JPH0638746A - Alkali lipase, microorganism producing the same and detergent containing alkali lipase - Google Patents

Abstract

PURPOSE:To provide the new alkali lipase having specific natures, highly stable to detergent components such as surfactants and proteases, little in the activity inhibition of detergent components, capable of increasing the washing force of the detergent, and useful for detergent compositions. CONSTITUTION:The alkali lipase has following characteristics; action and optimal pH: the action pH of 4-11.5 and the optimal pH of 7.0-9.5, when a triolein emulsion is used as a substrate; action and optimal temperature: action temperature of 10-80 deg.C and optimal temperature of 55-65 deg.C, when a triolein emulsion is used as a substrate; mol. wt.: 2800+ or -1.5 measured by a SDS-polyacrylamide electrophoresis method; isoelectric point; 4.5+ or -1.5 measured by an isoelectric point polyacrylamide gel electrophoresis method; the lipase activity inhibition with a detergent component: sodium linear alkylbenzenesulfonate-inhibition of <=50%. The lipase is obtained by culturing Pseudomonas.mendocina-SD70 strain (FERM P-12944).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkaline lipase, a microorganism producing the same, and its use. More specifically, it contains a novel lipase having an optimum pH on the alkaline side produced by Pseudomonas bacteria, a microorganism producing it, and an enzyme capable of hydrolyzing lipids in the alkaline region, and at the time of washing. It relates to a detergent composition in which the solution has an alkaline pH.

[0002]

2. Description of the Related Art Lipase is used for food processing enzymes for flavoring dairy products, medical enzymes as digestive agents, diagnostic enzymes for measuring blood lipids, and hydrolysis and modification of fats and oils. It is widely used as an industrial enzyme, etc. In recent years, the use of lipase as a detergent blending composition has been attracting more attention. It has been conventionally known that a protease is added to a detergent composition to decompose and remove protein and other dirt attached to an object to be washed. In addition, the addition of cellulase to remove the dirt attached to the cellulose fiber to be washed, or the addition of a glycolytic enzyme such as amylase to decompose and remove the sugar and other dirt attached to the washed material. Is also known. Further, in recent years, it has been known that lipase can be blended to decompose and remove lipids adhering to an object to be cleaned, thereby improving cleaning efficiency. This application is described by H. Andree et al., "Lipase as detergent components" (Journal of Applied Biochemistry, 2 , 21).
8-229 (1980)) etc.

A preferred detergent-containing lipase has a pH of a washing liquid in an alkaline region under normal washing conditions,
It is an alkaline lipase that functions at alkaline pH. Further, it is generally known that lipid stains are relatively easily removed under high temperature and high alkali conditions, but lipid stains cannot be sufficiently removed by washing at low temperature (60 ° C. or lower). The washing temperature tends to be lowered in Europe and the United States as well as in Japan where low-temperature washing has been mainly performed, and therefore, a preferred lipase for mixing a detergent is one which sufficiently functions at a low temperature. Further, a preferable lipase for blending detergent is one which can stably exhibit its function during washing even in the presence of detergent components such as a surfactant and protease contained in many detergents. In addition, the activity of a detergent component such as a surfactant is not significantly inhibited, and the composition is stable when stored in a detergent. It is desired to develop a detergent composition having a high cleaning effect on lipid stains, which is prepared by incorporating a lipase having the above-mentioned preferable properties.

The lipase produced by microorganisms is Pseudomonas spp., Alcaligene.
s) the genus Achromobacter (Achromobacter) genus Mucor (Mucor) genus Candida (Candida) spp., it is known that derived from Humicola (Humicola) genus, and the like. However, most of the lipases obtained from these have an optimum pH of neutral to slightly alkaline and have low stability against anionic surfactants. Furthermore, Achromobacter (Achromobacter) genus Candida (Candida) spp, Mucor (Mucor) genus Humicola (Humicola) genus each lipase is strongly inhibited its activity in the presence of an anionic surfactant .

