JP4137526B2 - Keratinase and production method thereof - Google Patents

Description

【図面の簡単な説明】
【図１】図１は、本発明によるアルカリプロテアーゼの最適pHを示すグラフである。
【図２】図２は、本発明によるアルカリプロテアーゼの安定pHを示すグラフである。
【図３】図３は、本発明によるアルカリプロテアーゼの最適温度を示すグラフである。
【図４】図４は、本発明によるアルカリプロテアーゼの安定温度を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel alkaline protease having high keratin resolution, a method for producing the same, and a novel alkaliphilic actinomycetes having the ability to produce the alkaline protease.
[0002]
[Prior art]
Alkaline protease is an enzyme that specifically hydrolyzes peptide bonds of proteins in the alkaline region, and is widely used in food, fiber, leather, detergent and other industries. Such an alkaline protease is known to be widely produced by microorganisms such as filamentous fungi, yeast, and bacteria, and is also produced by a group of microorganisms called so-called alkaliphilic microorganisms.
[0003]
As the alkaline proteases produced from the alkaliphilic microorganisms described above, there are already many enzymes obtained from so-called alkaliphilic Bacillus bacteria (for example, JP 7-63366, JP 7-63367, JP 7-63368, etc.). It is known and is being developed mainly for detergents. In addition, as thermostable enzymes obtained from the same alkaliphilic Bacillus genus, alkaline protease produced by AH-101 strain (JP-A-2-55087) and alkaline protease produced by B18-1 strain (Japanese Patent Publication No. 7-63368) Etc. are known. However, little is known about proteases produced by alkaliphilic actinomycetes, which are the same alkaliphilic microorganisms, and a few enzymes from the genus Streptomyces (Agr. Biol. Chem., 38 , (1), 37- 44, 1974), alkaline protease produced by Thermoactinomyces sp. HS682 (Biosci. Biotech. Biochem., 56 , (2), 246-250, 1992), and alkali produced by Nocardiopsis Dassonvillei OPC-210 Only proteases (J. Appl. Bacteriol., 69 , 520-529, 1990) have been reported.
[0004]
On the other hand, when protease is applied to detergents, it has been pointed out that an enzyme that works well against insoluble proteins such as keratin is desirable (Minakawa, Motoshi: 26 , 322, 1985). In addition, when protease is applied to a detergent for a bathtub, bath tub, bath bed drain, circulation bathtub piping, toilet bowl or vanity drain, it retains sufficient activity at medium temperature around 30 ° C, The ability to strongly decompose insoluble proteins such as keratin such as dirt or dirt is required. In addition, since the pH of current detergents and cleaning agents is in the alkaline region due to the compositional composition, alkaline protease is the optimum enzyme to be blended with them.
[0005]
Furthermore, hair, feathers, and the like whose main protein is keratin are important as raw materials for producing cysteine, an amino acid that cannot be chemically synthesized. However, under the present circumstances, since a significant amount of cysteine is decomposed due to the extreme reaction conditions and the yield is extremely poor, a mild hydrolysis method such as protease treatment has been desired. Furthermore, cows, buffalo horns, and feathers that are discarded in large quantities as unused resources in developing countries are composed of useful amino acids. If peptide solutions and amino acid solutions can be produced by mild hydrolysis, It is extremely useful for these countries as a pharmaceutical or nutritional supplement. In these cases, alkaline protease is the most suitable enzyme as the hydrolyzing agent because of the property that keratin swells in a highly alkaline region and is easily affected by the enzyme.
[0006]
In addition to these applications, detergents such as kitchen detergents, dishwasher detergents and house detergents, hair removal of fur (raw leather), refining of silk, wool and leather (improving texture), hair removal creams, bathing agents and contact lenses Alkaline proteases that are excellent in degradability of insoluble proteins are extremely useful for quasi-drugs such as detergents, food processing such as meat softeners, medicines, and reagents.
