JP2843777B2 - New protease NH and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a novel protease NH obtained by culturing a strain of the genus nas) and a method for producing the same. More specifically, the present invention relates to a novel protease NH having a high modifying effect on keratin protein-based fiber products represented by wool and a method for producing the same.

[0002]

2. Description of the Related Art Proteases are widely used not only in pharmaceuticals but also in fields such as detergents, food processing, and the leather industry. In recent years, research on the modification of keratin protein-based fiber products, particularly wool fibers, using this protease has been actively conducted, and with this, a commercially available enzyme capable of efficiently and effectively modifying wool fiber products. Selection and reports on wool processing methods and their effects (Kitano et al .: Textiles & Fashion, Vol. 6,
No.1, 13-34) (Osaka Institute of Industrial Science, Advanced Technology Joint Research 1990 (Heisei 2) Result Report C-70).

[0003]

However, the conventional protease was not developed for modifying keratin protein-based fiber products, but is an enzyme originally developed as a cleaning agent for food processing and protein stains. At present, this is diverted to fiber modification. Enzymes are biocatalysts with high substrate specificity.For example, even a protease having a high effect of degrading milk casein protein cannot necessarily degrade keratin protein similarly, and in many commercial proteases, keratin Activity on proteins is not satisfactory. In order to improve textile products using these commercially available proteases and to expect remarkable effects, it is necessary to treat textile products for a long time with a large amount of protease. Is expensive and its processing cost is high, so that it cannot be used in large quantities for fiber modification. Therefore, development of a protease specific to keratin protein and development of a mass production method for supplying it at low cost have been expected.

An object of the present invention is to provide a novel keratin protein-specific protease capable of efficiently modifying keratin protein fiber products. Another object of the present invention is to provide a method for producing a protease specific to a keratin protein in large quantities at low cost.

[0005]

Means for Solving the Problems In order to achieve the above objects, the present inventors have widely searched for a keratin protein-specific protease-producing strain from nature. as a result,
It was found that one strain of the genus Aeromonas isolated from industrial wastewater collected in Aichi Prefecture degraded keratin proteins well and produced a large amount of extracellular proteases. In addition, as a result of conducting various studies on the protease, it was found that the enzyme was composed of at least two components having protease activity by gel filtration and polyacrylamide gel electrophoresis. Then, as a result of separating and purifying these components, the protease NH was successfully obtained as a single component electrophoretically, thereby completing the present invention.

That is, the novel protease NH of the present invention comprises:
It has the following enzymatic properties: (1) Action: Hydrolyze various proteins to produce oligopeptides or amino acids. (2) Substrate specificity: It has good activity on soluble proteins such as casein and insoluble proteins such as keratin. (3) Optimum pH: When the reaction is carried out at 37 ° C. using casein as a substrate, the optimum pH is 7.0 to 9.0. (4) pH stability: 5 at various pHs using casein as a substrate
When treated at 5 ° C for 20 minutes, it is extremely stable in the pH range of 4.5 to 9.5. (5) Optimum temperature: When the reaction is performed at pH 7.5 using casein as a substrate, the optimum temperature is 50 to 60 ° C. (6) Heat resistance: casein as a substrate, pH 7.5, 40
When treated at 10 ° C. for 10 minutes, or when treated with 5 mM Ca 2+ at 50 ° C. for 10 minutes, it has a residual activity of 90% or more. (7) Effect of inhibitor: diisopropylfluorophosphate (D
FP), phenylmethanesulfonyl fluoride (PM
SF), parachloromercury benzoic acid (PCMB)
Does not inhibit activity. Ethylenediaminetetraacetic acid (ED
TA) inhibits the activity. (8) Effect of metal ion: When casein is used as substrate, C
The activity is inhibited by u2 +. Protease NH, which has lost its activity due to ethylenediaminetetraacetic acid (EDTA), is reactivated by Zn2 +, Ca2 +, Mn2 +, and Co2 +. (9) Molecular weight: about 34,500 (by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis).

