JPH09299082A - Low-temperature alkali protease x, microorganism capable of producing the same, production of the same, and detergent composition containing the enzyme and enzyme preparation for food processing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme, capable of hydrolyzing casein, also acting on hemoglobin or keratin, and useful for detergents, cosmetics, food modification, as a digestive auxiliary, antiinflammatory agent, etc., by culturing Xanthomonas sp. KSM-T66. SOLUTION: This enzyme, a new low-temperature alkali protease X, is obtained by culturing Xanthomonas sp. KSM-T66 (FERM P-15460) and collecting the product, being useful for detergent compositions, enzyme preparations for food processing, etc. This new enzyme has the following characteristics: acting at least 0-55 deg.C; optimum temperature is 18-22 deg.C; acting at pH5-11; optimum pH is about 9; capable of thoroughly hydrolyzing casein; also acting on hemoglobin, keratin; apparent estimated molecular weight determined by gel chromatography using Sephacryl S-200 is 23,000±1,000; and subject to inhibition by Hg<2+> , ethylenediaminetetraacetic acid, phenylmethanesulfonyl fluoride, chymostatin, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel low temperature alkaline protease, a microorganism producing the same, a method for producing the same,
It also relates to a detergent composition containing the enzyme and an enzyme preparation for food processing.

[0002]

Background Art Proteases of various origins are used as detergents for clothes, detergents for automatic dishwashers, detergents for contact lenses,
Bath agents, keratin-removing cosmetics, food modification (baking, softening meat, marine products processing), beer fining agents, leather tanning agents, photographic film gelatin removal, digestive aids, anti-inflammatory agents, etc. It is widely used in many fields.

Among them, the alkaline protease for detergents is the most industrially produced and has a large market size.
Alcalase, Sabinase (Novo Nordisk), Maxacar (Gist Brocades), AP
I-21 (manufactured by Showa Denko KK), Blap (manufactured by Henkel), Protease K (KAP; manufactured by Kao), etc. are known. However, since these enzymes have an optimum temperature on the high temperature side, it cannot be said that the enzyme characteristics are sufficiently exhibited when washing clothes in a low temperature region such as the temperature of tap water. Further, most of the above-mentioned application fields of proteases are under the conditions of body temperature, room temperature and low temperature, and therefore the use of high temperature optimum enzyme is not suitable. In addition, the high temperature treatment process using a high temperature optimum enzyme is not preferable from the viewpoint of energy saving.

On the other hand, the low temperature optimum protease is used for tanning leather, processing wheat protein in bread, or in a case where heat cannot be applied to the reaction system, that is, for aging cheese, softening meat, and improving food. It is valid.

[0005] When considering the use of protease in products such as detergents and the use in industrial processes, finding an enzyme that effectively acts in the low temperature range from room temperature is sufficient for energy saving as well as enzyme function. It is an indispensable condition in order to make full use. So far, proteases produced by protease-producing bacteria isolated from cold climate soil, organisms living in cold environments, seawater, milk in refrigeration, etc. are not necessarily low-temperature enzymes, but there are many reports.

[0006] In other words, Pseudomonas sp. (Ps
eudomonas sp.) No. 548 strain ( Agric . Biol . Chem ., 3
6, p. 1185, 1970), a strain of Escherichia freundii ( Eur . J.
Biochem., 44, pp. 87 pp., 1974), Xanthomonas Marutofira (Xanthomonas maltophila) 047
/ 08 strains ( FEMS Microbiol . Lett ., 79, 257,
1991), Pseudomonas fluorescens ( Pseudo
monas fluorescens ) T16 strain ( Appl . Environ . Microb
iol ., 46, 333, 1983), Pseudomonas fluorescens AFT36 strain ( Biochim . Biophys .
Acta , 717, 376, 1982), Pseudomonas sp. 145-2 strain ( Microbios ., 36, 7)
Page, 1982), Aeromonas salmonicida ( Aero
monas salmonicida ) 9016/02 strain ( J. Appl . Bact
eriol., 53, pp. 289 pp., 1983), Pseudomonas paucimobilis (Pseudomonas paucinomobilis)
049/03 strain, 178/13 strain, 189/06 strain and Bacillus sp. 172/15 strain ( J.
Basic Microbiol ., 31, 377, 1991),
Vibrio sp. SA1 strain ( Antonie
van Leeuwenhoek , 44, 157, 1978), Pseudomonas fluorescens NCDO 2085 strain ( J.
Dairy Res., 53, pp. 457 pp., 1986), Pseudomonas fluorescens OM228 strain and Serratia marcescens (Serratia marcescens) OM1192 shares (J. Dairy Res., 53, pp. 97 pp., 1986), Pseudomonas fluorescens GR83 strain ( Lebensm .- Wis
s. u .- Technol., 23, pp. 106 pp., 1990), Paecilomyces Marukuandi (Paecilomyces marquandi
i (WO88 / 03948), Xanthomonas sp. S-1 strain (JP-A 5-2118)
Microorganisms that can grow at low temperatures, such as No. 68), produce various proteases.