[0007]
For such applications, conventional alkaline proteases are inadequate particularly in terms of degrading ability of insoluble proteins such as keratin, and development of a novel alkaline protease having stronger keratin degrading ability has been desired.
[0008]
From such a viewpoint, the present inventors have already discovered a novel alkaline protease and have applied for a patent (Japanese Patent Laid-Open No. 2000-060547). This alkaline protease has a low molecular weight and an excellent proteolytic activity. However, the productivity of this enzyme varies greatly from culture to culture, and the enzyme becomes unstable rapidly at pH 10.0 or higher. Therefore, it is desired to develop an alkaline protease capable of stably producing an enzyme and stable even in a high alkaline region.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel alkaline protease having excellent keratin resolution and stability in a high alkaline region.
Another object of the present invention is to provide a novel microorganism that produces the alkaline protease, and a stable production method of the alkaline protease using the microorganism.
[0010]
[Means for Solving the Problems]
Under the circumstances as described above, the inventors searched for microorganisms producing alkaline protease having strong keratin degradability mainly focusing on alkalophilic actinomycetes. As a result, one strain of actinomycetes belonging to the genus alkalophilic Nocardiopsis However, it has been found that the desired novel alkaline protease can be produced efficiently and stably by aerobic culture, and the present invention has been completed.
[0011]
That is, the present invention provides a novel alkaline protease having the following physicochemical properties.
[0012]
(a) Action and substrate specificity: acts on proteins and peptides and cleaves their peptide bonds by an endo-type mechanism to produce low molecular weight oligopeptides and amino acids. In addition, it exhibits strong activity against insoluble proteins such as keratin.
(b) Stable pH: Stable at pH 1.5 to 12.0 under treatment conditions of 30 ° C. for 24 hours.
(c) Molecular weight: About 20,000 by SDS electrophoresis, and average molecular weight from amino acid sequence is 19,150.
(d) Isoelectric point: 10.0 or more (isoelectric focusing method).
(e) Specific activity: about 1,100 (PU / mg protein) when casein is used as a substrate and about 3,300 (KU / mg protein) when keratin is used as a substrate.
[0013]
The novel alkaline protease production method according to the present invention comprises a step of culturing a microorganism belonging to the genus alkalophilic Nocardiopsis and having the ability to produce the alkaline protease, and the novel alkaline protease from the culture obtained in the step. Comprising the step of separating. Furthermore, the novel strain having the ability to produce the novel alkaline protease according to the present invention is Nocardiopsis sp. TOA-1 strain (FERM P-18676).
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The alkaline protease provided by the present invention is an enzyme having the following physicochemical properties in addition to the properties (a) to (e).
[0015]
(f) Optimum pH: The optimum pH is 11.0 to 11.5 when casein is used as a substrate, and 12.0 or more when keratin is used as a substrate.
(g) Optimal temperature: The optimal working temperature is 70 to 75 ° C. when casein is used as a substrate, and 65 to 70 ° C. when keratin is used as a substrate.
(h) Stable temperature: Stable up to 60 ° C under the treatment conditions of pH 7.0 and 10 minutes regardless of the addition or absence of calcium.
(i) Inhibition: The activity is not inhibited by EDTA (ethylenediaminetetraacetic acid), but is inhibited by PMSF (phenylmethanesulfonyl fluoride) and SSI (Streptomyces subtilisin inhibitor).