[0007] The method for producing a novel protease NH of the present invention is obtained by culturing a microorganism belonging to the genus Aeromonas which produces a protease specific to keratin protein and secretes the protease out of the cells. In this method, protease is separated and obtained from the culture solution or culture supernatant.

Hereinafter, the present invention will be described in detail. The bacteriological properties of the isolate producing the protease of the present invention are described below. A. Morphological properties 1) Cell shape and size: 0.5 × 1.5-2.0 μm bacilli 2) Motility: Yes (with flagella) 3) Spores: Not formed 4) Gram stain: Negative Physiological properties 1) Oxidase: + 2) Catalase: + 3) Production of soluble brown pigment: -4) OF test: Fermentability 5) Acid production from glucose: + 6) Production of indole: + 7) MR Test: -8) VP test: +9) Utilization of citric acid: +10) Production of hydrogen sulfide: +11) Urease: -12) Arginine dehydrase: +13) Liquefaction of gelatin: +14) Hydrolysis of starch Degradation: +15) DNA hydrolysis: +16) Formation of yellow pigment: -17) Growth at 37 ° C: + W 18) Hydrolysis of esculin: +19) Reduction of nitrate: +20) Hydrolysis of Tween 80: +21) Production of gas from glucose: +22) Production of 2-keto-gluconic acid: +23) Production of 3-keto-lactic acid: -24) Production of acid and gas from sugars Salicin, sucrose, D- Mannitol,: + tray Loose, fructose, glucose, D-galactose, maltose, dextrin, L-arabinose D-xylose, D-arabinose, D-sorbitol,-: raffinose, inulin, lactose, p-hydroxybenzoic acid 25) Use of organic acids L- Histidine, L-arginine, L-glutamic acid: + L-aspartic acid, succinic acid, malonic acid, L-leucine, L-tyrosine adipic acid:-From the above mycological properties, BERGEY'S MANUAL of Systemati
c Based on Bacteriology Volume 1, this strain is considered to belong to Aeromonas hydrophila. Therefore, this strain was named Aeromonas hydrophila WPH22 and deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. The deposit number is FERM P-14748. The microorganism used in the present invention is the aforementioned Aeromonas hydrophila (Ae
romonas hydrophila) WPH22 strain (FEW deposited FE)
RM P-14748) and Aeromona (Aeromona
s) Any microorganism that belongs to the genus and that produces the protease of the present invention can be used.

In the present invention, the medium for producing protease may be any medium used for culturing ordinary microorganisms, and is not particularly limited as long as the microorganism to be used grows. As the carbon source, for example, starch, gelatin, dextrin, glucose, sucrose and the like can be used. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate and urea, and organic nitrogen sources such as polypeptone, yeast extract, meat extract, and soybean powder. In addition to a carbon source and a nitrogen source, an inorganic salt such as a magnesium salt, a phosphate, or a calcium salt can be added to the medium. The culture temperature may be any temperature suitable for the growth of the microorganism used, and is preferably adjusted to 20 to 35 ° C.
The pH of the medium used is preferably 6.5 to 9.0.
The culturing time is continued for a time sufficient for the growth of the bacterium and the production of the protease, but usually the protease is accumulated in the culture solution by culturing for 24-72 hours. After culturing in this way, the culture solution is centrifuged or filtered to remove cells and insolubles, thereby obtaining a crude enzyme solution. Protease is obtained from the crude enzyme solution by ammonium sulfate precipitation or solvent precipitation. The enzyme having improved specific activity may be used as it is or by appropriately combining ordinary enzyme purification means such as dialysis and column chromatography.