The protease produced by psychrophilic bacteria is summarized in a review by Fairbain et al. ( J. Dai
ry Res ., 53, 139, 1986). Since some of these proteases have an optimum temperature in the low temperature region, they can be expected to be used in the above-mentioned applications.

[0008] Recently, Asgeisson et al., Reported on elastase of Alaska pollack ( Biochim . Biophys . Acta , 116.
4, p. 91, 1993), Feller et al., A protease produced by the psychrophilic bacterium Bacillus TA41 strain ( J.
Biol . Chem ., 269, 17453, 1994).
And α-produced by the psychrophilic bacterium Alteromonas haloplanctis A23 strain
Amylase ( J. Biol . Chem ., 267, 521
Pp. 7, 1992), from the catalytic chemistry of each,
It is suggested that these enzymes may adapt to low temperature conditions. Also, Jackman et al. ( Appl . Environ . Microbiol ., 4
Volume 6, page 6, 1983) or Patel et al. ( Appl . Env
Iron . Microbiol ., 46, 333, 1983), the protease produced by Pseudomonas fluorescens, which is a psychrophilic bacterium, has a lower activation energy than the protease derived from mesophilic bacterium.

[0009]

As described above, various low-temperature proteases have been heretofore known, but development of a high-performance low-temperature protease is desired in order to utilize it as a detergent and the like. Therefore, the present invention provides a novel low-temperature alkaline protease that retains high activity even under low-temperature conditions, and has an optimum pH of the reaction on the alkaline side, and a microorganism that efficiently produces such an enzyme extracellularly and It is an object to provide a method for producing a low temperature alkaline protease using the microorganism.

[0010]

Under such circumstances, as a result of searching the low temperature alkaline protease from the natural world, the present inventors have found that the optimum temperature is 18 to 22 ° C. from hot spring drainage, and the temperature is low (for example, 0 ° C.). The present invention has been completed by finding a bacterium of the genus Xanthomonas that produces an alkaline protease that also sufficiently acts in the following.

That is, the present invention provides a low temperature alkaline protease X having the following enzymatic properties, a microorganism producing the same, a method for producing the same, a detergent composition containing the enzyme and an enzyme preparation for food processing. It is a thing.

1) Working temperature and optimum temperature Operates at least 0 to 55 ° C., and optimum temperature is 18 to 22.
° C. Approximately 50-6 of activity at optimal temperature at 10 ° C
It retains 0%, about 25-35% of the activity at 0 ° C at the optimum temperature. When Ca ²⁺ is present, the optimum temperature is 28-
Move to 32 ° C.

2) Temperature stability It is stable up to about 28 to 32 ° C. under a treatment condition of pH 10.0 for 15 minutes and has a maximum activity value of about 70 to 8 even at 40 ° C.
Retains 0% activity. Approximately 48-5 when Ca ²⁺ is present
Stable up to 2 ° C.

3) Working pH and optimum pH It works at least at pH 5 to 11, and the optimum pH is about 9.
It retains 50-60% of the maximum activity even at pH 11.

4) pH stability It is stable in the pH range of 5 to 10 under the treatment conditions of 20 ° C. and 15 minutes. Approximately 60-70% of maximum activity value at pH 11
Retain the activity of.

5) Molecular weight The apparent molecular weight estimated by gel chromatography using Sephacryl S-200 is 23,000 ± 1,0.
00.

6) Substrate specificity Casein is well hydrolyzed and also acts on hemoglobin and keratin. Synthetic substrate Suc-Ala-Ala-Pro-Ph
e- p Na, Glt-Ala-Ala-Pro-Leu- p Na, Suc-Ala-Ala-Pro-Va
l- p Na, Z-Gly-Gly-Phe- p Na, Suc-Ala-Ala-Ala- p Na, Suc
-Ala-Pro-Ala- p Na and Suc-Ala-Ala-Phe- p Na act to release p- nitroaniline (Suc is a succinyl group, Glt is a glutaryl group, Z is carbobenzoyl). Group, p Na represents a p -nitroanilino group).

7) Effect of metal ions It is inhibited by Hg ²⁺ .

8) Inhibitors ethylenediaminetetraacetic acid (EDTA), ethyleneglycolbis (2-aminoethylether) tetraacetic acid (EGT)
A), phenylmethanesulfonyl fluoride (PM
SF) and chymostatin.

9) Effect of surfactant Sodium dodecyl sulfate (SDS), sodium α-olefin sulfonic acid (AOS), sodium alkane sulfonic acid (SAS), α-sulfo fatty acid ester (α-)
SFE), polyoxyethylene alkyl ether (A
E), Sodium straight-chain alkylbenzene sulfonic acid (L
It is stable to AS), sodium polyoxyethylene alkyl ether sulfate (ES) and the like.

[0021]

Cold alkaline protease X of the present invention DETAILED DESCRIPTION OF THE INVENTION, for example Xanthomonas sp (Xanthomona
s sp.) KSM-T66 is cultivated and harvested from the culture.

Xanthomonas sp. KS of the present invention
The medium used for classification and identification of M-T66 is shown below (shown by weight%).