[0016]
The alkaline protease according to the present invention strongly hydrolyzes not only soluble proteins such as casein but also insoluble proteins such as keratin that are difficult to be degraded by conventional proteases. Therefore, it is added to detergents and softeners for clothes and various detergents for the purpose of enhancing the cleaning effect, and is added to clogging removers such as bathtubs, bathtubs, bathroom drains, circulation bathtub pipes, toilets, and vanity drains. Is extremely effective. Furthermore, it can also be applied to the production of amino acids such as cysteine and other peptides from hair, feathers, cow horn and the like containing keratin as the main protein. Further, it is possible to perform fur removal (raw leather), refining silk, wool, leather, etc. (improving the texture) under milder conditions. Furthermore, it can be used in an extremely wide range of industrial fields such as chemicals such as hair removal creams, bathing agents, meat softeners, medicines, reagents and the like. In addition, since the strain according to the present invention efficiently secretes the alkaline protease outside the cells, it is advantageous in that it can be produced efficiently with a simple process.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
The novel alkaline protease according to the present invention can be produced using microorganisms. Particularly preferably, the alkaline protease according to the invention is produced by the alkalophilic Nocardiopsis genus, in particular the Nocardiopsis sp. TOA-1 strain. This strain was isolated from a general house in Chigasaki City, Kanagawa Prefecture by the inventors. This strain is an alkalophilic actinomycete and has the following mycological properties. Since this strain is an alkaliphilic microorganism and does not grow in normal neutral medium or grows very poorly, an alkaline medium supplemented with 1.0% sodium carbonate is used for the examination of the following mycological properties. It was.
[0018]
Morphological properties
1) Branching method and morphology of spore-forming mycelium: simple branching / straight
2) Spore formation method: aerial hyphae break up and chain
3) Spore morphology and size: Straw-shaped, smooth, about 0.5 μm x 1.0 μm
4) Presence or absence of flagella: None
5) Presence or absence of spores: None [0019]
Physiological properties
1) Growth temperature range / pH: 15-40 ° C / pH 7.5-13
2) Gelatin liquefaction: Yes (liquefaction in 3 days)
3) Starch hydrolysis: Hydrolyzes
4) Coagulation and peptization of defatted milk: No coagulation: Peptone in 4 days
5) Generation of melanin-like pigment: Not generated
6) Utilization of each carbon source (+, assimilate;-, not assimilate)
a) L-arabinose: +
b) D-xylose: +
c) D-glucose: +
d) D-fructose: +
e) Sucrose: +
f) Inositol: +
g) L-rhamnose: +
h) Raffinose: +
i) D-man knit: +
7) Growth status in each medium (growth status, village surface color, village back surface color, diffusion dye)
a) Sucrose nitrate agar medium: good, white, colorless, light skin to pink
b) Glucose / asparagine agar medium: good, white, colorless, none
c) Glycerin / asparagine agar medium: good, white, colorless, none
d) Starch / inorganic salt agar medium: good, white, colorless, light brown
e) Tyrosine agar medium: good, white, colorless, light brown, no melanin
f) Nutrient agar medium: good, white, colorless, no
g) Yeast / malt agar medium: good, white, colorless, no
h) Oatmeal agar medium: good, white, colorless, light skin to pink [0020]
This strain TOA-1 has the above-mentioned mycological properties, in particular, morphological characteristics of actinomycetes. Therefore, the genus was searched according to “Classification and Identification of Actinomycetes” (Japan Actinomycete Society, 2001). First, since this strain contains only meso-diaminopimelic acid in the cell wall, it is considered that this strain belongs to a genus other than Streptomyces and Kineosporia. In addition, although this strain had little fragmentation of the basic hyphae and zigzag hyphae, the aerial hyphae fragmentation was vigorous and was considered to be a nocardio-form actinomycete.
[0021]
Therefore, 16S rDNA of this strain was prepared according to the adult book (same as above), and the homology with each actinomycete was searched. As a result, the strain was found to have 94.7-97.6% homology with each species of the genus Nocardiopsis, but no completely identical species was found. The most homologous was Nocardiopsis alba. Moreover, it was 96.7% with Nocardiopsis dasson belay, which is alkalophilic like this strain, and the homology among Nocardiopsis was not so high.
[0022]
In other words, considering that this strain TOA-1 is alkalophilic, etc., it is judged that it is a close relative to Nocardiopsis alba and named Nocardiopsis sp. TOA-1 (Nocardiopsis sp. TOA-1) did. This strain has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-18676.
