JPH0898685A - New aspartic proteinase - Google Patents

Abstract

PURPOSE: To obtain an aspartic proteinase stable in acidic region, decomposing proteins in the region and, accordingly, useful as a substitute for a digestive enzyme such as pepsin and enterokinase. CONSTITUTION: This new aspartic proteinase is produced by Rhizopus hangchow. It has a molecular weight of about 37,600 (SDS-PAGE), an isoelectric point of 4.5, an optimum pH of 3-4 and a stable pH of 2-7. The N-terminal amino acid sequence of the enzyme is Ser-Gly-Ser-Gly-Val-Val-Phe-Met-Thr-Asp-Tyr- Glu-Tyr-Asp-Ile-Glu-Tyr-Tyr-Gly. Since the enzyme has an αs -casein decomposition activity, it can be used in the production of a protein raw material effective for preventing the cow's milk allergy of neonate and the decomposition of proteins inducing food allergy. It is also useful as a protein-decomposition enzyme for the production of seasonings, foods having decreased allergen content, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to Rhizopus hansho.
( Rhizopus hangchow ) produced aspartic proteinase.

[0002]

BACKGROUND OF THE INVENTION Aspartic proteinase is a generic term for proteinases having an optimum pH for action on the acidic side and having an aspartic acid residue at the active site. It is used as an auxiliary agent or a substitute for digestive drugs and koji, and as a seasoning. It is also used when manufacturing liquids and peptides. As the origin of this aspartic proteinase, pepsin in animal gastric juice and rennin in calf stomach are widely known. Also, Aspergillus (Aspergillus) genus, penicillin <br/> um (Penicillium) genus, Rhizopus (Rhizopus) genus, Muko <br/> Le (Mucor) genus Saccharomyces (Saccharomyces) microorganisms such as genus also aspartokinase Chick proteinase Known as the origin.

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have conducted extensive research on various enzymes originating from microorganisms, and found that Rhizopus hangchow
Have been found to produce aspartic proteinase, and have completed the present invention. Therefore, the present invention
An object is to provide an aspartic proteinase produced by Rhizopus hangchow .

[0004]

Means for Solving the Problems The present invention comprises culturing Rhizopus hangchow , which has an aspartic proteinase-producing ability, and producing and accumulating aspartic proteinase in cells, followed by separation,
It is an aspartic proteinase obtained by purification, has a molecular weight of about 37,600 (SDS-PAGE), and has the following N-terminal amino acid sequence. Ser-Gly-Ser-Gly-Val-Val-Phe-Met-Thr-Asp-Tyr-Glu-Ty
r-Asp-Ile-Glu-Tyr-Tyr-Gly-

Next, a method for producing the aspartic proteinase of the present invention will be described. Rhizopus disprove used in the production of aspartate Chick proteinase of the present invention (Rhizopus hangchow), as long as it produces aspartase tic proteinase of the present invention may be any strain, such as a strain, Rhizopus disproved (Rhizopus hangchow ) IFO-4749 strain. This IFO-4749 strain is a fermentation research institute (Institu
te for Fermentation, Osaka) can be obtained without any restrictions.

[0006] Rhizopus hansho ( Rhizopus) used in the production of the aspartic proteinase of the present invention
The medium used for culturing hangchow ) may be any medium used for culturing ordinary Rhizopus hangchow , and may be a liquid medium or a solid medium. Moreover, an inducer for producing the aspartic proteinase of the present invention is not particularly required. The culture temperature is preferably around 25 ° C,
The culture time may be about 4 days.

[0007] Thus, Rhizop
purification of the aspartic proteinase produced and accumulated in the cells of Us hangchow ) is carried out by appropriately combining the methods usually used for the purification of the enzyme. For example, in liquid culture, cells are separated from the culture by treatment such as filtration or centrifugation, and then the cells are crushed by enzymatic or physical treatment, and aspartic proteinase is eluted to obtain a crude enzyme fraction. To get In solid-state culture, a solvent such as a buffer may be used to
The crude enzyme fraction of proteinase is extracted. Then, the crude enzyme fraction is purified by a treatment such as ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography and the like.