Medium 1. Nutrient Broth, 0.
8: Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 medium 2. Nutrient broth, 0.8 medium 3. Nutrient Broth, 0.8; Gelatin,
2.0; agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 medium 4. Bactolithomas milk, 10.5 medium 5. Nutrient Broth, 0.8; KNO ₃₀ .
1 medium 6. Bactopeptone, 0.7; NaCl, 0.5; glucose, 0.5 medium 7. SIM agar medium (manufactured by Eiken Chemical Co., Ltd.), indicated amount of medium 8. TSI agar medium (manufactured by Eiken Chemical Co., Ltd.), indicated amount medium 9. Bactopeptone, 1.5; yeast extract, 0.
5; soluble starch, 2.0; K ₂ HPO ₄ , 0.1; MgSO ₄ .7H ₂ O, 0.02; agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 medium 10. Koser's medium (Eiken Chemical Co., Ltd.), indicated amount of medium 11. Christensen's medium (Eiken Chemical Co., Ltd.), indicated amount of medium 12.1. Yeast extract, 0.05; glucose 1.0;
KH ₂ PO ₄ , 0.1; Na ₂ SO ₄ , 0.1 2. Yeast extract, 0.05; glucose, 1.0; KH ₂ P
_{O 4, MgSO 4 · 7H 2} O, 0.02; CaCl 2 · 2H 2 O, 0.05;
FeSO ₄ .7H ₂ O, 0.001; MnSO ₄・ 4-6H ₂ O, 0.001 As the nitrogen source, sodium nitrate, sodium nitrite, ammonium chloride and ammonium phosphate are used as described in 1 and 2 above.
It was used in addition to the medium. Medium 13. King A medium "Eiken" (manufactured by Eiken Chemical Co., Ltd.), indicated amount of medium 14. King B medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount of medium 15. Urea medium "Eiken" (manufactured by Eiken Chemical Co., Ltd.), indicated amount of medium 16. Cytochrome oxidase test paper filter paper (manufactured by Nissui Pharmaceutical Co., Ltd.) Medium 17.3% hydrogen peroxide water Medium 18. Bactopeptone, 1.5; yeast extract, 0.
_{5; KH 2 PO 4, 0.1} ; glucose, 1.0; MgSO ₄ · 7H
₂ O, 0.02 medium 19. Penassay broth (manufactured by Difco), indicated amount of medium 20. Bactopeptone, 2.7; NaCl, 5.5; glucose, 0.5; K ₂ HPO ₄ , 0.1; bromthymol blue, 0.06; agar powder, (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 Medium 21. (NH ₄ ) ₂ HPO ₄ , 0.1; KCl, 0.02; MgSO ₄ ·
7H ₂ O, 0.02, yeast extract, 0.05; sugar, 1.0 medium 22. Casein, 0.5; yeast extract, 0.05; glucose, 1.0; KH ₂ PO ₄ , 0.1; MgSO ₄ .7H ₂ O, 0.
02; Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5

Xanthomonas SP KSM-T6
The taxonomic properties of 6 are shown below.

(A) Results of Microscopic Observation It is a rod-shaped bacterium having a size of 0.8 to 0.9 μm × 2.3 to 3.8 μm, has polar flagella, and is motile. No spore formation is observed.

(B) Gram stainability Negative.

(C) Growth state in various media 1. Meat broth agar plate culture (medium 1) Good growth condition. The shape of the settlement is circular, and the surface is smooth and glossy. The color of the village is pale yellowish white and semi-transparent. 2. Gravy agar slant culture (medium 1) Grow. 3. Broth liquid culture (medium 2) Growth is good and no pellicle formation is observed. 4. Meat broth gelatin stab culture (medium 3) Growth is good and liquefaction of gelatin is observed. 5. Litmus milk medium (medium 4) Acid production is not observed.

(D) Physiological properties 1. Nitrate reduction and denitrification reaction (medium 5) Negative. 2. VP test (medium 6) negative. 3. Formation of indole (medium 7) Negative. 4. Production of hydrogen sulfide (medium 8) Negative. 5. Hydrolysis of starch (medium 9) Negative. 6. Utilization of citric acid (medium 10, 11) Positive. 7. Use of inorganic nitrogen source (medium 12) No ammonium salt or nitrate is used. 8. Pigment formation (medium 13, 14) Negative. 9. Urease (medium 15) negative. Ten. Oxidase (medium 16) positive. 11. Catalase (medium 16) positive. 12． Growth pH Range (Medium 18) The growth pH range is 5-11. The optimum pH range for growth is 8
~ 9. 13. Growth temperature range (medium 1, 19) The growth temperature range is 10 to 35 ° C, and the most vigorous growth temperature is between 20 to 28 ° C. 14. Attitude toward oxygen (medium 20) Facultative anaerobic. 15. OF test (medium 21) Oxidized type. 16． Utilization of sugar D-xylose, D-glucose, D-fructose,
Maltose, sucrose, etc. can be used. 17． Growth in medium containing salt (in medium 1) It grows at a salt concentration of 5% but cannot grow at 7%. 18． Casein degradation (medium 22) Positive.

[0029] Based on the above-mentioned examination of mycological properties,
Bergey's Mannual of Systematic Bacte
As a result of a comparative examination with reference to Riology) 8th edition, this strain was determined to be a kind of the genus Xanthomonas. Therefore, this strain was named Xanthomonas sp. KSM-T66 and deposited as FERM P-15460 at the Institute of Biotechnology, National Institute of Biotechnology.