[0005] In addition, Pseudomonas (Pseudomonas) is a microorganism belonging to the genus is known it is widely to produce lipase, Pseudomonas (Pseudomonas) genus is a strain Pseudomonas fluorescens (Ps. Fluorescen
s ), Pseudomonas cepacia ( Ps. cepacia ),
Pseudomonas fragi (Ps. Fragi), Pseudomonas Alcaligenes (Ps. Alcaligenes), there is a Pseudomonas aeruginosa (Ps. Aeruginosa).
However, the known enzymes obtained from these strains also do not satisfy the above characteristics.

[0006]

SUMMARY OF THE INVENTION Therefore, the present invention has an optimum pH on the high alkaline side, hardly inhibits the activity by detergent components, and is stable to detergent components such as surfactants and proteases. It is intended to provide a high alkaline lipase. Further, the present invention provides an enzyme-containing detergent composition containing such alkaline lipase. Furthermore, the present invention provides an enzyme-containing detergent composition containing two or more enzymes, which contains a protease in addition to the alkaline lipase.

[0007]

[Means for Solving the Problems] The inventors of the present invention isolated and cultured a large number of microorganisms in order to obtain alkaline lipase having the above-mentioned properties. As a result, the strain was isolated from soil in Tokyo. Pseud
omonas mendocina) SD702 Pseudomonas typified strain (Pseudomonas) strains belonging to the genus were found to produce an effective novel alkaline lipases as detergent composition formulation. In addition, the present inventors have found that Pseudomonas mendocina NCIMB1.
It has been found that lipases obtained from cultures of strain 0541, NCIMB10542 or NCIMB10543 also have the same properties as the above-mentioned alkaline lipases, and are effective for blending detergent compositions.

Conventionally, a lipase and a lipase-containing detergent composition produced by various strains are known, but a cleaning composition containing an alkaline lipase derived from Pseudomonas mendocina and an alkaline lipase derived from Pseudomonas mendocina is known. unknown. The present inventors have found that a novel alkaline lipase produced by Pseudomonas mendocina is extremely effective as an enzyme for blending detergents, and completed the present invention.

[0009]

[Production Bacteria] The microorganism used for producing the alkaline lipase of the present invention is a Pseudomonas ( Pseudomona) capable of producing an alkaline lipase having the properties described below.
s ) It is not particularly limited as long as it is a genus bacterium. Such a bacterium can be selected from among preserved strains or microorganisms newly isolated from the natural world. Natural or artificial mutants of these bacteria are naturally included as long as they have the ability to produce alkaline lipase having the characteristics described below. Bacterial strains can be isolated from soil or other sources by conventional methods. Selection of the target strain is carried out by culturing the test microorganism in, for example, an ordinary bacterial culture medium, and then selecting a high pH in the culture (medium or bacterial cells).
It can be performed by measuring the lipase activity under normal temperature conditions according to a conventional method.

As an example of a strain belonging to the genus Pseudomonas producing the novel lipase of the present invention, SD70 isolated from the soil under the present inventors by the present inventors
There are two strains.

The SD702 strain has the following properties. (A) Morphology (1) Cell shape and size: 0.5-0.7 x 2.2-3.3 micron straight bacilli. (2) Cell polymorphism: None. (3) Motility: Yes, with very monochrystal hair. (4) Spores: None. (5) Gram stain: Negative. (6) Anti-acid: None.

(B) Growth state (1) Meat broth agar plate culture: The colony is diffusive and smooth, and no pigment is produced. (2) Meat broth agar slope culture: Fungal moss is diffusive, the surrounding area is smooth, and no pigment is produced. (3) Broth liquid culture: Grows in the entire medium and precipitation is observed. (4) Meat broth gelatin stab culture: It grows only on the upper part of the medium and no liquefaction is observed. (5) Litmus milk: slightly alkaline, no coagulation is observed.