[0023]
Culturing conditions Since the above strain is an alkalophilic actinomycete, it is necessary to perform the culturing in an alkaline region. The method for making the medium alkaline is not particularly required, and for example, sodium carbonate or sodium hydrogen carbonate may be added to a normal medium. As a carbon source, glucose, soluble starch, monosaccharides such as cellulose, polysaccharides and the like can be used. As a nitrogen source, urea, peptone, dry yeast, yeast extract, skim milk, soybean flour, corn steep liquor, casein, meat extract, amino acid, and the like are used as well as inorganic substances such as nitrate and ammonium salt. In addition to these carbon source and nitrogen source, various inorganic salts such as magnesium salt, potassium salt, sodium salt, phosphate and the like may be added as necessary. As an alkali source to be added to the medium, about 0.5 to 2.0% of sodium carbonate, sodium hydrogen carbonate or the like carbonate, sodium hydroxide, ammonia or the like can be used, and the pH of the medium is preferably about 8.0 to 11.0. Culturing is performed in such a medium at a culture temperature of 20 to 40 ° C., preferably 30 to 35 ° C. for 2 to 7 days under aerobic stirring or shaking. The novel alkaline protease according to the present invention is mainly secreted and accumulated in the culture medium under the culture conditions as described above.
[0024]
Collection of enzyme In order to collect and purify the enzyme according to the present invention from the culture solution, known purification methods can be used alone or in combination. Since this enzyme is mainly secreted outside the bacterial cells (in the culture solution), a crude enzyme solution can be easily obtained by removing the bacterial cells by, for example, filtration or centrifugation. This crude enzyme is further purified by known purification methods such as salting out with ammonium sulfate, etc .; precipitation with organic solvents such as methanol, ethanol and acetone; adsorption method with keratin etc .; ultrafiltration; gel filtration chromatography; It can be purified by hydrophobic chromatography and other various chromatographies alone or in combination.
[0025]
A preferred purification method is as follows. First, 80% saturated ammonium sulfate is added to the culture filtrate for salting out, and the resulting precipitate is dissolved in a buffer solution. Subsequently, by performing ion exchange chromatography using CM-Toyopearl 650M (manufactured by Tosoh Corporation) and DEAE-Toyopearl 650M (manufactured by the same company), a purified enzyme uniform by SDS electrophoresis can be obtained.
[0026]
Properties of the enzyme The properties of the alkaline protease according to the present invention are as follows. In the following, the activity measurement method refers to the following method.
[0027]
Protease activity measurement method 1.9 ml of 50 mM glycine / NaCl / NaOH buffer solution (pH 11.0) containing 0.6% casein is mixed with 0.1 ml of enzyme solution, reacted at 30 ° C for 10 minutes, and then 2 ml of 0.11 M trichloroacetic acid. After adding the solution and allowing to stand at 30 ° C. for 30 minutes, the solution is filtered with a filter paper N0.5C manufactured by Advantech. Next, 0.5 ml of this filtrate was added to 2.5 ml of 0.5M sodium carbonate solution, 0.5 ml of a further 3-fold diluted phenol reagent was added, and the mixture was stirred and then allowed to stand at room temperature for 30 minutes to obtain an absorbance at 660 nm. taking measurement. The amount of enzyme that increases the absorbance corresponding to 1 μg of tyrosine per minute under the above measurement conditions is defined as one unit of protease activity (1 PU).
[0028]
Keratin degradation activity assay
Add 2.9 ml of 50 mM glycine / NaCl / NaOH buffer (pH 12.0) to 60 mg of keratin powder (Tokyo Kasei), add 0.1 ml of enzyme solution, and shake at 30 ° C for 1 hour (120 rpm). React. Next, 2.5 ml of 0.11M trichloroacetic acid is added to stop the reaction, and the mixture is allowed to stand at 30 ° C. for 30 minutes, followed by filtration through Advantech filter paper NO.5C. Add 0.5 ml of 0.5 M sodium carbonate solution to 0.5 ml of the filtrate, add 0.5 ml of a 3-fold diluted phenol reagent, and then stir for 30 minutes at room temperature, and measure the absorbance at 660 nm. The amount of enzyme that increases the absorbance corresponding to 1 μg of tyrosine in 10 minutes under the above measurement conditions is defined as 1 unit (1KU) of keratin degradation activity.