To obtain the low temperature alkaline protease X of the present invention using the above strain, the medium may be inoculated with the strain and cultured according to a conventional method.

It is desirable that the medium used for culture contains an appropriate amount of assimilable carbon source and nitrogen source. The carbon source and the nitrogen source are not particularly limited, and examples thereof include glucose, fructose, xylose, maltose, sucrose, molasses and assimilated organic acids such as citric acid.
As the nitrogen source, corn gluten meal, soybean flour,
Organic nitrogen sources such as corn steep liquor, casamino acid, yeast extract, pharma media, meat extract, tryptone, soyton, polypeptone, soybean meal, cottonseed oil cake, cultivator and zest are effective. Inorganic salts such as phosphates, magnesium salts, calcium salts, manganese salts, zinc salts, cobalt salts, sodium salts, potassium salts, and, if necessary, inorganic or organic micronutrients and vitamins are appropriately added to the medium. It can be added.

The culture temperature is 10 to 35 ° C., especially 20 to 2
Around 8 ° C is preferable, and the initial pH of culture is 5 to 11, especially pH 8
To 9 are preferred. Culturing is normally completed under these conditions in 1 to 3 days.

To collect low-temperature alkaline protease X, which is the target enzyme, from the culture broth thus obtained,
It may be performed according to a general method for collecting an enzyme. That is, after culturing, bacterial cells are removed from the culture solution by a usual 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 such as ultrafiltration and precipitation, and powdered by an appropriate method. It can also be purified by a general means for enzyme purification, for example, a combination of a suitable cation exchange resin, anion exchange resin, chromatography with hydroxyapatite, etc., affinity chromatography, hydrophobic chromatography, gel filtration and the like.

The enzymatic properties of the low temperature alkaline protease X of the present invention will be described below.

[Enzyme activity measurement method] 5 containing 1% of casein
1 ml of 0 mM various buffers was mixed with 0.1 ml of enzyme solution,
After reacting at 25 ° C for 15 minutes, the reaction stop solution (0.11
M trichloroacetic acid-0.22 M sodium acetate-0.3
2 ml of 3M acetic acid) was added and the mixture was allowed to stand at 30 ° C. for 20 minutes. Then, it was filtered through a filter paper (Whatman, No. 2), and the protein degradation product in the filtrate was subjected to the Foreign Lowry method (Lowry, OH).
et al ., J. Biol . Chem ., 193, 265, 195.
1 year). Under the above reaction conditions, 1
The amount of the enzyme that produces an acid-soluble proteolytic product corresponding to 1 mmol of tyrosine per minute was defined as 1 unit (U).

When the hydrolysis reaction of the synthetic substrate is carried out, 5
50 mM in 5.5 mM carbonate buffer (pH 10.0, 0.9 ml)
Various synthetic substrate solutions of (dissolved in dimethyl sulfoxide)
0.05 ml was mixed and kept warm at 25 ° C. for 5 minutes, and then 0.
An enzyme solution (05 ml) was added and the mixture was reacted at 25 ° C for 5 minutes. After adding 2 ml of the reaction stop solution (5% citric acid), the absorbance at 420 nm was immediately measured using a spectrophotometer to quantify the released p -nitroaniline. One unit (U) of enzyme was defined as the amount of enzyme required to release 1 μmol of p -nitroaniline in 1 minute under the above reaction conditions.

[Enzymatic properties] (1) Substrate specificity Various protein substrates were added to 50 mM carbonate buffer (pH 10.0) at a concentration of 0.
After adding 1% or 1%, an appropriate amount of enzyme was added and the reaction was carried out at 25 ° C. for 15 minutes. Table 1 shows the decomposition activity for each substrate, where the decomposition activity when casein was used as the substrate was 100. As is clear from this result, this enzyme showed good degrading activity for casein, hemoglobin and keratin.

[0038]

[Table 1]

Table 2 shows the results of examining the degradation activity of these using a synthetic oligopeptide substrate to which p- nitroaniline was bound. Tested N -succinylated Ala-Al
a-Pro-Phe, Ala-Ala-Pro-Val, Ala-Ala-Ala, Ala-Pro-A
la and Ala-Ala-Phe, N -glutalylated Ala-Ala-Pro
Release of p -nitroaniline from synthetic substrates such as -Leu and N -carbobenzoylated Gly-Gly-Phe was observed.

[0040]

[Table 2]

(2) Working temperature and optimum temperature This enzyme was added to a 50 mM carbonate buffer (pH 10.0) containing 0.91% casein as a substrate, and the reaction was carried out at each temperature for 15 minutes. As is clear from FIG. 1, the optimum temperature of this enzyme was 18 to 22 ° C. In the presence of calcium ( ₂ mM as CaCl ₂ ), the optimum temperature shifted to about 28 to 32 ° C., and about twice as much activity promotion was observed as compared to the optimum temperature in the absence of calcium. In addition, this enzyme shows about 50 to 60% of the activity at the optimum temperature even at 10 ° C, and the activity is 0 ° C (in ice water).
However, it shows about 25 to 35% of the activity at the optimum temperature, and it can be seen that it works sufficiently even under low temperature conditions.

