cysteine, 2-mercaptoethanol (2-ME), dithiothreitol (DTT)) and SH reagents (N-ethylmaleimide (NEM), MIA (monoiodo) Acetic acid) and r-chloromercuribenzoic acid (PCMB)) were considered to be neither metal endopeptidase nor cysteine endopeptidase. These results strongly suggested that this enzyme is a kind of trypsin-like serine protease that specifically cleaves the peptide bond on the carboxyl terminal side of the lysine residue or arginine residue of the substrate.

This enzyme is activated by 1.0 mM Co ²⁺ , Hg ²⁺ , Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , Ba ²⁺ , Cd ²⁺ , Ni ²⁺ , Sr ²⁺ , Cu ²⁺ and Fe ²⁺ It was not affected. Inhibition of trypsin-like serine protease on metal salts is reported to be 20% activated by 1.0 mM concentrations of Co ²⁺ , Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , Cd ²⁺ , and Zn ²⁺ However, in the case of this enzyme, the above activation phenomenon was not observed. In addition, since this enzyme is weakly inhibited by 1.0 mM Zn ²⁺ , it was suggested that the enzyme has different properties from the trypsin-like serine proteases reported so far. Acrosin is known as a trypsin-like serine protease that is inhibited by Zn ²⁺ .

  In addition, this enzyme was more resistant to denaturing agents such as urea, guanidine hydrochloride, and SDS than that of bovine and porcine pancreatic trypsin. In particular, in the resistance to urea, the concentration at which 50% residual activity was observed was 2.0 M for bovine and porcine trypsin, whereas it was 5.0 M for this enzyme. In addition, the molecular weight of this enzyme estimated from SDS-PAGE was found to be considerably higher than that of other microorganism-derived trypsin-like proteases (molecular weight 16,500-20,000).

  Furthermore, the enzyme showed 62.5% identity with goat-derived acrosin at the N-terminal amino acid sequence of 16 residues. Next, trypsin (56.3%) derived from Streptomyces griseus, trypsin (50.0%) from crayfish, Factor Xa from bovine, thrombin B from pig (43.8%), and porcine kallikrein (37.5%) in this order. It was. However, the identity with bovine-derived trypsin was 31.25%, which was considerably lower than the identity with other trypsin-like serine proteases. Furthermore, this enzyme includes N-terminal first residue isoleucine (Ile), which is commonly found in various trypsin-like serine proteases, as well as third and fourth residue glycines (Gly) and tenth residue. The glycine (Gly), the 13th proline (Pro), and the 16th residue valine (Val) were well conserved compared to any trypsin-like serine protease.

  The enzyme also showed high identity with the goat-derived acrosin in the N-terminal 16-residue amino acid sequence. Acrosin has long been known to exist in the apical membrane of mammalian spermatozoa as an enzyme essential for sperm passage through the egg membrane during fertilization, and as a protease exhibiting substrate specificity similar to trypsin, Among these peptide bonds, it is known to hydrolyze arginine (Arg) -X bond and lysine (Lys) -X bond. Acrosin derived from goat, which has been confirmed to have 62.5% identity in the N-terminal 16-residue amino acid sequence, consists of two polypeptide chains, consisting of an H chain with a molecular weight of 40,000 and an L chain with 3,700. Of these, the H chain showed high identity with this enzyme. Acrosin is known to be inhibited by diisopropyl fluorophosphate (DFP), tosyl lysyl chloromethyl ketone (TLCK) and zinc, and these properties were very similar to this enzyme.

  From the above results, this enzyme is a kind of trypsin-like serine protease that is inhibited by DFP, PMSF, TLCK and leupepsin, and the N-terminal amino acid sequence is identical to animal and microbial trypsin-like serine proteases. It became clear that it was expensive. Nevertheless, compared to known commercially available bovine and porcine trypsin, it has a higher resistance to urea, a higher molecular weight, a broad pH stability of 4.5-10.5, and an optimum temperature. It became clear that the difference was as high as 70 ℃.

