JP2000102382A - Carboxyl protease, microorganism producing the same, and production of carboxyl protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new carboxyl protease derived from microorganisms belonging to the genus χ-proteobacteria, manifesting high protein-degrading activities even in a low pH region, capable of improving a low-pH environment of a compost by the formation of ammonia, and useful for composting or the like of an organic waste. SOLUTION: This carboxyl protease is the new carboxyl protease MB8 capable of hydrolyzing casein and hemoglobin well, acting at least at pH 1.5-5, having an optimum pH of about 3-4, keeping about 5-15% activity value of the maximum activity value at pH 5, keeping about 25-35% activity value of the maximum activity value at pH 2, acting at least at 10-70 deg.C, having an optimum temperature of 50-60 deg.C, keeping about 50-60% activity value of the activity value at the optimum temperature at 40 deg.C about 25-35% activity value thereof at 30 deg.C, and about 15-25% activity value thereof at 70 deg.C, and having about 61,000±1,000 molecular weight (SDS-PAGE). The protease has a high protein-degrading ability even at a low pH region, and useful for improvement or the like of a low pH environment of a compost. The enzyme is obtained by culturing χ-proteobacteria sp.MB8 (FERM P-16948).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel carboxyl protease, a microorganism producing the same, and a method for producing the carboxyl protease.

[0002]

2. Description of the Related Art Composting organic waste by aerobic fermentation using microorganisms is a technique that has been practiced since ancient times at agricultural sites. High-speed composting facilities and bio-type garbage disposal devices that apply this principle to engineering are now widely used. In particular, bio-type garbage disposal equipment
Instead of the conventional large-scale centralized recovery and collective treatment, individual decentralized processing, such as promptly processing organic waste at the place where it is generated, has become possible. This individual dispersion treatment has greatly contributed to improving the hygiene of the storage place until the collection of organic waste and reducing the cost of collection and transportation. In addition, aerobic fermentation treatment using microorganisms is not only a resource-saving method with low running cost and low energy consumption, but also can reduce fermentation treatment products to compost as soil, Enabled the recycling of municipal waste. However, the aerobic fermentation treatment method using microorganisms can function only under environmental conditions in which microorganisms, which are the main components of the decomposition reaction of organic substances, can actively grow. Therefore, it is necessary to adjust the environmental conditions under inappropriate environmental conditions. Many of the microorganisms involved in the fermentation process have an optimal pH for growth from neutral to weakly alkaline, and their growth activity decreases in an acidic environment,
At pH 4-5 or lower, many do not show any growth at all. For this reason, it is known that if the pH is lowered in the fermentation process, the decomposition reaction rate is reduced, causing a significant delay or stoppage of the process (see Journal of Sanitary Engineering, 20
Vol. 175, 1984). Such a decrease in pH during the fermentation process is mainly caused by generation and accumulation of acidic substances such as organic acids. This organic acid generation is likely to occur when the concentration of oxygen decreases during a rapid decomposition reaction and becomes anaerobic, and operations such as stirring and turning back are stopped due to an accident such as a power failure, resulting in insufficient oxygen supply. The same phenomenon occurs in the case where it is performed.

In order to neutralize such an acidic substance, a mechanism for measuring the pH of the fermented substance using a measuring device in an organic substance treatment apparatus, or an alkaline agent such as slaked lime is introduced when the pH value is out of an appropriate range. A mechanism has already been proposed (JP-A-55-113688, JP-A-1-145388).
No.). However, if a mechanism for measuring pH or a mechanism for introducing an alkaline agent is installed in the processing apparatus, the apparatus becomes complicated and costs increase. Difficult to apply. In addition, the exact pH of the fermentation product, which is a solid-phase reaction system,
The measurement is difficult. If the supply of the alkali agent is insufficient, not only the effect cannot be obtained, but also the supply of an excessive amount of the alkali agent raises the pH too much and conversely causes a decrease in the activity of microorganisms. In addition, in the fermenter of the organic matter treatment device, the pH for maintaining the hydrogen ion concentration from neutral to weakly alkaline is set.
A method has been proposed in which an organic substance is subjected to high-speed fermentation by filling a regulator and a moisture regulator in which microorganisms that decompose organic substances are fixed (Japanese Patent Application Laid-Open No. 7-303781). However, this moisture regulator takes more than a week to make,
There is a problem that the manufacturing cost is increased.