(3) Temperature stability The enzyme was added to 50 mM carbonate buffer (pH 10.0), heated at each temperature for 15 minutes, and then cooled with ice. Residual activity was determined at 25 ° C. using casein as a substrate, and the results are shown in FIG. This enzyme was stable up to 28 to 32 ° C in the absence of Ca ²⁺ , and retained an activity of about 70 to 80% of the maximum activity value even at 40 ° C. It was stable up to 48-52 ° C in the presence of 2 mM Ca ²⁺ . It is generally known that when a low temperature enzyme is exposed to a temperature higher than the optimum temperature, it is inactivated, but it can be seen that this enzyme has a characteristic of exhibiting extremely stable properties above the optimum temperature.

(4) Working pH and optimum pH Casein was added to Britton-Robinson wide-range buffer (50 mM) to a final concentration of 0.91%, and the reaction was carried out at 25 ° C. for 15 minutes to obtain the activity at each pH. Was measured. As is clear from FIG. 3, the optimum pH of this protease is found around 9. The working pH is at least pH 5 to 1
As wide as 1. 50 to 60 of the maximum activity value even at pH 11
Retains% activity.

(5) pH stability The present enzyme was added to Britton-Robinson wide-range buffer (25 mM, each pH), and the mixture was allowed to stand at 20 ° C for 15 minutes, and the residual activity was measured at 25 ° C using casein as a substrate. As shown in FIG. 4, this enzyme was stable in a wide range of pH 5-10. It retains about 60-70% of the maximum activity even at pH 11.

(6) Molecular Weight The molecular weight of this enzyme was measured by gel chromatography using Sephacryl S-200. As the molecular weight markers, albumin (molecular weight: 66,000), ovalbumin (molecular weight: 43,000), carbonic anhydrase (molecular weight: 29,000), chymotrypsinogen of a low molecular weight marker kit (manufactured by Bio-Rad) is used. (Molecular weight: 25,000), Ribonuclease (Molecular weight: 13,700) and Cytochrome c (Molecular weight: 1)
2,400). As apparent from FIG. 5, the apparent estimated molecular weight of this protease is 23,000 ± 1,
000.

(7) Effect of metal ion This enzyme solution was added to a 20 mM carbonate buffer solution (pH 10.0) containing various metal salts at a concentration of 1 mM, and the solution was added at 20 ° C. for 20 minutes.
Let stand for a minute. Then, 50 mM carbonate buffer (pH 10.
The residual activity was measured by appropriately diluting with 0). The residual activity of the treated group was determined by setting the enzyme activity treated in the same manner in the system without addition of a metal salt as 100%. This enzyme activity was inhibited only by Hg ²⁺ (Table 3).

[0047]

[Table 3]

(8) Effect of inhibitor Various inhibitors were added to 10 mM phosphate buffer (pH 7.0; containing 2 mM CaCl ₂ ) to a predetermined concentration, the enzyme was added, and the mixture was allowed to stand at 20 ° C. for 20 minutes. After that, the residual activity was measured. As is clear from Table 4, EDT which is a chelating agent
This enzyme is considered to be a metal-dependent serine protease because it is inhibited by A and EGTA and serine enzyme inhibitors PMSF and chymostatin.

[0049]

[Table 4]

(9) Effect of surfactant The present enzyme was added to 50 mM Tris-hydrochloric acid buffer solution (pH 7.0; containing 2 mM CaCl ₂ ) containing 0.2% of the surfactant, and the enzyme was added at 20 ° C. for 20 minutes. After leaving for a minute, the residual activity was measured. As is clear from Table 5, this enzyme was used for SDS and AO.
S, SAS, α-SFE, Softanol 70H, LAS
It can be seen that even if it is contacted with a surfactant such as (each 0.2% concentration) for a long time, it is hardly deactivated, and that it has strong surfactant resistance.

[0051]

[Table 5]

Thus, the low temperature alkaline protease X of the present invention is a novel enzyme that has not been reported in the past. Recently,
A Bacillus species isolated from Antarctic seawater has been reported to produce a thermolabile subtilisin-type enzyme (Davail et al., J. Biol . Chem ., 269, 1744).
8 (1994)) The optimum temperature is around 40 ° C., and it is clearly different from the enzyme of the present invention by comparing other properties.

The amount of the low temperature alkaline protease X to be incorporated in the detergent composition of the present invention is not particularly limited as long as the low temperature alkaline protease exhibits activity, but it is 0.1 to 5000 U per 1 kg of the detergent composition. , Especially 1-50
0 U is preferred.

Further, the detergent composition of the present invention may be blended with a known detergent component, and examples of the known detergent component include the following.