[0029]
(1) Action and substrate specificity Acts on proteins and peptides and cleaves the peptide bonds by endo-type mechanism to produce low molecular weight oligopeptides and amino acids. In addition, it exhibits strong activity against insoluble proteins such as keratin.
[0030]
(2) Optimum pH and stable pH
Based on the above activity measurement method, the effect of pH on this enzyme was examined. As buffers, HCl / KCl (pH 1.0-1.5), glycine / NaCl / HCl (pH 2.0-3.0), acetic acid (pH 4.0-5.0), phosphoric acid (pH 6.0-7.0), Tris-HCl (pH 7.0-9.0), glycine / NaCl / NaOH (pH 9.0-12.0), KCl / NaOH (pH 12.0-13.0) were used. FIG. 1 shows the relative activity at each pH when the maximum value of activity is 100. FIG. 1 shows that the optimum pH of this enzyme is 11.0 to 11.5 when casein is used as a substrate at 30 ° C. and 12.0 or more when keratin is used as a substrate. Similarly, the pH stability of this enzyme is shown in FIG. After the enzyme was kept in a buffer solution at each pH at 30 ° C. for 24 hours, the remaining protease activity was shown as a relative activity with the untreated enzyme activity as 100. FIG. 2 shows that this enzyme is stable in a very wide pH range from pH 1.5 to 12.0 under the above treatment conditions.
[0031]
(3) Optimum temperature and stable temperature According to the above activity measurement method, the influence of temperature on this enzyme was examined. FIG. 3 shows the relative activity at each temperature when the maximum activity is 100. FIG. 3 shows that the optimum temperature of this enzyme is 70 to 75 ° C. when casein is used as a substrate, and 65 to 70 ° C. when keratin is used as a substrate. Further, this enzyme was added to 100 mM Tris-HCl buffer (pH 7.0) and held for 10 minutes under each condition in the range of 40 to 80 ° C., and then the remaining protease activity was measured. The results are shown in FIG. FIG. 4 shows that this enzyme is stable up to 60 ° C. In addition, regarding the temperature stability of this enzyme, the effect of calcium addition (10 mM) was not recognized.
[0032]
(4) Molecular weight The molecular weight of the enzyme was measured by SDS electrophoresis, and the molecular weight was about 20,000. Further, the average molecular weight calculated from the amino acid sequence described in the sequence listing and SEQ ID NO: 2 described later was 19,150.
[0033]
(5) Isoelectric point The isoelectric point of the enzyme was measured by isoelectric focusing, and the isoelectric point was 10.0 or more.
[0034]
(6) Specific activity The specific activity of this enzyme was measured according to the activity measurement method. The protein concentration was calculated by hydrolyzing the enzyme with hydrochloric acid and quantifying the produced amino acid by the ninhydrin method. As the enzyme, an electrophoretically uniform purified sample was used. As a result, the specific activity of this enzyme was about 1,100 (PU / mg protein) when casein was used as a substrate, and about 3,300 (KU / mg protein) when keratin was used as a substrate.
[0035]
(7) Inhibition Regarding the general enzyme inhibitors PMSF (phenylmethanesulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), and SSI (Streptomyces subtilisin inhibitor), the effects of these on the activity of this enzyme Examined. Each inhibitor was dissolved in 50 mM Tris-HCl buffer (pH 9.0) to a predetermined concentration, followed by treatment at 30 ° C. for 30 minutes after the addition of this enzyme. Next, a fixed amount was taken from the treatment solution, and the residual activity was measured according to the activity measurement method. As a result, this enzyme was inhibited by PMSF and SSI, and not inhibited by EDTA. From this, it was found that the alkaline protease is a serine protease.