Next, an example of a method for separating and purifying the protease of the present invention will be described. First, the microorganism culture solution is centrifuged to obtain a supernatant from which the cells have been removed. The supernatant was subjected to ammonium sulfate salting out, and the resulting precipitate was washed with Tris-HCl buffer (pH
Dissolve in 7.5). Next, this solution is subjected to gel filtration chromatography, eluted with a Tris-HCl buffer containing sodium chloride, the active fraction is collected, and the obtained active fraction is dialyzed against a Tris-HCl buffer (pH 7.5). The dialysate is subjected to ion exchange chromatography,
Elution is performed with a gradient of M sodium chloride, and the active fraction is collected.

The thus purified protease NH of the present invention has the following enzymatic properties. In the following, the enzyme activity was measured by the following method. [Enzyme activity measurement method] 1 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 0.6% (w / v) casein (HAMMARSTEN; manufactured by Merck) was incubated at 37 ° C for 20 minutes, and then 0.1 mM The enzyme solution was added to start the reaction. 37
After reacting at 10 ° C. for 10 minutes, a mixed solution of trichloroacetic acid (0.
11 M trichloroacetic acid, 0.22 M sodium acetate,
The reaction was stopped by adding 1 ml of 0.33 M acetic acid) and left at room temperature for about 20 minutes. After standing, the precipitate formed by centrifugation was removed, 5.0 ml of a 0.5 M sodium carbonate solution was added to 1.0 ml of the filtrate, 1.0 ml of a 2-fold diluted Foreign reagent was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes. Then, the absorbance at 660 nm is measured. 1 under the above conditions
The amount of enzyme that liberates 1 μmol of tyrosine per minute was defined as 1 unit (U).

(1) Action Various proteins such as casein, soybean protein and keratin are hydrolyzed.

(2) Substrate Specificity The specificity of protease NH for various substrates was compared with other commercially available proteases. The substrates used were casein,
Skim milk, soybean-derived casein, animal hair keratin, and collagen. 0.05M Tris-HCl buffer (pH
To 7.5), 1% of each substrate was added, 0.5 U of the enzyme solution was added, and the reaction was carried out at 40 ° C. for 30 minutes. Protease NH
Table 1 shows the relative activities of each of the substrates with the activity of casein as 100.

[0014]

[Table 1]

As is apparent from Table 1, protease N
H showed superior degradability, especially to animal hair keratin, as compared to a commercially available protease which is said to be effective for fiber modification. However, it did not show any degradability for collagen.

(3) Optimum pH 7.7 U of protease NH is added to 1 ml of Britton-Robinson's wide-range buffer containing 0.6% casein,
The reaction was performed for 0 minutes, and the activity at each pH was measured. FIG. 1 shows the relative activity at each pH, where the activity at the optimum pH is 100. As a result, the optimal pH of the protease NH was 7.0 to 9.0, and furthermore, p
H had an activity of 60% or more in the range of 6.0 to 10.0.

(4) pH stability 2 mM CaC was added to the above-mentioned Britton-Robinson buffer.
The system to which l2 was added and the system to which no l2 was added were respectively prepared, and 4.69 U of protease NH was added thereto.
Left for 0 minutes. The activity of this treated solution was measured at 37 ° C. in a 0.05 M Tris-HCl buffer (pH 7.5).
This activity is shown in FIG. 2 as a relative activity when the enzyme activity at pH 7.5 before treatment was set to 100. As a result, C
When aCl2 was not added, the pH was in the range of 5.0 to 9.0.
When added, it was stable in the pH range of 4.0 to 10.0.

(5) Optimum temperature 5.35 U of protease NH was added to 0.05 M Tris-HCl buffer (pH 7.5) containing 0.6% casein as a substrate, and the mixture was heated at a temperature of 20 to 80 ° C. for 10 minutes. The reaction was performed, the activity was measured, and the optimum temperature was determined. FIG. 3 shows the relative activity at each temperature, where the activity at the optimum temperature is 100. As a result, the optimum temperature was 60 ° C. Also 4
High activity was exhibited in the range of 0 to 70 ° C.