(1) Surfactant A linear alkylbenzene sulfonate having an alkyl group having an average carbon number of 10 to 16 and a linear or branched alkyl group having an average carbon number of 10 to 20 and having 1 molecule in one molecule. 0.5 on average
Alkyl ethoxy sulphate having 8 to 8 mol of ethylene oxide added thereto, alkyl sulphate having an alkyl group having an average carbon number of 10 to 20, olefin sulfonate having an average carbon number of 10 to 20, alkane sulfone having an average carbon number of 10 to 20 An acid salt, an α-sulfofatty acid methyl or ester salt having an average carbon number of 10 to 20, a higher fatty acid salt having an average carbon number of 8 to 20, and a linear or branched alkyl group having an average carbon number of 10 to 20. Anionic surfactants such as alkyl ether carboxylates having an average of 0.5 to 8 mol of ethylene oxide added to the molecule; an alkyl group having an average of 10 to 20 carbon atoms, and 1 to 20 mol of ethylene oxide. Non-ion such as added polyoxyethylene alkyl ether, higher fatty acid alkanolamide or its alkylene oxide adduct Surfactants: Other betaine-type amphoteric surfactants, sulfonic acid-type amphoteric surfactants, phosphate ester-based surfactants, amino acid-type surfactants, cationic surfactants and other surfactants, alone or in combination. Can be used.

These surfactants are contained in the detergent composition in an amount of 5%.
It is preferably blended in an amount of from 60 to 60% by weight (hereinafter simply referred to as%), particularly from 10 to 45% with respect to the powdery detergent composition, and from 20 to 55% with respect to the liquid detergent composition.
When the detergent composition of the present invention is a bleaching agent, the surfactant is generally added in an amount of 1 to 10%, preferably 1 to 5%.

(2) Divalent metal ion scavenger Condensed phosphate such as tripolyphosphate, pyrophosphate, orthophosphate, aluminosilicate such as zeolite, synthetic layered crystalline silicate, nitrilotriacetate, Ethylenediaminetetraacetate, citrate, isocitrate, polyacetalcarboxylate and the like can be optionally used.

The divalent metal ion scavenger is 0 to 50.
%, Preferably 5-40%. It is more preferable to use a phosphorus-free divalent metal ion scavenger.

(3) Alkaline agent and inorganic salt A silicate, a carbonate, a sesquicarbonate, a sulfate, an alkanolamine and the like can be optionally used. These are 0
80% blended.

(4) Anti-redeposition agent Polyethylene glycol, polyacrylic acid salt, polyacrylic acid copolymer, polyvinylpyrrolidone, carboxymethylcellulose, etc. can be optionally used. The anti-redeposition agent is blended in an amount of 0 to 10%, preferably 1 to 5%.

(5) Enzymes Any enzyme such as cellulase, amylase, pectinase, lipase, hemicellulase, β-glucosidase, glucose oxidase, cholesterol oxidase, protease other than low temperature alkaline protease X can be used. When the low temperature alkaline protease X is used in combination with an enzyme such as cellulase, pectinase or lipase, the detergency can be further improved.

(6) Scavenger or reducing agent for available chlorine in tap water As an effective chlorine scavenger, ammonium sulfate, urea, guanidine hydrochloride, guanidine carbonate, guanidine sulfamate, thiourea dioxide, monoethanolamine, diethanolamine, triethanol. Examples include amines, amino acids typified by sodium glycintamitamate and proteins such as bovine serum albumin and casein, and further hydrolysis of proteins, meat extracts, fish meat extracts and the like.

As the reducing agent, thiosulfates, sulfites,
Examples thereof include alkali metal salts such as dithionite salts, alkali soil metal salts, Rongalit C and the like.

(7) Bleaching agent Any percarbonate, perborate, sulfonated phthalocyanine zinc salt or aluminum salt, hydrogen peroxide or the like having a bleaching action may be used.

(8) Fluorescent dye A fluorescent dye that is usually used in detergents.

(9) Solubilizing agent In the case of a liquid detergent, the following solubilizing agents can be used. Lower alcohols such as ethanol, benzene, sulfonates, lower alkylbenzene sulfonates such as p -toluene sulfonate, polyols such as propylene glycol, and the like.

(10) Others In addition to the above, ingredients commonly used in detergents such as fragrances, anti-caking agents, enzyme activators, antioxidants, preservatives, pigments, bluing agents and bleach activators are required. Can be blended accordingly.

The form of the detergent composition of the present invention can be selected according to the intended use, and can be liquid, powder, granule or the like. Further, the cleaning composition of the present invention can be used as a cleaning agent for clothes, a cleaning agent for automatic dishwashers, a drain pipe cleaning agent, a denture cleaning agent, a bleaching agent and the like.

The low temperature alkaline protease X of the present invention can also be used for food processing such as bread wheat protein processing, meat softening, cheese aging, and skin tanning.

When the low temperature alkaline protease X of the present invention is used for food processing, the low temperature alkaline protease X can be used as it is or as a powdery or granular enzyme preparation for food processing. In addition to the low temperature alkaline protease X, starch, proteins, sugars, seasonings, esters and the like can be added to the enzyme preparation for food processing.

[0071]

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 Hot spring drainage was smeared on a protease production judging plate having the composition shown below, and cultured at 20 ° C. for 5 to 7 days.
Protease-producing bacteria were isolated around the grown communities using the zona pellucida produced by the decomposition of keratin as an index. From the obtained isolates, low temperature protease productivity was investigated,
Xanthomonas SP KSM-T66 was selected.

