JP4382890B2 - Carboxyl protease, microorganism producing the same, and method for producing carboxyl protease - Google Patents

Description

【００１７】

【００１８】
【発明の効果】
本発明によれば、低pH域でも高性能のタンパク質分解能をもち、直接的ではないにしろアンモニアを生じせしめて、コンポストの低pH環境を改善することができる、酵素並びにかかる酵素を菌体外に効率よく生産する微生物及び当該微生物を用いたカルボキシルプロテアーゼの製造法が提供される。
【００１９】
【配列表】

【図面の簡単な説明】
【図１】本発明の酵素の作用pH 及び 最適pHを示す図である。
【図２】本発明の酵素のpH安定性を示す図である。
【図３】本発明の酵素の作用温度 及び 最適温度を示す図である。
【図４】本発明の酵素の温度安定性を示す図である。
【図５】本発明の酵素のSDS‐PAGEおよび分子量の算出を示す図である。
【図６】本発明の酵素に与える阻害剤の影響を示す図である。
【図７】本発明の微生物の生育を示す図である。
【図８】本発明の微生物の産出する酵素のプロテアーゼ活性を示す図である。
【図９】本発明の微生物の培地に与えるpH調整能を示す図である。
【図１０】本発明の酵素の精製過程（DEAE‐Sepharose CL‐6Bによるイオン交換クロマトグラム）を示す図である。
【図１１】本発明の酵素の精製過程（Sephadex G‐75によるゲル濾過クロマトグラム）を示す図である。
【図１２】本発明の酵素の一連の精製過程をまとめた図である。
【図１３】本発明の酵素の酵素学的性質（他の微生物起源のカルボキシルプロテアーゼとの比較）を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel carboxyl protease, a microorganism producing the same, and a method for producing the carboxyl protease.
[0002]
[Prior art]
Composting organic waste by aerobic fermentation using microorganisms, especially aerobic fermentation using microorganisms, is a resource-saving method with low running costs and low energy consumption. Instead, the fermented product can be reduced to soil as compost.
However, the aerobic fermentation treatment method using microorganisms can function only under environmental conditions in which microorganisms that are the main components of organic matter decomposition reactions 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 optimum pH for growth from neutral to weakly alkaline, and their growth activity decreases in an acidic environment, and no growth occurs at pH 4-5 or lower (discarding) Journal of Physical Society, Vol. 4, p. 35, 1993). For this reason, it is known that when the pH is lowered in the fermentation process, the decomposition reaction rate is lowered, and the treatment process is significantly delayed or stopped (Hygiene Engineering Research Papers, 20, 175, 1984). Year). In order to neutralize these acidic substances, there is already a mechanism for measuring the pH of fermented substances using a measuring device in an organic matter treatment apparatus, and a mechanism for adding an alkaline agent such as slaked lime when the pH value is outside the appropriate range. Have been proposed (Japanese Patent Laid-Open Nos. 55-113688 and 1-145388). However, if a mechanism for measuring pH or a mechanism for introducing an alkaline agent is installed in the processing equipment, the equipment becomes complicated and expensive, and it is particularly applicable to individual dispersion processing using a bio-type garbage processing equipment with a small equipment. Difficult to do. In addition, it is difficult to accurately measure the pH of the fermented product that is a solid phase reaction system, and if the supply of alkaline agent is insufficient, the effect cannot be obtained. There were problems such as causing a decrease in the activity of microorganisms.
In addition, there is a method of fermenting organic matter at high speed by filling a fermenter of an organic matter processing apparatus with a pH regulator that maintains the hydrogen ion concentration from neutral to weakly alkaline and a moisture regulator that fixes microorganisms that decompose organic matter. It has been proposed (Japanese Patent Laid-Open No. 7-303871). However, this moisture regulator has a problem that it takes a week or more for preparation and the production cost is increased.
In the process of fermentation of organic waste, the factor that lowers the pH is the accumulation of organic acids, but the factor that raises the pH is the generation of ammonia. As a good fermentation process proceeds, ammonia produced by protein degradation dissolves in water and becomes ammonium hydroxide, raising the pH. When the pH rises to some extent, ammonia is volatilized as ammonia gas, so the pH does not rise so as to hinder fermentation. Accordingly, there has been a demand for the realization of a system capable of sufficiently degrading proteins and raising the pH even in an acidic environment, in particular, the provision of an enzyme suitable for the system, more preferably the provision of a microorganism.