(6) Heat resistance 5 mM in 0.05 M Tris-HCl buffer (pH 7.5)
5. A system to which CaCl2 was added and a system to which CaCl2 was not added were adjusted.
00 U of protease NH was added, and treatment was performed at each temperature of 20 to 80 ° C for 10 minutes. After quenching in ice water, 37
It was determined by measuring the activity at a temperature of 7.5 ° C. and a pH of 7.5. FIG. 4 shows the relative activity at each temperature treatment when the activity at pH 7.5 before the treatment was set to 100. As a result, CaC
In the system without the addition of l2, the activity of 85% or more was maintained up to 50 ° C. The activity of the 50 mM to 70 ° C. treatment with the 5 mM CaCl 2 added system was improved by about 5% compared with the non-added system.

(7) Influence of Inhibitor 7.0 in 0.02 M Tris-HCl buffer (pH 7.5)
2 U of protease NH was added, various inhibitors were added to predetermined concentrations, and the mixture was treated at 30 ° C. for 30 minutes. Thereafter, the mixture was reacted at 37 ° C. for 10 minutes with a 0.05 M Tris-HCl buffer (pH 7.5) containing 0.6% casein as a substrate, and the residual activity was measured. Table 2 shows the relative activity when each inhibitor was added, where the activity when no inhibitor was added was 100.

[0021]

[Table 2]

In Table 2, EDTA means ethylenediaminetetraacetic acid, DFP means diisopropylpropylfluorophosphate, PMSF means phenylmethanesulfonyl fluoride, and PCMB means parachloromercury benzoic acid.

As is apparent from Table 2, protease N
H is inhibited by the metalloprotease inhibitors ethylenediaminetetraacetic acid (EDTA) and the serine protease inhibitors diisopropylfluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF) and SH
Since no inhibition of the protease inhibitor by parachloromercury benzoic acid (PCMB) was observed,
The protease was presumed to be a metal protease whose activity is controlled by the presence of a divalent cation bound to the enzyme molecule.

(8) Effect of metal ions The effects of various metal ions on protease NH were examined. Various metal salts were added to a 0.02 M Tris-HCl buffer (pH 7.5) to a concentration of 1 mM, 5.40 U of protease NH was added, and the mixture was treated at 30 ° C. for 20 minutes. Thereafter, the mixture was reacted at 37 ° C. for 10 minutes with a 0.05 M Tris-HCl buffer (pH 7.5) containing 0.6% casein as a substrate, and the residual activity was measured. Table 3 shows the relative activity when each metal salt was added, where the activity without metal salt addition was defined as 100.

[0025]

[Table 3]

As apparent from Table 3, protease N
H was about 70% inhibited by Cu2 +. There was almost no inhibition on other metal salts.

(9) Reactivation by Metal Ion In a 4.71 U protease NH solution, 1 m of EDTA was added.
M and treated at 30 ° C. for 60 minutes to inactivate protease activity. Next, various metal salts were added to this treatment solution to a concentration of 2 mM, and treatment was performed at 30 ° C. for 30 minutes. Using the activity of this treatment solution as a substrate, 0.05 M Tris-HCl buffer (pH 7.0) containing 0.6% casein.
The reaction was performed at 37 ° C for 10 minutes in 5), and the measurement was performed. Table 4 shows the relative activities of enzyme reactivation by various metal salts when the enzyme activity without EDTA treatment was taken as 100.

[0028]

[Table 4]

As is clear from Table 4, over 90% was reactivated by Zn2 +. In addition, it was reactivated by 70% or more by Ca2 + and Co2 +. However, Zr2 + and K +
Did not reactivate at all.