[0004] In the course of the fermentation treatment of organic waste, the factor that lowers the pH is the accumulation of organic acids, while the factor that raises the pH is the generation of ammonia. As a good fermentation process proceeds, ammonia produced by the decomposition of proteins dissolves in water and becomes ammonium hydroxide, raising the pH. When the pH rises to some extent, ammonia evaporates as ammonia gas, so that the pH does not rise enough to impair fermentation. Therefore, realization of a system capable of effectively decomposing proteins and improving pH in an acidic environment without using a special device, particularly providing an enzyme suitable for the system, and more preferably a microorganism is required. Was.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an enzyme capable of improving the low pH environment of compost by having high protein resolution even in a low pH range and producing ammonia if not directly. It is another object of the present invention to provide a microorganism capable of efficiently producing such an enzyme outside the cells, and a method for producing a carboxyl protease using the microorganism.

[0006]

Under such circumstances, the inventors of the present invention have continued active decomposition reactions despite the fact that the pH of the fermented product has dropped to around 4.5 during the course of its research. And found a specific phenomenon. Therefore, when the fermentation product was used as a separation source to search for acid-resistant microorganisms, a protein-utilizing microorganism that produces a novel carboxyl protease was found among them, and the present invention was completed.

That is, the present invention provides a carboxyl protease MB8 having the following enzymatic properties. (1) Substrate specificity: Good hydrolysis of casein and hemoglobin. (2) Working pH and optimum pH: at least pH 1.5
Working at ~ 5, the optimum pH is about 3-4. It retains about 5-15% of its maximum activity at pH 5, and about 25-35% of its maximum activity at pH 2. (3) pH stability: Stable at least in a pH range of 3.5 to 5.5 under a treatment condition of 4 ° C. for 12 hours. (4) Working temperature and optimum temperature: at least 10 to 70
It works at <RTIgt; 0 C, </ RTI> and the optimal temperature is 50-60C. 40 ℃
At about 50-60% of the activity at the optimum temperature, at 30 ° C. about 25-35% of the activity at the optimum temperature, and at 70 ° C., about 15-25% of the activity at the optimum temperature. (5) Temperature stability: pH 4.7, stable at least up to about 40 to 50 ° C. under 10 minute treatment conditions. (6) Molecular weight: The apparent molecular weight estimated by SDS-PAGE is about 61,000 ± 1,000. (7) Inhibitor: Not inhibited by pepstatin. The present invention also provides a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (N-terminal fragment) and SEQ ID NO: 2 (intermediate fragment) in the sequence listing. Further, the present invention relates to SEQ ID NO: 1
The present invention provides the above protein having an amino acid sequence in which 1 to 27 amino acids have been added, deleted or substituted in SEQ ID NO: 2, and having carboxyl protease activity. Furthermore, the present invention provides:
-It is intended to provide a microorganism which produces carboxyl protease MB8 belonging to Proteobacteria (Pro-Proteobacteria). The present invention also provides a microorganism that is named χ-Proteobacteria sp. MB8 and that is produced as FERM P-16948 and produces the carboxyl protease MB8. Further, the present invention provides a method for producing carboxyl protease MB8, which comprises culturing the microorganism in a medium and collecting carboxyl protease MB8 from the culture.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The carboxyl protease MB8 of the present invention is, for example, χ-proteobacterium sp.
(χ-Proteobacteria sp. MB8) is produced by culturing and collecting from the culture.