Experimental Example 2 Substrate specificity of the enzyme Experimental materials Enzyme (SDS-PAGE) homogenous enzyme sample purified in Example 1 was stored at 4 ° C in 10 mM CAPS buffer (pH 10.5) containing 10% glycerol. used. Bovine and porcine pancreas-derived trypsin were manufactured by Worthington Biochemical corporation (TRTPCK) and Wako Pure Chemical Industries, Ltd., respectively.
The following substrates were used.
Bz-Arg-pNA; Peptide Institute, Bz-Lys-pNA; Wako Pure Chemical Industries, Ltd.
Arg-pNA; Bachem, Lys-pNA; Merck,
Ac-Asp-pNA; Bachem, Ac-Glu-pNA; Bachem,
Suc-Ala-pNA; Peptide Institute, Ac-Phe-pNA; Bachem,
Ac-Leu-pNA; Bachem, Bz-Tyr-pNA; Peptide Institute,
AMC (7-amino-4-methylcoumarin); Peptide Institute,
Bz-Arg-MCA (4-methylcoumarin-7-amide);
Z-Phe-Arg-MCA; Peptide Institute, Boc-Leu-Arg-Arg-MCA; Peptide Institute,
Boc-Gln-Gly-Arg-MCA;
Boc-Leu-Ser-Thr-Arg-MCA; Peptide Institute,
α-MSH (alpha melanocyte stimulating hormone);
Angiotensin II (Human, 4001-v); Peptide Institute.
As other substrates, those prepared by a generally known method were used.

The following reagents were used.
Trifluoroacetic acid (TFA); Peptide Research Institute Dimethylsulfoxide (for automatic amino acid analysis); Wako Pure Chemical Industries, Ltd.
2-Propanol (for HPLC); Wako Pure Chemical Industries, Ltd.
Acetonitrile (for HPLC); Kanto Chemical Co., Inc.
Ninhydrin sample solution-L8,500 set; Wako Pure Chemical Industries, Ltd.
Polyoxyethylene Lauryl Alcohol Ether ； Nacalai Tesque
b-thiodiglycol (for automatic amino acid analysis); Wako Pure Chemical Industries, Ltd.
Caprylic acid (for automatic amino acid analysis); Wako Pure Chemical Industries, Ltd.
Trisodium citrate dihydrate ； Wako Pure Chemical Industries, Ltd.
Citric acid monohydrate ： Wako Pure Chemical Industries, Ltd.

2. experimental method
(1) Action of the enzyme on various pNA substrates The substrate specificity of the enzyme for various pNA substrates was examined. That is, various synthetic substrates (Bz-Arg-pNA, Arg-pNA, Bz-Lys-pNA, Lys-pNA, Boc-His-pNA, Ac-Asp-pNA, Ac-Glu-pNA, Suc- 200 mM including Ala-pNA, Ac-Phe-pNA, Ac-Leu-pNA, Z-Pro-pNA, Boc-Gln-pNA, Bz-Tyr-pNA, Z-Thr-pNA, Boc-Met-pNA) After incubating 1.45 ml of Tris-HCl buffer (pH 9.2) at 30 ° C. for 5 minutes, 0.05 ml of an enzyme solution containing 0.05 μg of enzyme was added to initiate the reaction. After 10 to 1,500 minutes, 0.5 ml of 45% acetic acid was added to stop the reaction, the absorbance at 405 nm was measured, and the substrate specificity of the enzyme for various pNA substrates was examined. The results are shown in Table 7 with the activity against Bz-Arg-pNA as 100.

As is clear from Table 7, this enzyme had the strongest amidase activity against Bz-Arg-pNA, and then the activity with strong activity against Bz-Lys-pNA. In addition, amidase activity against Arg-pNA and Lys-pNA was slightly observed. This indicates that the enzyme belongs to endopeptidase but also has weak exopeptidase activity.
The above results strongly suggested that it is a kind of trypsin-like serine protease.