(10) Molecular weight The molecular weight of protease NH is determined by using 12.5% polyacrylamide gel sodium dodecyl sulfate (SDS)-
It was measured by polyacrylamide gel electrophoresis. The molecular weight markers include the Electrophoresis Calibration Kit
[Phospholyase b (molecular weight 94,000) bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), carbonic anhydrolase (molecular weight 30,000), soybean trypsin inhibitor (molecular weight 20, 100), and α -A mixed sample of lactalbumin (molecular weight: 14,400): manufactured by Pharmacia] was used. By this method, the molecular weight of protease NH was determined to be about 34,500.

As described above, the properties of protease NH were clarified. It was found that the protease NH of the present invention exhibited high activity in neutral to weak alkaline conditions, and was also excellent in degrading keratin protein. Therefore, the protease NH of the present invention can be said to be an effective protease for modifying keratin protein-based fiber products.

[0032]

Next, embodiments of the present invention will be described in detail, but the following embodiments are merely examples and do not limit the technical scope of the present invention. <Example 1> Production of protease NH First, bactotripton: 10 per liter of tap water
g, yeast extract: 5 g, NaCl: 5 g, and glucose: 1 g were uniformly mixed and prepared at pH 7.2.
-50 ml of broth medium is placed in a 300 ml Erlenmeyer flask, sterilized at 121 ° C for 20 minutes, and then Aeromonas hydrophila WPH.
Twenty-two strains (FERM P-14748, deposited by Micro Engineering Laboratory) were inoculated and pre-cultured at 25 ° C. overnight to obtain a seed culture.

Next, per liter of tap water, polyppetone: 10 g, potassium phosphate: 2 g, magnesium sulfate: 2 g, ammonium sulfate: 0.5 g, zinc sulfate: 0.1 g, and calcium chloride: 0.1 g. One liter of a liquid medium for the production of protease NH of pH 7.5, prepared by uniformly mixing 5 g with 5 g, was placed in a 5 liter Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. This liquid culture medium was inoculated with the above-mentioned seed culture at a volume of 1%, and at 25 ° C and 200 rpm.
For 24 hours to obtain a culture solution containing protease NH.

Example 2 Purification of Protease NH One liter of the culture solution obtained in Example 1 was centrifuged (1
(000 rpm, 10 minutes) to remove the cells, and ammonium sulfate was added to the supernatant while cooling and stirring so as to reach 75% saturation, whereby protease NH was precipitated. The precipitate was collected by centrifugation (7,000 rpm, 10 minutes), and the precipitate was collected in 20 mM Tris-HCl buffer (pH
7.5) The solution was dissolved in 50 ml, and the solution was placed in a dialysis membrane (molecular weight cut off: 3,500), and dialyzed with the same buffer overnight. Next, a 20 mM Tris-HCl buffer (1
The obtained inner dialysis solution was applied to a column packed with Sephacryl S-200HR (manufactured by Pharmacia) equilibrated with 00 mM NaCl (pH 7.5), and eluted with the same buffer to elute the active fraction. Collected. The collected active fraction solution is put into a dialysis membrane (molecular weight cut off: 3,500) and
Dialysis was performed overnight with 0 mM Tris-HCl buffer (pH 7.5). The dialysis solution was previously equilibrated with 20 mM Tris-HCl buffer (pH 7.5).
After adsorbing to an itrap Q column (manufactured by Pharmacia), washing and eluting with the same buffer, concentration gradient elution was performed with the same buffer containing 0 to 0.5 M sodium chloride to collect an active fraction. By the above operation, uniform protease NH could be obtained from the crude purified protease solution by SDS-polyacrylamide gel electrophoresis.

<Example 3> The enzymatic and physical properties of the protease NH obtained in Example 2 were examined.