Χ-Proteobacteria sp. MB8 (χ-P
The taxonomic properties of roteobacteria sp. MB8) are shown below. (A) Results of microscopic observation 0.5-0.8 μm × 1.5-3.5 μm bacilli, which are motile. Produces a yellow pigment. No spore formation is observed. (B) Gram staining:-(c) Lysis with 3% KOH: + (d) Aminopeptidase activity: + (e) Sporulation ability:-(f) Motility: + (g) Catalase activity: + (h ) Oxidase activity: + (i) β-galactosidase activity:-(j) alcohol dehydrogenase activity:-(k) formation of nitrous acid from nitrate: + (l) urease activity:-(m) hydrolysis of gelatin:- (N) Hydrolysis of Tween 80: + (m) Hydrolysis of esculin:-(o) Availability: glucose: + phenlyacetat:-arabinose:-citrat:-malat:-adipat:-maltose: + mannit:-mannose : + N-acetylglucosamin: + Cellobiose: ++ β-hydroxybutyrat:-L-histidin:-

[0010] The partial sequence of 16S rDNA is Stenotroph
89.5% homology with omonas maltophilia, other χ-Pr
It showed about 80-88% homology with oteobacteria.
However, the fatty acid composition of the cells is clearly Stenotrophomonas
It was different from the genus. Based on the above mycological properties, this strain was identified by DSMZ in Germany as belonging to Stenotrophomona belonging to χ-Proteobacteria.
It was identified as a new species of a new genus closely related to the genus as. Therefore, this strain was transformed into χ-Proteobacteria sp. MB8 (χ-Proteo
bacteria sp. MB8) and FERM P-1694
No. 8 was deposited at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. Attach a receipt.

In order to obtain the carboxyl protease MB8 of the present invention using the above-mentioned strains, the carboxyl protease MB8 of the present invention can be obtained by inoculating the medium with the strain and culturing it in a conventional manner. It is desirable that the culture medium used for cultivation contains an appropriate amount of assimilable carbon source and protein.
When a nitrogen source other than protein is provided, the microorganism can grow, but the production of the enzyme of the present invention is reduced. The carbon source and the nitrogen source are not particularly limited, and examples thereof include, for example, glucose and organic acids that can be assimilated, but various starches are most preferable. Organic nitrogen sources such as casein, soy flour, and meat extract are effective as protein sources.However, when a nitrogen source rich in low-molecular peptides and amino acids is supplied, the production of the enzyme of the present invention is significantly reduced. I do. In addition, inorganic salts such as phosphate, magnesium salt, calcium salt, sodium salt and potassium salt, and if necessary, inorganic and organic trace components can be appropriately added to the medium. As such a medium, for example, ACS
A medium can be particularly preferably used. [ACS medium] (Soil Biol. Biochem. 7, 345,
(Modified based on 1975) Starch: 1%, casein: 0.3%, NaCl: 0.1%
2%, MgSO ₄ 7H ₂ O: 0.005%, CaC
O ₃ : 0.002%, FeSO ₄ 7H ₂ O: 0.001
%, KH ₂ PO ₄ : 0.3%, pH: 4.5. The culture temperature is preferably around 30 ° C.
Preferably around 5. Culture is usually completed under these conditions in 3-5 days.

In order to collect the target enzyme, carboxyl protease MB8, from the culture solution thus obtained, the enzyme can be obtained by performing the method according to a general enzyme collection method. That is, after the culturing, the cells are removed from the culture solution by ordinary separation means such as centrifugation and filtration to obtain a crude enzyme solution. This crude enzyme solution can be used as it is,
If necessary, it can be collected by means such as ultrafiltration, precipitation, or the like, and can be used after being pulverized using an appropriate method. The crude enzyme solution can be further purified by a common means of enzyme purification, for example, a combination of appropriate ion exchange chromatography, affinity chromatography, hydrophobic chromatography, gel filtration chromatography and the like. In addition, a suitable enzyme purification method is as follows. That is, the crude enzyme solution adjusted to pH 5.8 was added to DE.
AE ion exchange chromatography and gel filtration can be repeated as needed.