(2) Kinetic constants of the enzyme for various synthetic substrates The kinetic constants (Km, kcat) of the enzyme in the hydrolysis reaction for various synthetic substrates were determined from Lineweaver-Burk plots.
(i) Optimum pH of this enzyme for MCA (4-methylcoumarin-7-amide) substrate
Prior to measuring the amidase activity for various MCA substrates, the optimum pH of the enzyme for Bz-Arg-MCA (4-methylcoumarin-7-amide) substrate was measured. First, using various buffers, the amidase activity of this enzyme against Bz-Arg-MCA substrate when the pH was changed was measured.
In addition, the enzyme activity with respect to a MCA substrate was performed as follows. That is, 20 mM of various buffers previously prepared at a predetermined pH and various MCA substrates prepared to a final concentration of 100 nM were prepared to a total volume of 2,995 ml and incubated at 30 ° C. for 5 minutes. Thereafter, 0.05 ml of enzyme solution (enzyme; 5 pg) was added to start the reaction. The reaction was monitored with a recorder at an excitation wavelength of 380 nm and a fluorescence wavelength of 440 nm for exactly 5 minutes, and the fluorescence intensity of released AMC (7-amino-4-methylcoumarin) was measured. The amount of AMC released by enzyme action was calculated from the standard curve of standard AMC.

The obtained results are shown in FIG. FIG. 13 shows the activity value at each pH when the maximum activity value is 100, ○ is 20 mM Tris-HCl buffer, × is 20 mM AMP hydrochloric acid buffer, ■ is 20 mM glycine hydrochloride. The results when using a buffer solution are shown.
As a result, as in the case of the pNA substrate, the optimum pH of the MCA substrate of this enzyme was 9.2. Therefore, hereinafter, when measuring the amidase activity of the MCA substrate, the measurement was performed at a pH of 9.2.

(ii) Lineweaver-Burk plots of various enzymes against pNA substrate and MCA substrate As representative examples of hydrolysis reactions, this enzyme against Bz-Arg-pNA and Lineweaver-Burk plots of porcine pancreatic trypsin are shown in Fig. 14 (A: book). Enzyme, B: Trypsin derived from porcine pancreas) and plots of this enzyme and porcine pancreatic trypsin against Bz-Arg-MCA are shown in Fig. 15 (A: this enzyme, B: trypsin derived from porcine pancreas), respectively.
In addition, the enzyme activity measurement with respect to MCA substrate was performed by the method according to (i), and the amidase activity of the enzyme with respect to pNA substrate was measured as follows. Specifically, after adding 1.3 ml of 200 mM Tris-HCl buffer (pH 9.2) and 0.15 ml of various concentrations of substrate (0.01-0.40 mM) to a test tube (1.5 × 13 cm), incubating at 30 ° C for 5 minutes, 0.05 ml (0.05 μg) of enzyme solution was added and reacted. Exactly after a certain time (10-135 minutes), 0.5 ml of 45% acetic acid was added to stop the reaction, and p-nitroanilide released by enzymatic action was measured at an absorbance of 405 nm to obtain amidase activity.

(iii) Substrate specificity of the enzyme for various substrates By the above methods (i) and (ii), the kinetic constants of various enzymes for various Arg and Lys-containing pNA substrates and various Arg and Lys-containing MCA substrates ( Km, kcat) were calculated. The catalytic center activity (kcat) for various pNAs of this enzyme was calculated from the maximum activity (Vmax) using a molar extinction coefficient of 9,620 and the molecular weight of this enzyme as 29,000. In addition, kcat was calculated assuming that the molecular weights of commercially available bovine and porcine pancreatic trypsin used for comparison were 24,000 and 23,300, respectively.
The results of the kinetic constants (km and kcat) of the enzyme for various Arg and Lys-containing pNA substrates are shown in Table 8, and those of the enzyme for various Arg and Lys-containing MCA substrates are shown in Table 9.

  From the above results, it was found that the kcat / Km value for Bz-Arg-pNA of this enzyme was 1,920. This value was about 960 times and about 150 times that of bovine and porcine trypsin (about 2.0 and about 13.0). In addition, the kcat / Km values for Bz-Arg-pNA of trypsin-like serine proteases derived from Streptomyces griseus, Streptomyces fradiae and Streptomyces erytheus have been reported (about 260, about 170 and about 90) Compared with, it was 7-20 times higher.