Next, the physicochemical properties of the aspartic proteinase of the present invention will be shown. (1) Molecular weight: 37,600 by SDS-PAGE, fast protein liquid chromatograph using Superose
(FPLC) is 36,000. (2) Isoelectric point: It is 4.5 by isoelectric focusing. (3) Substrate specificity: two peptide bonds of Leu ¹⁵ -Tyr ¹⁶ and Tyr ¹⁶ -Leu ^{17 in} the insulin B chain are cleaved initially,
In addition, Leu ¹¹ -Val ¹² , Ala ¹⁴ -Leu ¹⁵ and Phe ²⁴ -Phe ²⁵
Disconnect. It also activates trypsinogen in the pH range of 2-5. (4) Optimum pH: pH 3 to 4 (see FIG. 1). (5) Thermostability: Treatment at 45 ° C for 5 minutes at pH 3.0 reduces enzyme activity, and treatment at 50 ° C for 5 minutes at pH 3.0 completely eliminates enzyme activity. (6) pH stability: It is extremely stable at pH 2 to 7 (see FIG. 2). In addition, about 50% of enzyme activity is reduced by overnight treatment at pH 9.0 at 5 ° C. (7) Inhibitor: The enzyme activity was completely eliminated by 1 μM of pepstatin A, and N-diazoacetylnorleucine methyl ester (N-diazoacetylnorleucinemethase) was incubated at 14 ° C for 40 minutes.
yl ester) (DAN) treatment reduces approximately 56% of enzyme activity. (8) N-terminal amino acid sequence: Ser-Gly-Ser-Gly-Val-Val-Ph
e-Met-Thr-Asp-Tyr-Glu-Tyr-Asp-Ile-Glu-Tyr-Tyr-Gly- (9) Structure: Circular dichroism spectrum measurement (see Figure 3)
Has about 90% β-structure.

The aspartic proteinase activity was measured as follows. 0.1M citrate buffer
1 ml of a 2% milk casein solution was added to 1 ml of the enzyme solution diluted with (pH 3.0) and reacted at 30 ° C. for 10 minutes. After the reaction was stopped by adding 2 ml of 0.4 M trichloroacetic acid, the mixture was allowed to stand for 20 minutes or longer, and filtered with Toyo filter paper No.2. To 1 ml of this filtrate
After adding 5 ml of 0.4 M sodium carbonate and thoroughly stirring, 1 ml of a 6-fold diluted Folin-Ciocalteu reagent was added and the mixture was allowed to stand at 30 ° C. for 30 minutes to measure the absorbance at 660 nm. Separately from this, a blind test was performed using an enzyme solution that had been inactivated in advance. The enzyme activity is
When an enzyme reaction was performed using a 2% milk casein aqueous solution as a substrate at 30 ° C and pH 3.0, 1 mole of tyrosine equivalent to 1 second was obtained.
The amount of enzyme that liberates a trichloroacetic acid-soluble degradation product equivalent to the absorbance at 280 nm was set to 1 katal. Moreover, the specific activity was shown by the number of katal per 1 kg of enzyme protein.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Preparation of crude enzyme To 1,800 g of wheat bran was added 1,500 ml of water for sterilization, and then Rhizopus hangchow IFO-4749 strain was inoculated and cultured at 25 ° C. for 4 days. After culturing, 10,000 ml of 20 mM phosphate buffer (pH 7.0) was added to the obtained koji, and the mixture was extracted overnight in a low temperature room to obtain 7,350 ml of a crude enzyme solution. After centrifuging this crude enzyme solution, hollow fiber (AIL-1010,
Asahi Kasei Co., Ltd.), and freeze-dried to obtain crude enzyme powder.
15.5 g was obtained. The specific activity of this crude aspartic proteinase enzyme powder was 7.9 × 10 ⁻³ katal / kg protein. The yield of aspartic proteinase activity was 68%.

Purification of aspartic proteinase 10 g of the crude enzyme powder obtained as described above was suspended in 100 ml of 10 mM citrate buffer (pH 5.2) containing 2 mM calcium acetate, stirred for 2 hours to dissolve, and then centrifuged. Separation (15,000 ×
g, 20 minutes), and the resulting supernatant was dialyzed against the same buffer. After dialysis, centrifugation (15,000 xg, 20 minutes) was performed, and the supernatant was used as a crude enzyme solution. Ammonium sulfate was gently added to this supernatant to a concentration of 35%, centrifuged (10,000 xg, 20 minutes),
The supernatant was used as a 35% ammonium sulfate fraction supernatant. Specific activity 8.7 × 10
^{It was -3} katal / kg protein.