The carboxyl protease MB8 of the present invention
The enzymatic properties of are described below. (Enzyme activity measurement method) Mcll containing 1.33% of hemoglobin
vaine buffer (pH 3.0) (J. Biol. Chem. 49,
183, 1921) (acid component: 0.1 M citric acid,
Base component: 0.2 MKH ₂ PO ₄ ) 0.700 ml
Then, 0.250 ml of the enzyme solution was added thereto and reacted at 37 ° C. for 10 minutes (hereinafter referred to as “reaction solution”). Reaction stop solution (0.44
M trichloroacetic acid) (1.0 ml), and the mixture was allowed to stand at 30 ° C. for 20 minutes. Next, the mixture was centrifuged (1000 G × 5 minutes). To 0.5 ml of the supernatant, 2.5 ml of 0.44 M sodium carbonate was further added, and 0.5 ml of 1N Folin-Ciocalteu reagent (manufactured by Sigma) was added. After standing for 20 minutes at, the absorbance at 660 nm is measured. Under the above reaction conditions, the amount of the enzyme that produces an acid-soluble proteolysate corresponding to 1 μg of tyrosine per minute (in the reaction solution) is defined as 1 unit (U). (Protein Content Measurement Method) In the chromatography operation, a measured value of absorbance (280 nm) was used. In each purification step, the fraction of the active fraction collected was Brad
Bovine serum albumin (manufactured by Nacalai Tesque) was measured by the ford method (Anal. Biochem., vol. 72, p. 248) as a standard protein. (Enzymatic properties) (1) Action and substrate specificity Final concentration 1.0% in McIlvaine buffer (pH 3.5)
Hemoglobin (Sigma, Bovine) or casein (Wako Pure Chemical Industries, Hamarsten's method)
Was added, and an enzyme reaction was performed at 37 ° C. for 10 minutes, and the activity was measured. As a result, it was found that this enzyme decomposes hemoglobin and casein well. (2) Working pH and optimum pH Clark-Lubs buffer (pH 1-2) (J. Bact., Volume 2, 19)
1, page 1917) (acid component: 0.2 M HCl, base component: 0.2 M KCl) and McIlvaine buffer (pH
Hemoglobin (Bovine, manufactured by Sigma) was added to 2-8) to a final concentration of 1.0%, and an enzymatic reaction was performed at 37 ° C. for 10 minutes, and the activity was measured. As shown in FIG.
Works at least at pH 1.5-5, optimal pH about 3 ~
4 (Clark-Lubs buffer is represented by solid circles, McIlvaine buffer is represented by open circles). The maximum activity value of about 5 at pH 5
~ 15%, about 25 ~ 35% of the maximum activity value at PH2
Keep activity. (3) pH stability The enzyme was added to Clark-Lubs buffer (pH 1-2) and McIlvaine buffer (pH 2-8), and left at 4 ° C. for 12 hours. PH 3.5, 37 ° C using hemoglobin as a substrate
The enzyme reaction was carried out for 10 minutes under the conditions described above, and the residual activity was measured. As shown in FIG. 2, it is stable at least in the range of pH 3.5 to 5.5 under the treatment conditions of 4 ° C. and 12 hours (Clark-Lubs buffer is indicated by a black circle, McIlvaine buffer is indicated by a white circle). . (4) Working temperature and optimum temperature Final concentration 1.0% in McIlvaine buffer (pH 3.5)
Hemoglobin was added so as to give an enzyme reaction at each temperature for 10 minutes, and the activity was measured. As shown in FIG.
Works at least 10-70 ° C, optimal temperature is 50-6
0 ° C. Approximately 50 activities at optimum temperature
~ 60%, about 25-3 of activity at optimum temperature at 30 ° C
5%, about 15-25% of activity at optimum temperature at 70 ° C
% Activity. (5) Temperature stability The enzyme was allowed to stand in a 20 mM citrate buffer (pH 4.7) at each temperature for 10 minutes and then cooled on ice. Using hemoglobin as a substrate at pH 3.5 and 37 ° C., 1
The enzyme reaction was performed for 0 minutes, and the residual activity was measured. As shown in FIG. 4, the enzyme is stable up to at least about 40 to 50 ° C. under a treatment condition of pH 4.7 for 10 minutes. (6) Molecular Weight Standards (Low Range) (manufactured by Bio-Rad, phosphorylase b (97.4 kD), serum albumin (66.2 kD), ovalbumin (45.0)
kD), carbonic anhydrase (31.0 kD), trypsin inhibitor (21.5 kD), lysozyme (14.4)
kD)) as a molecular weight marker and 10% SDS
-The estimated molecular weight measured by PAGE is about 61,00
0 ± 1,000 (FIG. 5, open circles indicate the present protein). (7) Inhibitor Using porcine pepsin (Sigma) as a control, the sensitivity to pepstatin, a specific inhibitor of carboxyl protease, was examined. McIlvaine buffer (pH 4.
This enzyme was added to 0) at a final concentration of 10 (μg / ml), and pepstatin at each concentration was allowed to act at 37 ° C. for 10 minutes. After the action, pH 3.
The enzymatic reaction was carried out at 5, 37 ° C. for 10 minutes, and the residual activity was measured. As shown in FIG. 6, it can be seen that this enzyme is not inhibited by pepstatin (open circles indicate this enzyme; black circles indicate conventional carboxyl proteases).