  In addition, the kcat / Km value (13,800) for Bz-Arg-MCA of this enzyme was about 4,000 times and about 3,600 times higher than those of bovine and porcine trypsin (3.4 and 3.8). In addition, the kcat / Km value of this enzyme against Z-Phe-Arg-MCA, Boc-Leu-Arg-Arg-MCA, Boc-Gln-Gly-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA ( About 41,000, about 32,000, about 41,000 and about 53,000) were 2-4 times higher than Bz-Arg-MCA. In these MCA substrates, Boc-Leu-Ser-Thr-Arg-MCA with 3 residues extended from the P1 position to the N-terminal side showed the highest kcat / Km value of about 53,000, and the bovine and porcine pancreas Compared with those of derived trypsin (6,000 and 3,500), it was about 9 times and about 15 times higher.

From the above results, this enzyme specifically cleaves Arg-containing pNA substrate and MCA substrate, and also shows very high affinity for both substrates compared to trypsin derived from bovine and porcine pancreas. It was. This strongly suggested that this enzyme is a kind of trypsin-like serine protease.
In addition, the enzyme hydrolyzed Arg-pNA and Lys-pNA, indicating that the enzyme also belongs to endopeptidase but has weak exopeptidase activity.

(3) Action of the enzyme on angiotensin II The substrate specificity of the enzyme was examined using angiotensin II (8 residues) in which one arginyl peptide bond is present in the peptide chain.
That is, 100 μg (95.6 nmol) of angiotensin II was dissolved in 0.1 ml of 20 mM Tris-HCl buffer (pH 9.2) (0.1% concentration). Next, an enzyme equivalent to 1 / 2,000 of the substrate in a molar ratio (1.39 μg, 47.8 pmol) was added, and the reaction was carried out at 30 ° C. for 9 hours. After the completion of the reaction, the decomposition product was heated in boiling water at 100 ° C. for 3 minutes to stop the reaction and freeze-dried. The freeze-dried degradation product was dissolved in 100 μl of Millicue-treated ultrapure water containing 0.05% TFA, fractionated by HPLC, and purified. The results are shown in FIG. The fractionation was performed under the following HPLC conditions.
HPLC instrument: L-6300 (manufactured by Hitachi), stationary phase: Tosoh TSKgel Octyl-80Ts column (4.6 ID × 25 cm), mobile phase: solution A; 0.05% TFA aqueous solution, solution B; Isopropanol / Acetonitrile = 7 / 3 (v / v) (including 0.05% TFA)
Table 10 shows the amino acid composition, N-terminal amino acid sequence and recovery rate of the five fractionated peaks (AT-1, -2, -3, -4 and -5). The numbers in parentheses in Table 10 represent the number of known amino sequences in the peptide.

  The amino acid composition was analyzed as follows. That is, a 6 N hydrochloric acid solution with a constant boiling point was transferred to a test tube for hydrolysis (vacuum hydrolysis tube, 10 x 15.0 cm), sealed under reduced pressure, acid hydrolyzed at 110 ° C for 22 hours, and then allowed to cool after acid hydrolysis. Then, it was again dried under reduced pressure in a desiccator containing sodium hydroxide. To this dried solid, 0.02 N hydrochloric acid amino acid analysis diluent (pH 2.2) was added to a concentration of 2 nmol / 20 μl, and identified using a Hitachi L-8800 amino acid analyzer.

  Further, the N-terminal amino acid sequence analysis was performed as follows. In other words, each peptide fractionated by HPLC was dispensed into a pitch-type test tube (1.3 x 10.5 cm), dried under reduced pressure, diluted with millicue-treated ultrapure water to 1.0 nmol / ml, and updated. The N-terminal amino acid sequence was identified using a Biosystem 494 protein sequencer.

From Table 10, AT-1, -2, -3, -4 and -5 are Asp ¹ -Arg ² , Val ³ -Tyr ⁴ , Asp ¹ -Tyr ⁴ , Ile ⁵ -Phe ⁸ and Val ³ -Phe, respectively. It was found to be ⁸ peptides.

From the above results, in addition to the Arg ² -Val ³ bond, which is an arginyl peptide bond in Angiotensin II, this enzyme was hydrolyzed by the enzyme with a Tyr ⁴ -Ile ⁵ bond, which is a tyrosyl peptide bond. (See Figure 17).