[0036]
(8) Amino terminal sequence The amino terminal sequence of this enzyme was determined using a gas phase protein sequencer (manufactured by Shimadzu Corporation, PPSQ-21). The sequence from the amino terminus of the enzyme to the 25th position is shown below.
Ala-Asp-Ile-Ile-Gly-Gly-Leu-Ala-Tyr-Thr-Met-Gly-Gly-Arg-Cys-Ser-Val-Gly-Phe-Ala-Ala-Thr-Asn-Ala-Ser
[0037]
(9) Base sequence and amino acid sequence documents (for example, J. Sambrook, EF Fritsch, T. Maniatis: Molecular Cloning. A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory Press, 1989, etc.) and equipment and reagents used The gene and amino acid sequences of this enzyme were determined according to a protocol such as a kit. First, the internal sequence of this enzyme was determined for the purpose of designing primers. The purified enzyme was treated with urea and then decomposed with lysyl endopeptidase (Wako Pure Chemical Industries), and the amino acid sequence of the resulting fragment was determined with a gas phase protein sequencer. Two types of oligonucleotide primers were synthesized by the phosphoramidite method from the appropriate internal sequence and amino terminal sequence obtained here. The gene was amplified by PCR (Biometra, T-Gradient Thermoblock 050-801) using this primer. As a result, a specific amplified fragment was observed around 0.5 kbp. Using this fragment as a probe, a full-length gene encoding this enzyme was screened from the genomic library of this strain TOA-1. The DNA sequencer based on the dideoxy method (F. Sanger et al., Proc. Natl. Acad. Sci., 74 , 5463-5467, 1977) was used as the principle of the base sequence of the obtained clone (LICOR, LICOR-4000) Determined by. The base sequence (564 bp) is shown in Sequence Listing, SEQ ID NO: 1. Further, the amino acid sequence (188 amino acids) was determined based on the base sequence, and the amino acid sequence is shown in Sequence Listing, SEQ ID NO: 2.
[0038]
【Example】
Next, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
[0039]
Example 1 Preparation of Crude Enzyme Powder Nocardiopsis SP TOA-1 strain 50 ml of pre-culture solution (30 ° C, shaking culture for 3 days), skim milk 0.5%, yeast extract 0.1%, and carbonate added after further sterilization A small jar fermenter containing 4000 ml of a medium containing 1.0% sodium (pH 10.5) was inoculated and cultured at 30 ° C. for 3 days at an aeration rate of 1 v / v / min and a rotation speed of 200 rpm. After completion of the culture, the culture was centrifuged at 8000 rpm for 10 minutes to remove the cells. As a result, approximately 3,800 ml of a 30 PU / ml crude enzyme solution was obtained.
[0040]
Example 2 Preparation of Purified Enzyme A small jar fermenter containing 4000 ml of the same medium as in Example 1 was inoculated with 50 ml of Nocardiopsis sp. TOA-1 preculture. This was cultured in the same manner as in Example 1, and then 3,790 ml of culture supernatant was obtained by centrifugation. The protease activity of this supernatant at pH 11.0 was 29 PU / ml. Next, ammonium sulfate powder was added to the supernatant until 80% saturation, and the mixture was left standing in the dark at 5 ° C. for 1 day and then centrifuged at 8000 rpm to collect the precipitate. This precipitate was dissolved in 10 mM phosphate buffer (pH 7.0) and dialyzed against the same buffer using a cellulose tube. After dialysis, the enzyme was adsorbed through a CM-Toyopearl 650M column equilibrated with 10 mM MOPS buffer (pH 7.5) and eluted with a NaCl concentration gradient of 0 to 0.5M. After dialysis of the active fraction, the active fraction was passed through a DEAE-Toyopearl 650M column equilibrated with 10 mM Tris-HCl buffer (pH 9.0) to adsorb the impure protein. By the above series of purification, 17 ml of 1940 PU / ml purified alkaline protease (specific activity: 1,100 PU / mg) was obtained, and the recovery rate of enzyme activity was 30%. This fraction showed a single band by SDS electrophoresis and was confirmed to be uniform as an enzyme protein.