[0014] Thus, the carboxyl protease MB8 of the present invention is a novel enzyme which has not been reported in the past. For carboxyl proteases produced by mesophilic bacteria, see Pseudomonas sp. 101 (Biochimica et Biophyl.
sica Acta, 923, 463, 1987. J. Bio
Chem., 269, 26518, 1994) and Xanthomonas sp. (Agric. Biol. Chem., 51,
3073, 1987. J. Biochem., Volume 120, 5
64, 1996). This enzyme is
Although these Pseudomonas sp. 101 and Xanthomonas sp. Are considered to be a kind of pepstatin-insensitive carboxyl protease together with the enzymes produced by them, differences can be observed in the molecular weight of the microorganisms and enzymes from which they originate.
The other properties are clearly different from the enzyme of the present invention even when compared.

The microorganism of the present invention undergoes a series of protein assimilation processes and finally produces ammonia by a deamination reaction, which results in an increase in the pH of the growing environment.

The carboxy protease MB8 of the present invention
In addition to the organic waste fermentation treatment step, it can be effectively used as a detergent for removing protein stains by adding it to an acidic detergent, or as a detergent for production lines of acidic foodstuffs.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. (Example 1) The present inventors have found a specific phenomenon in the course of research that an active decomposition reaction continues despite the fact that the pH of the fermentation product has dropped to around 4.5 by chance. . Therefore, an attempt was made to isolate the strain of the present invention from the fermented product. First, as a primary screening, acid-resistant microorganisms were separated by selective culture in an acidic medium (SPYN medium). [SPYN medium] (J. Gen. Appl. Microbiol. 41
Volume, p. 175, 1995) Soluble Starch: 1%, Pepton: 0.1%, Yeast extract
: 0.01%, Nutrient broth: 0.05%, KNO ₃ : 0.2%, NaC
l: 0.2%, MgSO ₄ 7H ₂₀ : 0.005%, CaCl ₂ 2H ₂ O: 0.002
%, FeSO ₄ 7H ₂ O: 0.001%, (pH: 4.5) The above fermentation product was added to SPYN liquid medium, and 30 ° C.
Aerobic culture was performed, and after 1 to 7 days of culture, the generated strain was isolated using a SPYN agar medium as appropriate. The obtained strain was purified by repeating streaking on SPYN agar medium. Next, secondary screening based on the presence or absence of protease activity was performed. A strain having protease activity was identified as AC
When cultured on an S agar medium, a clear zone (halo) is formed around the colony. Using this property as an index, protein-assimilating microorganisms were separated. When the carboxyl protease activity of the obtained isolate was quantified,
It had the enzyme activity. Thus, χ-Proteobacteria sp. MB8
) The strain was selected.

(Example 2) Next, Δ- obtained in Example 1 was used.
Production of carboxyl protease of Proteobacterium sp. Strain MB8 and pH associated with protein degradation
The effect of the improvement was verified. This strain was cultured in an ACS medium at 30 ° C. for 5 days to grow the cells,
H and protease activity were measured. [Method of Measuring Cell Growth] The cell growth was measured by measuring the ATP concentration and used as an index. The ATP concentration was measured using an ATP measurement reagent kit Lucifer LU (manufactured by Kikkoman Corporation). The growth of the bacterial cells, the protease activity secreted into the medium, and the change over time in the pH value of the medium are shown in FIGS. 7-9. It can be seen that the present strain actively produces protease with its growth. In addition, proteases are produced by the growth of bacterial cells, and the pH value of the medium increases to near neutrality. As shown in FIG. 10, plotting the relationship between the protease activity in the medium and the pH value accompanying the growth of the microorganism of the present invention shows that when the protease is produced and the degradation of the protein proceeds, the pH value is clearly increased. Was shifted to the neutral side, indicating that the object of the present invention was met.