That is, the recovery rates of AT-3 (Asp ¹ -Tyr ⁴ ) and AT-4 (Ile ⁵ -Phe ⁸ ), which were hydrolyzed at one Tyr ⁴ -Ile ⁵ bond in the peptide chain, were 25.29% and It was 29.59%, the highest among the five peaks, and almost the same yield, suggesting that it was hydrolyzed primarily. In addition, AT-1 (Asp ¹ -Arg ² ) and AT-5 (Val ³ -Phe) produced by hydrolysis of the Arg ² -Val ³ bond, which was estimated from the specificity results for the synthetic substrate (see Table 7). recovery of ⁸⁾ as low as 11.92% and 6.17%, the recovery of ^{aT-2 (Val 3 -Tyr 4} ) was the lowest recovery rates 2.02%. From these results, it was suggested that this enzyme hydrolyzes the Tyr ⁴ -Ile ⁵ bond first, and simultaneously Arg ² -Val ³ bond to Angiotensin II. Next, the resulting Arg ² -Val ³ bond in the AT-3 (Asp ¹ -Tyr ⁴ ) peptide or the Tyr ⁴ -Ile ⁵ bond in the AT-5 (Val ³ -Phe ⁸ ) peptide was weakly hydrolyzed by this enzyme. It was considered that AT-2 (Val ³ -Tyr ⁴ ) was produced by decomposition.
This enzyme cleaved the Tyr ⁴ -Ile ⁵ bond in the peptide chain, even though it did not hydrolyze the synthetic substrate Bz-Tyr-pNA. This result was different from the known trypsin in which the cut points of the synthetic substrate and the peptide substrate coincided.

(4) Action of the enzyme on α-MSH (melanocyte-stimulating hormone) Substrate of the enzyme using α-MSH (15 residues), each containing arginyl peptide bonds and lysyl peptide bonds in the peptide chain. Specificity was examined.
That is, 100 μg (60.0 nmol) of α-MSH was dissolved in 0.1 ml of 20 mM Tris-HCl buffer (pH 9.2) (0.1% concentration). Next, an enzyme (0.87 μg, 30.0 pmol) equivalent to 1 / 2,000 of the substrate by molar ratio was added, and the reaction was carried out at 30 ° C. for 18 hours. After completion of the reaction, the decomposed product was heated in boiling water at 100 ° C for 3 minutes to stop the reaction and freeze-dried. The freeze-dried degradation product was dissolved in 100 μl of Millicue-treated ultrapure water containing 0.05% TFA, fractionated by HPLC, and purified. The results are shown in FIG. Table 11 shows the amino acid composition, N-terminal amino acid sequence and yield of the eight fractionated peaks (α-1, -2, -3, -4, -5, -6, -7 and -8). It was. The numbers in parentheses in Table 11 represent the number of known amino sequences.

From Table 11, α-1, -2, -3, -4, -5, -6, -7 and -8 are Pro ¹² -Val ¹³ , His ⁶ -Arg ⁸ , Glu ⁵ -Arg ⁸ and Trp, respectively. ⁹ -Lys ^11, it was confirmed that a peptide of ^{Trp 9 -Val 13, Ser 1 -Glu} 5, Ser 1 -Met 4 and Ser ¹ -Arg ^8.
Based on the above results, this enzyme is capable of binding methionyl peptide bonds in addition to Arg ⁸ -Trp ⁹ bonds, which are arginyl peptide bonds in α-MSH, and Lys ¹¹ -Pro ¹² bonds, which are lysyl peptide bonds. It was shown that a certain Met ⁴ -Glu ⁵ bond and a glutamyl peptide bond Glu ⁵ -His ⁶ bond were hydrolyzed by this enzyme (see FIG. 19).