(C) Physiological properties (1) Reduction of nitrate: Positive. (2) Denitrification reaction: Negative. (3) MR test: negative. (4) VP test: negative. (5) Indole formation: negative. (6) Generation of hydrogen sulfide: Negative. (7) Starch hydrolysis: negative. (8) Use of citric acid: Positive. (9) Use of inorganic nitrogen source: Use nitrates and ammonium salts. (10) Dye formation: A water-insoluble dye is formed, and neither a water-soluble dye nor a fluorescent dye is formed. (11) Urease: Negative. (12) Oxidase: Positive. (13) Catalase: Positive. (14) Growth pH range: 5 to 12.5. (15) Temperature range for growth: 5 to 43 ° C. (16) Attitude toward oxygen: aerobic. (17) OF test: Oxidation. (18) Acid and gas production from sugars: D-glucose,
Produces acid from D-fructose and glycerin but not gas; from L-arabinose, D-xylose, D-mannose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, inosit and starch. Produces neither acid nor gas.

(D) Other properties (1) Tolerance of sodium chloride: Grows in the presence of NaCl up to 7%. (2) Decomposition of fat: Decomposes Tween 80, tributyrin, triacetin, vegetable oil, etc. (3) Assimilation of carbon compounds: Utilizes glucose and geraniol. Does not decompose starch. Do not liquefy gelatin. (4) Vitamin requirement: Negative. (5) Accumulation of PHB: Negative. (6) Arginine dihydratase: Positive. (7) Phenylalanine deamino acid reaction: negative.

The taxonomic properties of the bacterium having the above-mentioned mycological properties are described by Bergey's Mannual of Systematic.
As a result of comparison with other strains by referring to Bacteriology, 1984), this strain was Pseud.
omonas mendocina ). This strain has been deposited in the Institute of Microbiology, Institute of Industrial Science and Technology as Microorganism Research Institute No. 12944.

[0016]

[Culturing Method] To produce the alkaline lipase of the present invention, the alkaline lipase-producing bacterium belonging to the genus Pseudomonas is cultured in a medium. As the nutrient source of the medium, those commonly used for culture can be widely used. The carbon source may be any assimilable carbon compound or a compound containing the same, and examples thereof include glucose, glycerin, oils and fats, corn steep liquor, and Tween-based surfactants. As long as it is a nitrogen compound that can assimilate as a nitrogen source, or one containing this,
For example, ammonium salt, nitrate, soybean powder, meat extract, corn steep liquor, pharmacomedia and the like are used. Further, salts such as phosphates and magnesium salts are used as the inorganic salts.

Although the culture conditions may vary depending on the medium composition,
Select the most favorable conditions for the production of alkaline lipase, which is the target product. The culturing temperature is in the range of 10 to 40 ° C., preferably 20 to 37 ° C., and the culturing time is 8 hours to 1
It is about 00 hours, and the culture may be terminated when the alkaline lipase production reaches the maximum. The pH of the medium is 7-9.
5 is suitable for alkaline lipase production. By such culturing, the desired alkaline lipase is mainly obtained outside the cells (in the culture solution).

[0018]

[Separation and Purification Method] The alkaline lipase can be collected from the culture broth thus obtained by separating and purifying it according to a conventional method for collecting lipase. That is, a supernatant or a filtrate obtained by separating bacterial cells and medium solids from the culture solution by a known appropriate method such as a filtration method or a centrifugation method is separated, and the separated solution is concentrated or concentrated. Salting out method of adding soluble salts to precipitate the enzyme without any action, organic solvent precipitation method of adding hydrophilic organic solvent to precipitate the enzyme or contaminants, adsorption / desorption method using ion exchange resin, gel filtration Alkaline lipase can be obtained by using the method, spray-drying method with or without stabilizing aid, separation or purification means such as freeze-drying method, alone or in combination.