(Example 3) Next, a method for purifying the enzyme obtained by the present invention will be described. Χ- obtained in Example 1
Proteobacteria sp. MB8 strain was inoculated into an ACS liquid medium (pH 4.5) and cultured at 30 ° C. for 4 days. After the culture, 20 mM Na was added to the supernatant from which the cells were removed by centrifugation.
₂ HPO ₄ was added and the pH was adjusted to 5.8. This crude enzyme solution was previously added to a 20 mM citrate buffer (pH 5.8).
Was added to DEAE-Sepharose CL-6B (manufactured by Pharmacia, column diameter 1.84 cm × length 15.0 cm), which was eluted with a concentration gradient of 0-0.4 M sodium chloride (FIG. 11). Fractions having high protease activity were collected and concentrated by Collodion Vac (Sartorius). The concentrate is diluted with 2 M containing 0.2 M sodium chloride.
Bi equilibrated with 0 mM citrate buffer (pH 5.8)
o-Gel P-100 (manufactured by Bio-Rad, column diameter 1.7 cm)
× 81 cm in length) and eluted with the same buffer to collect an active fraction (FIG. 12). By the above operation, an enzyme preparation showing a single band electrophoretically was obtained. As a result, the enzyme was purified to about 13 times. Each purification step is summarized in the purification table in Table 1 below. It is noted that the specific activity of the crude enzyme solution obtained using the ACS medium as a culture medium shows an extremely high value of 649 (U / mg), and this medium is suitable for production and purification of the enzyme. This is a medium. Therefore, the use of the present medium has made it possible to easily purify the present enzyme.

[0020]

[Table 1]

[0021] Protein sequencer (Applied Bios
As a result of determining the amino acid sequence of the present enzyme using 477A manufactured by ystem, it was found that the present enzyme was a protein containing the following N-terminal fragment and intermediate fragment.

[N-terminal fragment] Ala Ser Gly Ser Ala Thr Gly His Lys Met Thr Asp Ph
e Ala Gln Ile Tyr AsnAla Gly

[Intermediate fragment] Thr Ser Gly Thr Thr Trp Thr Asn Glu Thr Val Trp As
n Asn Leu Asn Glu AsnGlu Gly Ala Thr Gly Gly Ser P
ro Ser Thr Phe Glu Pro Gln Pro Ser Trp GlnAsn Gly
Val Gly Gln

[0024]

Industrial Applicability According to the present invention, an enzyme and an enzyme capable of improving the low pH environment of compost by having high-performance protein resolution even in a low pH range and producing ammonia, if not directly, And a method for producing carboxyl protease using the microorganism.

[0025]

[Sequence List] SEQUENCE LISTING <110> ASAHI DENKA KOGYO KK ADEKA CLEAN AID KK <120> CARBOXYL PROTEASE, MICROORGANISM PRODUCING SAME AND METHOD FOR PRODUCING CARBOXYL PROTEASE <130> K20010 <160> 2 <210> 1 <211> 20 <212 > PRT <213> χ-Proteobacteria sp.MB8 <220> <221> UNSURE <223> The amino acid sequence has not been completely analyzed at this stage. <400> 1 Ala Ser Gly Ser Ala Thr Gly His Lys Met Thr Asp Phe Ala Gln Ile 1 5 10 15 Tyr Asn Ala Gly 20 <210> 2 <211> 41 <212> PRT <213> χ-Proteobacteria sp.MB8 <220> <221> UNSURE <223> Another partial sequence has been identified in the middle of the overall amino acid sequence. <400> 2 Thr Ser Gly Thr Thr Trp Thr Asn Glu Thr Val Trp Asn Asn Leu Asn 1 5 10 15 Glu Asn Glu Gly Ala Thr Gly Gly Ser Pro Ser Thr Phe Glu Pro Gln 20 25 30 Pro Ser Trp Gln Asn Gly Val Gly Gln 35 40

[Brief description of the drawings]

FIG. 1 is a view showing the pH characteristics of the protease MB8 of the present invention.