The supernatant of this 35% ammonium sulfate fraction was preliminarily used as a 10 mM citrate buffer (pH 5.2) containing 35 mM ammonium sulfate at a concentration of 2 mM calcium acetate.
It was used for Butyl-TOYOPEARL 650M (manufactured by Tosoh Corporation) equilibrated with. Then, the active fraction was eluted with a linear concentration gradient of 35 to 0% ammonium sulfate, and the eluate was diluted with Butyl-TOYOPEAR.
It was used as an L 650M elution active fraction. Specific activity is 9.1 × 10 ^-3 katal /
It was kg protein.

This Butyl-TOYOPEARL 650M elution active fraction was thoroughly dialyzed against 10 mM citrate buffer (pH 5.0) containing 2 mM calcium acetate, and then DEAE-TOY equilibrated with the same buffer.
It was used for OPEARL 650S (manufactured by Tosoh Corporation). And 0
The active fraction was eluted with a linear gradient of ~ 0.5 M sodium chloride, and the eluate was used as the DEAE-TOYOPEARL 650S eluting active fraction. The specific activity was 15.8 × 10 ^-3 katal / kg protein.

The DEAE-TOYOPEARL 650S elution active fraction was thoroughly dialyzed against 10 mM citrate buffer (pH 3.0) containing 2 mM calcium acetate, and then equilibrated with SP-TOYOP.
It was used for EARL650M (manufactured by Tosoh Corporation). And 0-
The active fraction was eluted with a linear gradient of 0.75M sodium chloride, and the eluate was designated as SP-TOYOPEARL 650M eluting active fraction.

This SP-TOYOPEARL 650M elution active fraction was thoroughly dialyzed against Milli-Q water and freeze-dried to obtain 212 mg of purified aspartic proteinase. The purified aspartic proteinase thus obtained was electrophoretically single and had a specific activity of 11 × 10 ⁻³ katal / kg protein. The yield of aspartic proteinase activity was 8%.

[0016]

TEST EXAMPLE 1 The trypsinogen activation reaction of the aspartic proteinase obtained in Example 1 was examined.
25 μl of a solution of trypsinogen dissolved in 2.5 mM hydrochloric acid to a concentration of 0.5 mg / ml, 10 mM sodium citrate buffer
(pH 3.0) 50 μl and enzyme solution 25 μl were mixed, and after activation reaction at 30 ° C., 0.3 M Tris-HCl buffer (pH 8.3) 0.5
The reaction was stopped by adding ml and 0.9 ml of water. Then, 10 μM Bz-Arg-MCA (5 μl) was added to this solution and reacted at 30 ° C., and then using a Fluorescence Spectrophotometer F-3000 (manufactured by Hitachi, Ltd.), an excitation wavelength of 360 nm and a fluorescence wavelength of 440
The fluorescence intensity was measured at nm. Under these conditions, the amount of enzyme required to activate 1 mole of trypsinogen per second was 1 katal. As a result, the specific activity of the aspartic proteinase of the present invention in the trypsinogen activation reaction was 13.9 × 10 ⁻³ katal / kg protein.

[0017]

TEST EXAMPLE 2 The ability of the aspartic proteinase obtained in Example 1 for the allergen protein was examined. Using 5 μl of 2% α _S -casein (pH 3.0) and 5 μl of 2% β-lactoglobulin (pH 3.0) as a substrate, the enzyme solution (p
H 3.0) 5 μl was added to each well and reacted at 30 ° C, then 0.8M
The reaction was stopped by adding 5 μl of trichloroacetic acid. The reaction solution was allowed to stand at 30 ° C. for 20 minutes or longer and then centrifuged (15,000 × g, 20 minutes) to obtain a precipitate. And
After converting this precipitate into SDS, it was subjected to SDS-PAGE to observe the state of decomposition. The result is shown in FIG. It was found that the aspartic proteinase of the present invention decomposes α _S -casein but not β-lactoglobulin.