Protease production determination plate Yeast extract (manufactured by Difco) 2 g Monopotassium phosphate 1 g Magnesium sulfate 0.2 g Keratin 10 g Glucose 5 g Carboxymethyl cellulose 5 g Agar 15 g * Sodium carbonate 2 g Ion-exchanged water 1 liter *: Separate sterilization (pH 10. 0)

Example 2 Xanthomonas sp. KSM obtained in Example 1
-T66 strain aerobically at 20 ° C. in the liquid medium shown below, 2
Cultured for days. After culturing, centrifuge (12,000 xg,
The supernatant obtained after 20 minutes) was dialyzed against 10 mM Tris-hydrochloric acid buffer (pH 7.5; containing 2 mM CaCl ₂ ) at 5 ° C. overnight. The protease activity of the dialysate was measured as a casein substrate at 20 ℃ (in 50 mM carbonate buffer, pH.
10.0), the production of protease corresponding to 2-3 U / l culture was confirmed.

Liquid medium Polypeptone S 10 g Yeast extract 0.5 g Monopotassium phosphate 1.0 g Magnesium sulfate 0.2 g * Glucose 5 g * Sodium carbonate 2.6 g Ion-exchanged water 1.0 liter *: Separate sterilization (pH 9.3)

Example 3 Xanthomonas sp. KSM-T66 strain was inoculated into the liquid medium of Example 2 and cultured at 20 ° C for 40 hours. After culturing, 10 mM Tris-HCl buffer (pH 7.5; 2 mM CaC
was dialyzed overnight at 5 ℃ for l ₂ containing). The dialyzed solution was freeze-dried, and after drying, dissolved with a small amount of ion-exchanged water and dialyzed against the same equilibration buffer solution (pH 7.5; containing 2 mM CaCl ₂ ) at 5 ° C. overnight to give a concentrated solution. Gel chromatography was performed on a column of Sephacryl S-200 Superfine (Pharmacia) equilibrated with this concentrated solution. As a result, it was purified up to about 6 to 7 times (recovery rate about 8
0%).

Example 4 Cleaning Test Using the cleaning composition of the present invention, the following cleaning test was conducted. (1) Detergent A weak alkaline powdered clothing detergent having the composition shown in Table 6 was prepared and used.

[0078]

[Table 6]

(2) Contamination cloth i) Artificial pollution cloth A commercially available artificial pollution cloth EMPA116 (blood / milk / carbon stain) manufactured by the Swiss National Industrial Materials Testing Institute was used for the experiment. ii) Contaminated cloth of natural collar cloth A cotton gold cloth (# 2023 cloth) is sewn on the collar of the shirt, and the young man wears it for 3 days. 25 ° C, 65 after wearing
After left in% RH for 1 month, the degree of dirt is divided into three levels. Of these, the one with the most dirt is the one with symmetrical dirt with respect to the center point, and the cloth is cut in half at this symmetrical point of dirt. It was subjected to an experiment.

(3) Washing conditions and method i) Washing of artificially contaminated cloth EMPA116 was cut to a size of about 8 × 8 cm, and then a detergent for weakly alkaline powdered clothing having the composition shown in Table 6 was adjusted to 0.0833%. After adjusting with water of ° DH, immersion was carried out at 20 ° C for 1 hour. After the immersion, use a targot meter (Kamijima Seisakusho), 1 liter, 20 ° C, 100rp
m, washed for 10 minutes. After rinsing and drying, the lightness was measured with a color-difference meter CR-300 (manufactured by Minolta Co., Ltd.), and the ratio when the lightness by the enzyme-free washing system was 100 was expressed as a detergency index.

Ii) Washing of Naturally Contaminated Cloth with a Targot Meter After cutting a natural collar cloth-contaminated cloth to a size of 11 × 13 cm, 0.083 of a detergent for weakly alkaline powdered clothing having the composition shown in Table 6 was cut.
After adjusting with 4 ° DH water to 3%, 20 ° C, 1
Immersion was carried out for a period of time. After the immersion, use a targot meter (manufactured by Kamijima Seisakusho), 1 liter, 20 ° C, 100
Washing was performed at rpm for 10 minutes. After rinsing and drying, a pair of collar cloths (15 sets) were compared, and the degree of stain removal was visually judged by three skilled judges. The judgment method is 5 points when the dirt is almost completely removed, 1 point when the dirt is hardly removed, and the total evaluation point of 15 collar cloths is obtained. 1
The ratio when 00 was shown as a cleaning liquid index.

Ii) Washing of naturally contaminated cloth with a washing machine 9 × 30 cm of naturally contaminated cloth is cut in half at symmetrical positions, and 9 ×
One of a pair of 15 cm soiled cloths was washed with a non-enzyme added detergent which was a standard detergent, and one was washed with a detergent containing the low temperature alkaline protease X of the present invention which was a comparative detergent. First, 15 pieces of naturally contaminated cloth are sewn on a 50 × 50 cm cotton cloth, and 6 liters of a weak alkaline powdered cloth detergent 0.417% detergent solution having the composition shown in Table 6 is used to contaminate the contaminated cloth and cotton underwear. 1 kg were put together and immersed in a washing machine (galaxy) manufactured by Toshiba Corp. for 1 hour at 20 ° C., and the total amount was adjusted to 30 liters, followed by strong inversion washing for 10 minutes. After rinsing and drying, the semi-cut cloth washed with the standard detergent and the semi-cut cloth washed with the detergent containing the low temperature alkaline protease X of the present invention were evaluated by a paired comparison by visual inspection. Degree of dirt 10
The cleaning cloths were ranked on the basis of the standard stains ranked in stages. The detergency represents the detergency score of the detergent containing the low temperature alkaline protease X of the present invention when the detergency of the standard detergent is 100. The difference in detergency index is 0.
A difference of 5 or more can be regarded as a significant difference.