[0003]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide an enzyme capable of improving the low pH environment of compost, which has high-performance protein resolution even in a low pH range, and can generate ammonia, if not directly, to improve the low pH environment of compost. It is another object of the present invention to provide a microorganism that can be efficiently produced and a method for producing a carboxyl protease using the microorganism.
[0004]
[Means for Solving the Problems]
Under such circumstances, during the research by the present inventors, a specific phenomenon was discovered in which an active decomposition reaction continued by chance even though the pH of the fermented product was lowered to around 4.5. Thus, when microorganisms were separated from the fermented product, conventional microorganisms such as Pseudomonas sp. 101 (Oda et al. Biochimica et Biophysica Acta, 923, 463, 1987. Oda et al. J. Biol. Chem., 269, 26518, 1994), or Xanthomonas sp. (Oda et al. Agric. Biol. Chem., 51, 3073, 1987. Oda et al J. Biochem., 120, 564). Page, 1996) and a novel carboxyl protease produced by the microorganism was found and the present invention was completed.
[0005]
That is, the present invention is produced from a microorganism named as Burkholderia sp. MB-11 and commissioned as FERM P-16949, and SEQ ID NO: 1 (N-terminal fragment) and SEQ ID NO: 2 ( A carboxyl protease MB11 comprising the amino acid sequence represented by ( intermediate fragment) and having the following enzymatic properties (1) to (8) is provided.
(1) The substrate-specific casein is well hydrolyzed and acts on hemoglobin.
(2) Working pH and optimum pH
It operates at least at pH 2-6, and the optimum pH is about 3-4. It retains 20-30% of the maximum activity value at pH 5, and 10-20% of the maximum activity value at pH 2.5.
(3) pH stability It is stable in the pH range of 3 to 8 under the treatment conditions of 4 ° C. and 12 hours.
(4) Working temperature and optimum temperature The working temperature is at least 10 to 80 ° C, and the optimum temperature is 50 to 60 ° C. About 55-65% of activity at optimum temperature at 40 ° C, about 20-30% of activity at optimum temperature at 30 ° C, about 60-70% of activity at optimum temperature at 70 ° C, activity at optimum temperature at 80 ° C Retains about 30-40% activity.
(5) Temperature stability pH 5.8 Stable to at least about 50-60 ° C. under 10 minute processing conditions.
(6) Molecular weight The estimated molecular weight estimated by SDS-PAGE is about 36,000.
(7) The isoelectric point is 4.2.
(8) It is not inhibited by the inhibitors pepstatin, diazoacetylnorleucine methyl ester and 1,2-epoxy-3- (p-nitrophenoxy) propane .
Or the invention, Burkholderia sp (Burkholderia sp.) MB11 named, there is provided a microorganism producing the carboxyl protease MB11 which is commissioned as FERM P-16949.
Furthermore, the present invention provides a method for producing carboxyl protease MB11, which comprises culturing the microorganism in a medium and collecting the carboxyl protease MB11 from the culture.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The carboxyl protease MB11 of the present invention is produced, for example, by culturing Burkholderia sp. MB11 and collecting it from the culture.
[0007]
The taxonomic properties of Burkholderia sp. MB11 are shown below.
(a) Microscopic observation results 0.5 to 0.8 μm × 1.5 to 3.0 μm of Bacillus having flagella and motility. Spore formation is not observed.
(b) Gram stainability: −,
(c) Lysis with 3% KOH: +
(d) Aminopeptidase activity: +
(e) Spore-forming ability: −,
(f) Motility: +,
(g) Catalase activity: +,
(h) Oxidase activity: −,
(i) alcohol dehydrogenase activity:-,
(j) Urease activity: −,
(k) Gelatin hydrolysis:-,
(l) Production of nitrous acid from nitric acid: +,
(m) Denitrification: − (ng)
(n) Usability of sugar, etc .:
Glucose: +
Use of citrates: +,
Arabinose:-,
Inositol:-
Adonit: +
α-amylamin: ++
m-hydroxybenzoat: +
2,3-butylene glycol: +
ciraconat: ++
Tryptamine: +
lavulinat: +
(o) Growth at 41 ° C: +
[0008]
Based on the above-mentioned studies on mycological properties, the strain was determined by German DSMZ to be a strain with properties similar to those of the Burkholderia genus Burkholderia cepacia or Burkholderia pylosinia. It was. However, this strain had specific properties as genus Burkholderia, such as acid resistance and production of carboxyl protease. Therefore, this strain was named Burghorderia SP MB11 and deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology as FERM P-16949. Attach the certificate of trust.