[0018]

[Test Example 3] The substrate specificity of the aspartic proteinase obtained in Example 1 for the oxidized insulin B chain was examined. HPL of oxidized insulin B chain (manufactured by Sigma)
An enzyme reaction was carried out at 30 ° C. and pH 3.0 using the substrate purified by C TSK-gel ODS-120T. The reaction was stopped by adding 1/10 volume of 25% aqueous ammonia, dried under reduced pressure, dissolved in 0.1% TFA, and used as a sample. This sample
Peaks were collected by HPLC TSK-gel ODS-120T, and the amino acid sequence of each peak was analyzed by peptide sequencer (Applied
Biosystems 477A Protein sequencer (manufactured by Applied Biosystems) was used to determine the cutting points and the cutting order. The aspartic proteinase of the present invention initially cleaves two peptide bonds of Leu ¹⁵ -Tyr ¹⁶ and Tyr ¹⁶ -Leu ¹⁷ , and further, Leu ¹¹ -Val ¹² , Ala ¹⁴ -L.
It was found to cleave eu ¹⁵ and Phe ²⁴ -Phe ²⁵ .

[0019]

TEST EXAMPLE 4 The coagulant activity of the aspartic proteinase obtained in Example 1 was examined. 0.1μ per lane
15V / cm, using enzyme to give g, 0.5 μg, 1.0 μg
Electrophoresis was carried out for 2 hours. After the electrophoresis, the gel was immersed in 0.15 M acetate buffer (pH 5.3), allowed to stand at room temperature for 30 minutes, and then placed on a skim milk-agarose plate. After reacting at 37 ° C. for 2 hours, 0.02% amido black was added, stained for 20 minutes, washed with water, and then decolorized with 0.01 M acetate buffer (pH 5.8) containing 5% glycerol. As a comparative example, Aspergillus sait
The same test was carried out on the aspartic proteinase derived from ( oi ). The result is shown in FIG. The aspartic proteinase of the present invention comprises Aspergillus
It was found to have stronger milk-clotting activity than the aspartic proteinase from Cytoi (Aspergillus saitoi ).

[0020]

INDUSTRIAL APPLICABILITY The aspartic proteinase of the present invention is stable at pH 2 to 7 and decomposes proteins well in the acidic region. Therefore, it is useful as a substitute for pepsin secreted in gastric juice and at pH 3 It activates pancreatic-derived trypsinogen at -4, and thus is also useful as a substitute for enterokinase secreted in the duodenum. Further, the aspartic proteinase of the present invention is a digestive enzyme having two functions of pepsin and enterokinase, and since it has the ability to decompose α _S -casein, it is used as a digestive agent for neonates with immature gastrointestinal tract. As a result, it becomes possible to prevent allergies such as milk and eggs, which are a problem for newborns. Furthermore, since the aspartic proteinase of the present invention exhibits high activity in an acidic region that is unlikely to be contaminated by various bacteria, it is also most suitable as a proteolytic enzyme used when producing food materials such as seasonings and hypoallergenic foods. .

[Brief description of drawings]

FIG. 1 shows the effect of pH on trypsinogen activation of the aspartic proteinases of the present invention.

FIG. 2 shows the pH stability of the aspartic proteinases of the present invention against trypsinogen activation.

FIG. 3 shows the measurement results of the circular dichroism spectrum of the aspartic proteinase of the present invention.

FIG. 4 shows hydrolysis of allergen proteins (α _s -casein, β-lactoglobulin) by the aspartic proteinase of the present invention.

[Explanation of symbols]

LL 1, 6: marker proteins LL 2, 3, 4: α s - casein LL 7, 8, 9, 10 : β- lactoglobulin

FIG. 5 shows the milk- clotting activity of the aspartic proteinases and aspartic proteinases obtained from Aspergillus Saitoi of the present invention.

[Explanation of symbols]

LL 1, 2, 3: Cottling activity of the aspartic proteinase of the present invention LL 4, 5, 6: Cottling activity of the aspartic proteinase of Aspergillus cytoii

Front Page Continuation (72) Inventor Yukitaka Innobe 6883-8 Furuyaue, Kawagoe City, Saitama B2-205 (72) Inventor Kenichi Hirano 212 Inari Nishi, Inari Town, Iwakura City, Aichi Prefecture 11 (72) Inventor Hiroshi Ito Aichi 221 Inaricho, Iwakura City, Japan

Claims (2)

[Claims] 1. Rhizopus hang
chow ) has a molecular weight of about 37,600 (SDS-PAGE),
An aspartic proteinase having the following N-terminal amino acid sequence: Ser-Gly-Ser-Gly-Val-Val-Phe-Met-Thr-Asp-Tyr-Glu-Ty
r-Asp-Ile-Glu-Tyr-Tyr-Gly- 2. Rhizopus hang
chow ), but Rhizopus hangcho
w ) The aspartic protein according to claim 1, which is the IFO-4749 strain.
Proteinase.