[0041]
Example 3 Enzyme Productivity Using the same medium as in Example 1, shake culture using a flask and culture with a small jar fermenter were performed, and enzyme productivity for each culture batch was compared. First, 100 ml of medium was placed in a 500 ml shoulder flask, one platinum loop of TOA-1 strain spores was inoculated from the agar medium, and cultured with shaking at 30 ° C. and 130 rpm for 4 days. This culture was repeated 10 times, and the protease activity was measured for each culture solution in the flask. Separately, five cultures using the same small jar fermenter as in Example 1 were carried out, and the protease activity for each batch was measured. As a result, it was 38 to 41 PU / ml in the flask culture and 28 to 33 PU / ml in the small jar fermenter culture, and there was almost no variation in activity depending on the culture batch. From this, it was found that the productivity of this enzyme was stable.
[0042]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the optimum pH of an alkaline protease according to the present invention.
FIG. 2 is a graph showing the stable pH of the alkaline protease according to the present invention.
FIG. 3 is a graph showing the optimum temperature of the alkaline protease according to the present invention.
FIG. 4 is a graph showing the stable temperature of the alkaline protease according to the present invention.

Claims (9)

A keratinase having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having the following physicochemical properties:
(A) Action and substrate specificity: Acts on keratin and cleaves its peptide bond by an endo-type mechanism to produce low molecular weight oligopeptides and amino acids .
(B) Stable pH: Stable at pH 1.5 to 12.0 under the treatment conditions of 30 ° C. and 24 hours.
(C) molecular weight: about 20,000 in SDS electrophoresis, minutes Koryou from the amino acid sequence is 19,150.
(D) Isoelectric point: 10.0 ( isoelectric focusing method).
(E) Specific activity: about 1,100 (PU / mg protein) when casein is used as a substrate and about 3,300 (KU / mg protein) when keratin is used as a substrate. Furthermore, the keratinase of Claim 1 which has the following physicochemical property.
(F) optimum pH: optimum pH is when the casein as a substrate is 11.0 to 11.5, if you keratin as a substrate, the measurement range of pH 8 ~ 12, is 12.0.
(G) Optimal temperature: The optimal action temperature is 70 to 75 ° C. when casein is used as a substrate, and 65 to 70 ° C. when keratin is used as a substrate.
(H) Stable temperature: Stable up to 60 ° C. under the treatment conditions of pH 7.0 and 10 minutes irrespective of the addition or absence of calcium
(I) Inhibition: The activity is not inhibited by EDTA (ethylenediaminetetraacetic acid), but is inhibited by PMSF (phenylmethanesulfonyl fluoride) and SSI (Streptomyces subtilisin inhibitor). The gene shown in the sequence table and SEQ ID NO: 1 encoding the keratinase according to claim 1 or 2 . The keratinase according to any one of claims 1 to 3, which is obtained from an alkalophilic actinomycete Nocardiopsis genus. The keratinase according to claim 4, which is obtained from Nocardiopsis sp. TOA-1 strain ( FERM P-18676 ) . A method for producing keratinase according to any one of claims 1 to 5, comprising the steps of culturing Nocardioides Opsys sp (Nocardiopsis sp.) TOA-1 strain (FERM P-18676), obtained in about該工preparation keratinase comprising the step of separating the keratinase from obtained culture product. The method for producing keratinase according to claim 6, wherein the microorganism is cultured at a culture temperature of 15 to 35 ° C in an alkaline pH of 8.0 to 11.0. An alkaliphilic actinomycete Nocardiopsis sp. TOA-1 strain (FERM P-18676). A method for using an enzyme having the amino acid sequence shown in SEQ ID NO: 2 for degrading keratin.

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