The recovery rates of α-8 (Ac-Ser ¹ -Arg ⁸ ) and α-5 (Trp ⁹ -Val ¹³ -NH2) produced by hydrolysis of the Arg ⁸ -Trp ⁹ bond were 33.33% and 21.08, respectively. %, Which is higher among the 8 peptides obtained by HPLC (see Table 11), suggesting that this arginyl peptide bond is predominantly hydrolyzed by this enzyme. Furthermore, the recoveries of α-7 (Ac-Ser ¹ -Met ⁴ ) and α-3 (Glu ⁵ -Arg ⁸ ) generated by hydrolysis of the Met ⁴ -Glu ⁵ bond in the peptide chain were 19.17% and Since it is almost equal to 19.00%, the Arg ⁸ -Trp ⁹ bond of α-MSH was first hydrolyzed to obtain α-8 (Ac-Ser ¹ -Arg ⁸ ) peptide, and then the peptide chain It was thought that the Met ⁴ -Glu ⁵ bond was hydrolyzed. Similarly, the recovery rate of α-6 (Ac-Ser ¹ -Glu ⁵ ) and α-2 (His ⁶ -Arg ⁸ ) produced by hydrolysis of the Glu ⁵ -His ⁶ bond of α-MSH is 3.92. % And 3.41% are almost equal to each other and low. Therefore, the Arg ⁸ -Trp ⁹ bond of α-MSH is also hydrolyzed to produce α-8 (Ac-Ser ¹ -Arg ⁹ ). It was considered that the Glu ⁵ -His ⁶ bond in this peptide chain was hydrolyzed after the obtained peptide. However, since the recovery rate of both α-6 (Ser ¹ -Ac ¹ -Glu ⁵ ) and α-2 (His ⁶ -Arg ⁸ ) peptides is low, the specificity of this enzyme for glutamyl peptide binding is low. It was considered.
The enzyme cleaved the Met ⁴ -Glu ⁵ bond and Glu ⁵ -His ⁶ bond in the peptide chain even though it did not hydrolyze the synthetic substrates Boc-Met-pNA and Ac-Glu-pNA. . This result was different from the known trypsin in which the cut points of the synthetic substrate and the peptide substrate coincided.

  Thus, although the synthetic substrates Bz-Tyr-pNA, Boc-Met-pNA and Ac-Glu-pNA are not hydrolyzed, Tyr-Ile, Met-Glu and Glu-His bonds in the peptide chain This is the first trypsin-like serine protease to be hydrolyzed by this enzyme.

  From the above results, it was revealed that this enzyme hydrolyzes Ars-X bonds and Lys-X bonds including Lys-Pro bonds in synthetic substrates and peptide chains. In addition, peptide substrates hydrolyzed Tyr-X, Met-X, and Glu-X bonds that were not reported at all by known trypsin-like serine proteases. Therefore, compared to known serine proteases, this enzyme is expected to have a wider subsite in the active center, strongly suggesting that the trypsin-like serine protease is a novel protease.

It is a figure showing the culture curve (absorbance 660 nm) of Rhizobacter genus IB-9374 according to the present invention and the amidase activity at that time. It is a figure explaining each step at the time of refine | purifying an enzyme from Rhizobacter genus IB-9374. Example 1 The pH, absorbance at 280 nm, and enzyme (amidase) activity of the fraction (fraction) after electrophoresis obtained in (9) are shown. Example 1 It shows the result of electrophoresis of this enzyme obtained in (11) by SDS-PAGE. This is a graph of various molecular weight proteins and their relative variability as determined by SDS-PAGE. It is a graph showing the change of the amidase activity of this enzyme when using various buffers and changing pH. It is a graph showing the change of the amidase activity of this enzyme when temperature is changed. It is a graph showing the change of the amidase activity of this enzyme when using various buffer solutions and changing temperature. It is a graph showing the change of the amidase activity of this enzyme when temperature is changed. It is a graph showing the change of the amidase activity of various enzymes when a urea concentration is changed. It is a graph showing the change of the amidase activity of various enzymes when changing a guanidine hydrochloride density | concentration. It is the graph showing the change of the amidase activity of various enzymes when changing a sodium dodecyl sulfate (SDS) density | concentration. It is a graph showing the change of the amidase activity of this enzyme when using various buffers and using Bz-Arg-MCA as a substrate and changing the pH. The Lineweaver-Burk plots of this enzyme (A) and porcine pancreatic trypsin (B) against Bz-Arg-pNA are respectively shown. The Lineweaver-Burk plots of this enzyme (A) and porcine pancreatic trypsin (B) against Bz-Arg-MCA are shown, respectively. The liquid chromatograph when the degradation product of angiotensin II by this enzyme is fractionated by liquid chromatography is shown. It is a figure showing the cleavage site of angiotensin II of this enzyme. The liquid chromatograph when the degradation product of α-MSH (melanocyte stimulating hormone) by this enzyme is fractionated by liquid chromatography is shown. It is a figure showing the cleavage site of α-MSH (melanocyte stimulating hormone) of this enzyme.