[0009]
In order to obtain the carboxyl protease MB11 of the present invention using the above-mentioned strain, it can be obtained by inoculating the strain on a medium and culturing according to a conventional method.
It is desirable that the medium used for the culture contains an appropriate amount of assimilable carbon source and protein. However, when a nitrogen source other than protein is provided, microorganisms can grow but the production of the enzyme of the present invention decreases. .
Although the carbon source and nitrogen source are not particularly limited, examples of the carbon source include glucose and organic acids (for example, citric acid), but various starches are most suitable. As a protein source, organic nitrogen sources such as casein, soybean powder, and meat extract are effective. However, when a nitrogen source rich in low molecular weight peptides and amino acids is supplied, the production of the enzyme of the present invention is significantly reduced.
In addition, phosphate, magnesium salt, calcium salt, manganese salt, zinc salt, cobalt salt, sodium salt, potassium salt and other inorganic salts, and if necessary, inorganic, organic micronutrients and vitamins are appropriately added to the medium. Can be added.
Examples of such a medium include ACS medium (starch 1%, casein 0.3%, NaCl 0.2%, MgSO ₄ .7H ₂ O 0.005%, CaCO 3 0.002%, FeSO ₄ .7H ₂ O. 0.001%, KH ₂ PO ₄ 0.3%, pH 4.5) can be used particularly preferably.
The culture temperature is preferably around 30 ° C., and the initial culture pH is preferably 4.5. Under these conditions, the culture is usually completed in 3 to 5 days.
[0010]
In order to collect carboxyl protease MB11, which is the target enzyme, from the thus obtained culture broth, it can be obtained according to a general enzyme collecting means. That is, after culturing, the cells are removed from the culture solution by a normal separation means such as centrifugation or filtration to obtain a crude enzyme solution. This crude enzyme solution can be used as it is, but if necessary, it can be recovered by means of ultrafiltration, precipitation, etc., and powdered using an appropriate method. The crude enzyme solution is further purified by a combination of general means for enzyme purification, such as chromatography using an appropriate cation exchange resin, anion exchange resin, hydroxyapatite, etc., affinity chromatography, hydrophobic chromatography, gel filtration and the like. It can also be purified. A suitable enzyme purification method is as follows. That is, while repeating the concentration appropriately, for example, the crude enzyme solution adjusted to pH 5.8 can be subjected to cation exchange chromatography, and gel filtration can be repeated as necessary.
[0011]
The enzymatic properties of the carboxyl protease MB11 of the present invention are described below.
(Enzyme activity measurement method)
Mcllvaine buffer containing 1.33% hemoglobin (Mcllvaine, J. Biol. Chem., 49, 183, 1921, acid component: 0.1 M citric acid, base component: 0.2 M Na ₂ HPO ₄ ) (pH 3. 0) Bring 375 μl to 37 ° C., add 125 μl of enzyme solution, and react at 37 ° C. for 10 minutes. Add 0.5 ml of the reaction stop solution (0.44 M trichloroacetic acid) and let stand at 30 ° C. for 20 minutes. Next, centrifuge (1000G for 5 minutes), add 0.5ml of 0.44M sodium carbonate to 0.5ml of the supernatant, add 0.5mL of 1N Folin-Ciocalteu reagent (Sigma), and leave at 37 ° C for 20 minutes. Thereafter, the absorbance at 660 nm is measured. Under the above reaction conditions, the amount of enzyme that produces an acid-soluble proteolytic product corresponding to 1 μg of tyrosine per 1 ml of the reaction solution per minute is defined as 1 unit (U).
(Enzymological properties)
(1) Action and substrate specificity: Decomposes proteins and peptides such as casein, hemoglobin, albumin, meat protein, fish protein, and soy protein. In particular, casein is well hydrolyzed and acts on hemoglobin.