<Example 4> The keratin protein fiber (wool fiber) was modified using the partially purified protease NH solution obtained in Example 1. Wool cloth (30cm × 3
0 cm, double thread worsted yarn 2/48) is well immersed in 50 times of tap water of 20 times the weight of the wool cloth, and 6.15 U of the enzyme solution is added thereto. Was subjected to an enzyme treatment of wool fibers. Wool fibers were similarly treated with enzymes using a commercially available enzyme as a comparative sample. The treatment liquid was sampled at intervals of 15 minutes from treatment time 0 to 90 minutes, and the amount of degradation of the wool fiber by the protease was examined by colorimetric measurement of the amount of amino acids released into the solution using a foreign reagent. The results are shown in FIG. As is clear from FIG. 5, it can be said that in the treatment using the protease NH, the amount of released amino acids was larger than when the commercially available enzyme was used, and the wool fiber was degraded accordingly. It was also observed that the wool fiber was rapidly decomposed in a short time of 30 minutes.

[0037]

As described above, according to the novel protease NH of the present invention, keratin protein fiber products can be efficiently modified. According to the production method, a large amount of protease NH that efficiently modifies keratin protein fiber products can be produced at low cost. Specifically, the microorganism used has an excellent protease-producing ability, and by producing the protease outside the cells, the purification of the enzyme requires less labor and the protease can be obtained industrially stably. The protease NH of the present invention is extremely excellent as a keratin protein-based fiber modifying enzyme, and can be effectively used for fiber processing of wool products and the like.

[Brief description of the drawings]

FIG. 1 is a view showing an optimum pH of a protease NH of the present invention.

FIG. 2 shows the pH stability of the protease NH of the present invention.

FIG. 3 is a view showing an optimum temperature of protease NH of the present invention.

FIG. 4 is a diagram showing the heat resistance of protease NH of the present invention.

FIG. 5 is a graph showing the amount of wool fiber degraded by protease NH and commercially available protease according to an example of the present invention.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-4-349882 (JP, A) JP-A-4-16186 (JP, A) JP-A-1-101885 (JP, A) JP-A-2- 97384 (JP, A) JP-A-4-190790 (JP, A) JP-A-5-308967 (JP, A) JP-A 8-173168 (JP, A) Journal of General and Applied Microbiology, 40 (JP) 4) (1994), p. 339-348 Applied Environ. Microbiol. , 50 (4) (1985), p. 1031-1037 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A novel protease having the following enzymatic properties: NH (1) action Various proteins are hydrolyzed to produce oligopeptides or amino acids. (2) Substrate specificity It has good activity on soluble proteins such as casein and insoluble proteins such as keratin. (3) Optimum pH Optimum pH at 37 ° C using casein as substrate
H is 7.0 to 9.0. (4) pH stability When casein is used as a substrate and treated at 55 ° C. for 20 minutes at various pHs, it is extremely stable in the pH range of 4.5 to 9.5. (5) Optimum temperature When the reaction is carried out at pH 7.5 using casein as a substrate, the optimum temperature is 50 to 60 ° C. (6) Heat resistance When casein is used as a substrate and treated at pH 7.5 and 40 ° C. for 10 minutes, or treated with 5 mM Ca 2+ at 50 ° C. for 10 minutes, it has a residual activity of 90% or more. (7) Effect of inhibitor The activity is not inhibited by diisopropylfluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), or parachloromercury benzoic acid (PCMB). The activity is inhibited by ethylenediaminetetraacetic acid (EDTA). (8) Effect of metal ions When casein is used as a substrate, its activity is inhibited by Cu2 +. Protease NH that lost its activity due to ethylenediaminetetraacetic acid (EDTA) is Zn2 +, Ca2 +, Mn2.
+, Reactivated by Co2 +. (9) Molecular weight about 34,500 (sodium dodecyl sulfate (SDS)-
By polyacrylamide gel electrophoresis). 2. A method of culturing a microorganism belonging to the genus Aeromonas, accumulating the protease NH according to claim 1 in a culture solution, and collecting the protease NH according to claim 1 from the culture solution. For producing protease NH. 3. The microorganism belonging to the genus Aeromonas is Aeromonas hydrophila.
ydrophila) WPH22 (Ferment P-1
4748).