[0019]

[Method of measuring enzyme titer] In the present invention, the lipase activity is measured by triolein-polyvinyl alcohol (PV
A) TLC-FID measurement method using emulsion as a substrate,
Alternatively, the pH stat titration method using olive oil-PVA or olive oil-gum arabic as a substrate was used. The TLC-FID measurement method was specifically performed by the following method. Enzyme solution 0.1 ml, 100 mM Tris buffer (pH 9.
0) 0.4 ml and a mixed solution consisting of 0.5 ml of triolein emulsion were heated in a test tube equipped with a stopper at 37 ° C. for 10 minutes to cause reaction, and the reaction was stopped by using 0.2 ml of 1N hydrochloric acid as a reaction stopping solution. It was Here, as the triolein emulsion, a 2% aqueous solution of polyvinyl alcohol (PVA) (Poval PVA 117 (trade name of Kuraray Co., Ltd.):
Poval PVA205 (Kuraray Co., Ltd. product name) 9: 1)
To 15 ml, add 1.5 g of triolein and cool with ice.
What was homogenized at 000 rpm for 10 minutes was used. After the reaction was stopped, 2 ml of n-hexane, 2 ml of isopropyl alcohol, and 1 ml of distilled water were added, and the mixture was vigorously stirred. After standing, the hexane layer was sampled and the TLC-FID method (Minagawa
Et al., Lipids, 18 , 732, 1983) to quantify oleic acid. The unit of activity was defined as 1 unit (U) of the enzyme that produces 1 micromol of oleic acid per minute.

The pH stat titration method was specifically carried out by the following method. Olive oil-gum arabic emulsion as a substrate is 10 g of olive oil and 10 g of gum arabic.
100 g of water was added, and the mixture was homogenized at 25,000 rpm for 10 minutes while ice-cooling. An olive oil-PVA emulsion was prepared by adding 100 ml of the above PVA 2% aqueous solution to 10 g of olive oil and homogenizing at 18,000 rpm for 10 minutes while cooling with ice. Substrate emulsion 5 ml, 11
10 mM Tris buffer (pH 9.0) containing 0 mM-NaCl
A mixture of 5 ml, 4.5 ml of water and 0.5 ml of enzyme solution at 30 ° C, pH
The rate of fatty acid formation when reacted at 9 was 0.05 N N
It was determined from the titration rate of the aOH aqueous solution. The unit of activity is the amount of enzyme that produces 1 micromol of fatty acid per minute.

The protease activity is shown in Japanese Examined Patent Publication No. 60-551.
Measured by the titration method described in No. 18, the unit of activity is
It is indicated by nkatal (nanocatal; nkatal = 10 ⁻⁹ katal).

[0022]

[Enzyme Properties] As an example of the alkaline lipase belonging to the present invention, the above-mentioned Pseudom
The properties of alkaline lipase produced by onas mendocina ) SD702 strain are described below.

(1) Action It acts on glyceride to hydrolyze its ester bond.

(2) Substrate specificity It hydrolyzes various glycerides and esters over a wide range.

As the glyceride substrate, each glyceride-
A gum arabic emulsion was used. The emulsion used was 10 g of each glyceride, 10 g of gum arabic, and 10 g of water.
0 g was added and homogenized at 25,000 rpm for 10 minutes while cooling with ice. 5 ml of the emulsion, 110
A fatty acid production rate was 0.05 when a mixture of 5 ml of 10 mM Tris buffer (pH 9.0) containing mM NaCl and 26 mM CaCl ₂ .2H ₂ O, 4.5 ml of water and 0.5 ml of enzyme was reacted at 30 ° C. and pH 9. It was determined by the pH stat titration method using an aqueous solution of N NaOH. This fatty acid production rate was used as the decomposing power of each substrate. Decomposition power of triolein is 1
If 00, tributyrin 101, olive oil 10
The relative activity of 0, soybean oil 113, and cottonseed oil 101 is shown.

The decomposing power for the ester is pH 9.0, 3 with p-nitrophenyl fatty acid ester as a substrate.
It was determined from the colorimetric value (OD ₄₀₅ ) of p-nitrophenol generated by the hydrolysis reaction at 0 ° C. When the decomposing power of pNPP (p-nitrophenyl palmitate) is 100, pNPV (p-nitrophenyl valerate) 34,
The relative activity of pNPB (p-nitrophenyl butyrate) 17 is shown.