A series of samples was added to a final concentration of 1.0% in Mcllvaine buffer (pH 3.5), and the reaction was carried out at 37 ° C. for 10 minutes to measure the activity. Hemoglobin was manufactured by Sigma, Bovine, and casein was manufactured by Wako Pure Chemical Industries, Hamarsten method.
(2) Working pH and optimum pH (FIG. 1): It works at least at pH 2-6, and the optimum pH is about 3-4. It retains 20-30% of the maximum activity value at pH 5, and 10-20% of the maximum activity value at pH 2.5.
In Mcllvaine buffer (pH 2-8) and Clark-Lubs buffer (Clark, Lubs, J. Bact, Vol. 2, 191 pages, 1917 (acid component 0.2M HCl, base component 0.2M KCl) (pH 1-2) Hemoglobin was added to a final concentration of 1.0%, the reaction was carried out at 37 ° C. for 10 minutes, and the activity at each pH was measured.
(3) pH stability (FIG. 2): The treatment at 4 ° C. for 12 hours is stable in the pH range of 3-8.
The enzyme was added to Mcllvaine buffer (pH 2-8) and Clark-Lubs buffer (pH 1-2) and allowed to stand at 4 ° C. for 12 hours. The residual activity was measured under the conditions of pH 3.5 and 37 ° C. using hemoglobin as a substrate.
(4) Working temperature and optimum temperature (FIG. 3): It works at least at 10 to 80 ° C., and the optimum temperature is 50 to 60 ° C. About 55-65% of activity at optimum temperature at 40 ° C, about 20-30% of activity at optimum temperature at 30 ° C, about 60-70% of activity at optimum temperature at 70 ° C, activity at optimum temperature at 80 ° C Retains about 30-40% activity.
Hemoglobin was added to Mcllvaine buffer (pH 3.5) to a final concentration of 1.0%, and the reaction was carried out at each temperature for 10 minutes to measure the activity.
(5) Temperature stability (FIG. 4): Stable up to at least about 50-60 ° C. at a pH of 5.8 for 10 minutes.
This enzyme was allowed to stand at 20 ° C. for 10 minutes in a 20 mM citrate buffer and then cooled on ice. Using hemoglobin as a substrate, the residual activity was measured by reacting for 10 minutes under conditions of pH 3.5 and 37 ° C.
(6) Molecular weight (FIG. 5): As a result of 10% SDS polyacrylamide gel electrophoresis using Parfect Protein Markers (Novagen 150,000, 100,000, 75,000, 50,000, 35,000, 25,000, 15,000) as molecular weight markers. Has a molecular weight of about 36,000.
(7) Isoelectric point: The isoelectric point is 4.2.
The polyacrylamide gel was measured by isoelectric focusing. As a pI marker, a low pI calibration kit (pI 2.5-6.5) (Pharmacia) was used.
(8) Effect of inhibitor (FIG. 6): Not inhibited by pepstatin, DAN (diazoacetylnorleucine methyl ester), EPNP (1,2-epoxy-3- (p-nitrophenoxy) propane).
Using porcine pepsin (Sigma) as a control, sensitivity to an inhibitor specific for carboxyl protease was measured.
[Pepstatin] The enzyme was added to Mcllvaine buffer (pH 4.0) to a final concentration of 10 (μg / ml), and each concentration of pepstatin was allowed to act at 37 ° C. for 10 minutes. After the action, the residual activity was measured by enzyme reaction for 10 minutes under the conditions of pH 3.5 and 37 ° C. using hemoglobin as a substrate.
[DAN] This enzyme, 30 mM CuSO ₄ , 60 mM DAN was added to Mcllvaine buffer (pH 5.0) and allowed to act at 37 ° C. for 0-60 minutes. Final concentrations at the time of action were enzyme: 57 (μg / ml), Cu ²⁺ : 2.20 mM, and DAN 2.65 mM. After the action, the residual activity was measured by enzyme reaction for 10 minutes under the conditions of pH 3.5 and 37 ° C. using hemoglobin as a substrate.
[EPNP] This enzyme was added to Mcllvaine buffer (pH 4.0) to 57 (μg / ml), 10 mg / ml of EPNP was added, and the mixture was reacted at 30 ° C. for 0-76.5 hours. After the reaction, the residual activity was measured by enzyme reaction for 10 minutes under conditions of pH 3.5 and 37 ° C. using hemoglobin as a substrate.