Explanation of symbols

  In FIG. 1, -o- indicates a value (culture curve) of a culture solution obtained by culturing Rhizobacter IB-9374 at an absorbance of 660 nm, and -x- indicates amidase activity when Rhizobacter IB-9374 is cultured. Represents.

  In FIG. 3,-●-represents the absorbance of each fraction (fraction) at 280 nm, -O- represents pH of each fraction, and -x- represents amidase activity of each fraction.

  In FIG. 5, 1 is the result of phosphorylase B having a molecular weight of 94,000, 2 is the result of serum albumin having a molecular weight of 67,000, 3 is the result of ovalbumin having a molecular weight of 43,000, and 4 is the result of carboxylate anhydrase having a molecular weight of 30000. Represents the result of trypsin inhibitor having a molecular weight of 20100, 6 represents the result of α-lactalbumin having a molecular weight of 14400, and ● represents the result of the enzyme.

  In FIG. 6,-○-indicates the results when using glycine hydrochloride buffer, -X- indicates the results when using citrate buffer,-■-indicates potassium dihydrogen phosphate-hydrogen hydrogen phosphate. The results when using potassium buffer, the results when using-△ -Tris-HCl buffer,-●-is 2-amino-2-methyl-1,3-propanediol (AMP) -hydrochloric acid buffer -□-represents the results when using a glycine sodium hydroxide buffer, respectively.

  In FIG. 8,-○-indicates the results when using glycine hydrochloride buffer, -X- indicates the results when using citrate buffer,-■-indicates potassium dihydrogen phosphate-dipotassium hydrogen phosphate. The results when using buffer solution, the results when using-△ -Tris-HCl buffer solution,-●-is 2-amino-2-methyl-1,3-propanediol (AMP) -hydrochloric acid buffer solution -□-represents the results when using glycine sodium hydroxide buffer, respectively.

  In FIG. 10, —O— represents the amidase activity value of this enzyme, — ● — represents the amidase activity value of bovine pancreatic trypsin, and —Δ— represents the amidase activity value of porcine pancreatic trypsin.

  In FIG. 11, -O- represents the amidase activity value of this enzyme,-●-represents the amidase activity value of bovine pancreatic trypsin, and -Δ- represents the amidase activity value of porcine pancreatic trypsin.

  In FIG. 12, —O— represents the amidase activity value of this enzyme, — ● — represents the amidase activity value of bovine pancreatic trypsin, and —Δ— represents the amidase activity value of porcine pancreatic trypsin.

In FIG. 13,-○-indicates the results when using Tris-HCl buffer, and -X- indicates the results when using 2-amino-2-methyl-1,3-propanediol (AMP) -HCl buffer. -■-represents the results when using sodium glycine hydroxide buffer, respectively.

Claims (1)

Using the following possess the properties of a to e, trypsin-like serine protease and collected from the culture of a microorganism accession number FERM P-16928 belonging to Rizobakuta genus, arginine -X bond, lysine -X bond, tyrosine A hydrolysis method for cleaving a -X bond, a methionine-X bond and a glutamic acid-X bond (where X represents an amino acid).
a. pH: optimum pH; 7.5-11.0, stable pH range; 4.5-10.5
b. Temperature: Optimal temperature; 70 ° C, temperature stability range 10 ° C to 70 ° C
c. Molecular weight: 29,000 (SDS-PAGE method)
d. A sequence of 16 residues from the N-terminal amino acid:
Ile Ile Gly Gly Ile Asp Val Ala Asn Gly Lys Phe Pro Phe Met Val
e. Cleaves arginine-X bond, lysine-X bond, tyrosine-X bond, methionine-X bond and glutamate-X bond (where X represents an amino acid)

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