(4) Results The results of the above cleaning test are shown in Table 7.

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[Table 7]

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INDUSTRIAL APPLICABILITY The protease of the present invention has an optimum action temperature in a low temperature region, is hardly inhibited by a surfactant, and is stable in a wide range from acidic to highly alkaline solutions. Therefore, the present enzyme can be advantageously used at a low temperature as a blending component of a detergent composition. Further, it is also effective for improving foods such as tanning, processing wheat protein of bread, softening meat and aging cheese under low temperature conditions.

[Brief description of drawings]

FIG. 1 is a view showing a temperature-activity curve of low temperature alkaline protease X.

FIG. 2 is a graph showing the temperature stability of low temperature alkaline protease X.

FIG. 3 is a view showing a pH activity curve of low temperature alkaline protease X.

FIG. 4 shows low temperature alkaline protease XpH stability.

FIG. 5 is a diagram showing the measurement results of the molecular weight of low temperature alkaline protease X.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication (C12N 1/20 C12R 1:64) (72) Inventor Katsuhisa Saeki 2606 Kabane Stock Association, Kabayacho, Haga-gun, Tochigi In-house research institute (72) Inventor Toru Kobayashi 2606 Akabane Kabayashi Co., Ltd., Kaga-cho, Haga-gun, Tochigi Prefecture In-house research institute (72) In-house 2606 Akabane Kai-cho, Haga-gun, Tochigi, Kao Corporation

Claims (5)

[Claims] 1. A low temperature alkaline protease X having the following enzymatic properties. 1) Working temperature and optimum temperature Operates at least 0 to 55 ° C, and optimum temperature is 18 to 22
° C. Approximately 50-6 of activity at optimal temperature at 10 ° C
It retains 0%, about 25-35% of the activity at 0 ° C at the optimum temperature. When Ca ²⁺ is present, the optimum temperature is 28-
Move to 32 ° C. 2) Temperature stability It is stable up to about 28 to 32 ° C under the treatment condition of pH 10.0 for 15 minutes, and the maximum activity value is about 70 to 8 even at 40 ° C.
Retains 0% activity. Approximately 48-5 when Ca ²⁺ is present
Stable up to 2 ° C. 3) Working pH and optimum pH It works at least at pH 5 to 11, and the optimum pH is about 9.
It retains 50-60% of the maximum activity even at pH 11. 4) pH stability It is stable in the pH range of 5 to 10 under the treatment conditions of 20 ° C and 15 minutes. Approximately 60-70% of maximum activity value at pH 11
Retain the activity of. 5) Molecular weight Apparent estimated molecular weight by gel chromatography using Sephacryl S-200 is 23,000 ± 1,0.
00. 6) Substrate specificity It hydrolyzes casein well and acts on hemoglobin and keratin. Synthetic substrate Suc-Ala-Ala-Pro-Ph
e- p Na, Glt-Ala-Ala-Pro-Leu- p Na, Suc-Ala-Ala-Pro-Va
l- p Na, Z-Gly-Gly-Phe- p Na, Suc-Ala-Ala-Ala- p Na, Suc
-Ala-Pro-Ala- p Na and Suc-Ala-Ala-Phe- p Na act to release p- nitroaniline (Suc is a succinyl group, Glt is a glutaryl group, Z is carbobenzoyl). Group, p Na represents a p -nitroanilino group). 7) Effect of metal ions ^Inhibited by Hg ²⁺ . 8) Inhibitors ethylenediaminetetraacetic acid, ethylene glycol bis (2
-Aminoethyl ether) tetraacetic acid, phenylmethanesulfonyl fluoride and chymostatin. 9) Effect of surfactant Sodium dodecyl sulfate, sodium α-olefin sulfonic acid, sodium alkane sulfonic acid, α-sulfo fatty acid ester, polyoxyethylene alkyl ether, sodium straight chain alkylbenzene sulfonic acid, polyoxyethylene alkyl ether sulfate Stable against sodium etc. 2. The method of claim 1] Xanthomonas sp (Xanthomona
s sp.) KSM-T66, and FERM P-1
A microorganism producing the low temperature alkaline protease X according to claim 1, deposited as 5460. 3. The method for producing low temperature alkaline protease X according to claim 1, wherein the microorganism according to claim 2 is cultured and the low temperature alkaline protease is collected from the culture. 4. A detergent composition containing the low temperature alkaline protease X according to claim 1. 5. An enzyme preparation for food processing, which comprises the low temperature alkaline protease X according to claim 1.