[0012]
Thus, the carboxyl protease MB11 of the present invention is a novel enzyme that has not been reported previously. Although this enzyme is similar to the enzyme produced by Pseudomonas sp. 101 described above, it is a novel enzyme that can recognize differences in its molecular weight, optimum pH, and isoelectric point.
The microorganism of the present invention goes through a series of protein assimilation processes, and finally produces ammonia by a deamination reaction, which consequently increases the pH of the growth environment.
The carboxyprotease MB11 of the present invention can be effectively used as a detergent for removing protein stains or as a detergent for the production of acidic food products by adding it to an acidic detergent, in addition to a fermentation treatment step for organic waste. .
[0013]
【Example】
Hereinafter, although an example is given and explained in detail, the present invention is not limited to these.
Example 1
First, as a primary screening, acid-resistant microorganisms were separated by selective culture using an acidic medium (SPYN medium).
[SPYN medium] (J. Gen. Appl. Microbiol. 41, 175, 1995)
Soluble Starch: 1%, Pepton: 0.1%, Yeast extract: 0.01%, Nutrient broth: 0.05%, KNO ₃ : 0.2%, NaCl: 0.2%, MgSO ₄ 7H ₂ 0: 0.005%, CaCl ₂ 2H ₂ O: 0.002 %, FeSO ₄ 7H ₂ O: 0.001%, (pH: 4.5)
The fermented product was added to the SPYN liquid medium, and aerobic culture was performed at 30 ° C., and the generated strain was isolated using the SPYN agar medium in a timely manner after 1-7 days of culture. The obtained strain was purified by repeated streak culture using SPYN agar medium.
Next, secondary screening based on the presence or absence of protease activity was performed. When a strain having protease activity is cultured on an ACS agar medium, a zona pellucida (halo) is formed around the colony. Using this property as an index, protein-utilizing microorganisms were separated.
About the obtained isolate, when carboxyl protease activity was quantified, it had this enzyme activity. In this way, the Burkholderia SP MB11 strain of the present invention was selected.
[0014]
Example 2
Next, the production of carboxyl protease of Burkholderia sp. MB11 strain obtained in Example 1 and the effect of improving pH accompanying protein degradation were verified. This strain was cultured in an ACS medium at 30 ° C. for 5 days, and the growth of the cells, the pH of the medium, and the protease activity were measured.
[Method for Measuring Cell Growth] Cell growth was determined by measuring the ATP concentration. For the measurement of the ATP concentration, an ATP measurement reagent kit Lucifer LU (manufactured by Kikkoman Corporation) was used.
The growth of the bacterial cells (FIG. 7), the protease activity secreted into the medium (FIG. 8), and the change over time in the pH value of the medium (FIG. 9) are shown in the figure. It can be seen that this strain actively produces protease as its growth occurs. Protease is produced by the growth of the cells, and the pH value of the medium is increased to near neutrality. Looking at the relationship between protease activity in the medium and pH value, when protease is produced and protein degradation proceeds, the pH value is clearly shifted to the neutral side, which is consistent with the purpose of the present invention. It was shown that.
[0015]
Example 3
Burkholderia sp. MB-11 strain obtained in Example 1 was inoculated into ACS liquid medium (pH 4.5) and cultured at 30 ° C. for 72 hours. After culturing, 20 mM citric acid was added to the supernatant from which the cells had been removed by centrifugation, and the pH was adjusted to 5.8. This crude enzyme solution was added to DEAE-Sepharose CL-6B (Pharmacia, column diameter 1.8 cm × length 23.5 cm) previously equilibrated with 20 mM citrate buffer (pH 5.8). Elution was performed with a concentration gradient of -0.5 M sodium chloride (FIG. 10). A fraction having high protease activity was collected and concentrated by collodion bag (manufactured by Sartorius). The concentrated solution was added to Sephadex G-75 (Pharmacia, column diameter 3.3 cm × length 71 cm) equilibrated with 20 mM citrate buffer (pH 5.8) containing 0.2 M sodium chloride, Eluted with the same buffer. The active fraction was collected and concentrated by collodion bag (manufactured by Sartorius). Add the concentrated solution to Bio-Gel P-100 (Biorad, column diameter 1.7 cm x length 81 cm) equilibrated with the same buffer in advance, and elute with the same buffer to separate the active fraction. (FIG. 11). By the above operation, an enzyme preparation that electrophoretically shows a single band was obtained (FIG. 5). As a result, this enzyme was purified to about 9 times. The purification steps are summarized in the purification table (FIG. 12). Of particular note is that the specific activity of the crude enzyme solution obtained using the ACS medium as a culture medium shows an extremely high value of 147 (U / mg), and this medium is suitable for the production of this enzyme. It is a medium. For this reason, the use of this medium made it possible to purify the enzyme relatively easily.