FIG. 2 is a view showing the pH stability of the protease MB8 of the present invention.

FIG. 3 is a graph showing temperature characteristics of the protease MB8 of the present invention.

FIG. 4 shows the temperature stability of protease MB8 of the present invention.

FIG. 5: SDS-PAGE of protease MB8 of the present invention
FIG. 3 is a diagram showing estimated molecular weights by the following method.

FIG. 6 is a graph showing pepstatin sensitivity of protease MB8 of the present invention.

FIG. 7 is a view showing a growth curve of the microorganism of the present invention.

FIG. 8 is a diagram showing protease production by the microorganism of the present invention.

FIG. 9 is a graph showing a change in the pH of a medium accompanying the growth of the microorganism of the present invention.

FIG. 10 is a graph showing the relationship between the pH value of a medium and the production of protease in accordance with the growth of the microorganism of the present invention.

FIG. 11 is a view showing an ion exchange chromatogram (DEAE-Sepharose CL-6B) in the purification process of the enzyme of the present invention.

FIG. 12 is a view showing a gel filtration chromatogram (Bio-Gel P100) in the purification process of the enzyme of the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Ryo Takei 7-35, Higashiogu, Arakawa-ku, Tokyo Asahi Denka Kako Kogyo Co., Ltd. (72) Takefumi Oguri 7-35, Higashiogu, Arakawa-ku, Tokyo Asahi Denka Kogyo Co., Ltd. (72) Inventor Kohei Uno 7-35, Higashiogu, Arakawa-ku, Tokyo Asahi Denka Kogyo Co., Ltd. (72) Inventor Mamoru Honma 2-jo 5-9-1, Hanakawakita, Ishikari City, Hokkaido 10 (72) Inventor Hirokazu Matsui Hokkaido, Sapporo City, Kita-ku, 6-chome, 16-chome, 4-6-24 (72) Inventor Sakira 9-chome, Kitazawa, Minami-ku, Sapporo, Hokkaido F-term (reference) 4B050 CC01 DD02 LL10 4B065 AA01X BA22 CA33 CA54

Claims (6)

[Claims] 1. A carboxyl protease MB8 having the following enzymatic properties: (1) Substrate specificity: Good hydrolysis of casein and hemoglobin. (2) Working pH and optimum pH: at least pH 1.5
Working at ~ 5, the optimum pH is about 3-4. It retains about 5-15% of its maximum activity at pH 5, and about 25-35% of its maximum activity at pH 2. (3) pH stability: Stable at least in the range of pH 3.5 to 5.5 under the treatment conditions of 4 ° C. and 12 hours. (4) Working temperature and optimum temperature: at least 10 to 70
It works at <RTIgt; 0 C, </ RTI> and the optimal temperature is 50-60C. 40 ℃
At about 50-60% of the activity at the optimum temperature, at 30 ° C. about 25-35% of the activity at the optimum temperature, and at 70 ° C., about 15-25% of the activity at the optimum temperature. (5) Temperature stability: pH 4.7, stable at least up to about 40 to 50 ° C. under 10 minute treatment conditions. (6) Molecular weight: The apparent molecular weight estimated by SDS-PAGE is about 61,000 ± 1,000. (7) Inhibitor: Not inhibited by pepstatin. 2. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 (N-terminal fragment) and SEQ ID NO: 2 (intermediate fragment) in the sequence listing. 3. An amino acid sequence comprising 1 to 9 amino acids in SEQ ID NO: 1 and 1 to 27 amino acids in addition, deletion or substitution in SEQ ID NO: 2, and having carboxyl protease activity. 2. The protein according to 2. 4. A χ-proteobacterium (χ-Proteobact)
eria), a microorganism that produces carboxyl protease MB8. 5. The χ-proteobacteria sp. MB8 (χ)
-Proteobacteria sp. MB8) and FERM P-
The microorganism producing the carboxyl protease MB8 according to claim 1, which has been deposited as 16948. 6. The method according to claim 5, wherein the microorganism is cultured in a medium.
Collecting a carboxyl protease MB8 from the culture;
8. Manufacturing method.