As a result of determining the amino acid sequence of this enzyme using a protein sequencer (Applied Biocystems 477A), it was found that this enzyme is a protein containing the following N-terminal fragment and intermediate fragment. The enzymological properties were as shown in FIG.
[0016]

[0017]

[0018]
【The invention's effect】
According to the present invention, there is provided an enzyme capable of improving the low pH environment of compost, which has high-performance protein resolution even in a low pH range, and can generate ammonia, if not directly, to improve the low pH environment of compost. And a method for producing a carboxyl protease using the microorganism are provided.
[0019]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the working pH and optimum pH of the enzyme of the present invention.
FIG. 2 is a graph showing the pH stability of the enzyme of the present invention.
FIG. 3 is a graph showing the working temperature and optimum temperature of the enzyme of the present invention.
FIG. 4 is a graph showing the temperature stability of the enzyme of the present invention.
FIG. 5 is a diagram showing SDS-PAGE and molecular weight calculation of the enzyme of the present invention.
FIG. 6 is a graph showing the influence of an inhibitor on the enzyme of the present invention.
FIG. 7 is a diagram showing the growth of the microorganism of the present invention.
FIG. 8 shows the protease activity of the enzyme produced by the microorganism of the present invention.
FIG. 9 is a diagram showing the pH adjusting ability given to the microorganism culture medium of the present invention.
FIG. 10 is a diagram showing a purification process (ion exchange chromatogram by DEAE-Sepharose CL-6B) of the enzyme of the present invention.
FIG. 11 is a diagram showing a purification process (gel filtration chromatogram by Sephadex G-75) of the enzyme of the present invention.
FIG. 12 summarizes a series of purification steps of the enzyme of the present invention.
FIG. 13 is a diagram showing enzymological properties of the enzyme of the present invention (comparison with other microorganism-derived carboxyl proteases).

Claims (3)

Burkholderia sp. MB-11, produced from a microorganism deposited as FERM P-16949, with SEQ ID NO: 1 (N-terminal fragment) and SEQ ID NO: 2 (intermediate fragment) Carboxyl protease MB11 having the following enzymatic properties (1) to (8), comprising the amino acid sequence represented :
(1) The substrate-specific casein is well hydrolyzed and acts on hemoglobin.
(2) Working pH and optimum pH
It operates at least at pH 2-6, and the optimum pH is about 3-4. It retains 20-30% of the maximum activity value at pH 5, and 10-20% of the maximum activity value at pH 2.5.
(3) pH stability It is stable in the pH range of 3 to 8 under the treatment conditions of 4 ° C. and 12 hours.
(4) Working temperature and optimum temperature The working temperature is at least 10 to 80 ° C, and the optimum temperature is 50 to 60 ° C. About 55-65% of activity at optimum temperature at 40 ° C, about 20-30% of activity at optimum temperature at 30 ° C, about 60-70% of activity at optimum temperature at 70 ° C, activity at optimum temperature at 80 ° C Retains about 30-40% activity.
(5) Temperature stability pH 5.8, stable to at least about 50-60 ° C. under 10 minute processing conditions.
(6) Molecular weight The estimated molecular weight estimated by 10% SDS-PAGE is about 36,000.
(7) The isoelectric point is 4.2.
(8) It is not inhibited by the inhibitors pepstatin, diazoacetylnorleucine methyl ester and 1,2-epoxy-3- (p-nitrophenoxy) propane.   The microorganism producing the carboxyl protease MB11 according to claim 1, which was named Burkholderia sp. MB-11 and was commissioned as FERM P-16949. A method for producing carboxyl protease MB11, comprising culturing the microorganism according to claim 2 in a medium and collecting the carboxyl protease MB11 according to claim 1 from the culture.

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