(3) Working pH and optimum pH Using the triolein emulsion as a substrate, the above TLC
-By the titer measurement method by the FID method, provided that the pH is 4.0-
Range of 11.5 (however, as a buffer, 100 mM ε-aminocaproic acid, 100 mM bistris (Bis (2-hydroxyethyl) i
minotris (hydroxymethyl) methane), 100 mM TAPS
(N-Tris (hydroxymethyl) methyl-3-aminopropanesulfon
ic acid) mixed buffer solution was adjusted with hydrochloric acid or NaOH to adjust the pH at different pH, and the relationship between reaction pH and relative activity is as shown in FIG.
The optimum pH is 7.0 to 9.5.

(4) Range of operating temperature and optimum temperature Using triolein emulsion as a substrate, 30 to 80 ° C.
Except that the reaction temperature is in the range of
The relationship between the reaction temperature and the relative activity measured by the same method as the titer method by the FID method is as shown in FIG.
The working temperature is 10 to 80 ° C, and the optimum temperature is 55 to 65 ° C. At 40 ° C. and 70 ° C., a relative activity of about 50% of the activity at the optimum temperature is shown.

(5) Temperature stability Residual activity after heat treatment at pH 8 for 1 hour at different temperatures from 20 ° C to 70 ° C was measured by the above-mentioned TLC-F.
The relative activity determined by the titration method by the ID method is as shown in FIG. 3, and it is hardly inactivated by the treatment at 30 ° C. and 40 ° C. for 1 hour, but at 70 ° C. by the treatment for 1 hour. Almost deactivated.

(6) pH stability 1 at 37 ° C. under different pH ranges from pH 3 to 12.
The residual activity after the time treatment was measured by the TLC-FID method was as shown in FIG.
3 to 9 have a residual activity of 80% or more. However, the following buffer was used for the treatment (pH 3: glycine-
Hydrochloric acid; pH 4 to 5: acetic acid-sodium acetate; pH 6 to
7: Phosphoric acid; pH 8-9: Tris-hydrochloric acid; pH 10-1
2: Glycine-NaOH).

(7) Molecular weight SDS-polyacrylamide gel electrophoresis (molecular weight standard: cytochrome C (monomer, dimer, trimer,
The molecular weight obtained with the tetramer, hexamer)) is 28
It is 000 ± 2000.

(8) Isoelectric point The isoelectric point obtained by the isoelectric point polyacrylamide gel electrophoresis is 4.5 ± 1.5.

(9) Effect of Detergent Components Alkaline protease for detergents typical of various commercial detergents (4 types) at standard use concentration (1.2 to 5 g / liter) solutions and various commercial detergents (4 types) at standard use concentration solutions Is A
5 mM or 2.5 mM CaCl ₂ .2H in a solution containing PI-21 (Japanese Patent Publication No. 60-55118) at a concentration of 0.3 nkat / ml
The ₂ O was added, pH10,30 ℃ residual activity of TLC-FID after treatment alkaline lipase of the present invention under the conditions of
The result measured by the method is as shown in Table 1, and the stability in the detergent solution is high.

[0034]

[Table 1]

Also, sodium linear alkylbenzene sulfonate (LAS) 300 ppm, 5 mM CaCl ₂ · 2H
The activity of alkaline lipase produced by the SD702 strain at a pH of 10 and 30 ° C. in a solution of ₂ O was measured using olive oil-P.
The activity inhibition measured by the pH stat titration method using a VA emulsion as a substrate was 50% or less, and it was found that the activity inhibition by LAS was very small.

Furthermore, 5 mM or 2.5 mM CaCl ₂ was added to a solution prepared by adding alkaline protease API-21 at a concentration of 0.3 nkat / ml to a standard use concentration (1.2 to 5 g / liter) solution of various commercially available detergents (5 types). 2H ₂ O was added, and the activity inhibitory activity of alkaline lipase produced by SD702 strain was measured by pH stat titration method using olive oil-gum arabic emulsion as a substrate under the conditions of pH 10 and 30 ° C. The results are shown in Table 2, which shows little activity inhibition in various commercial detergent solutions.

[0037]

[Table 2]

[0038]

The lipase of the present invention has an optimum pH of 7.0-9.
It has a high level of 5, and is highly stable against detergent components such as surfactants and proteases, and its activity is less inhibited by detergent components. Can be enhanced.

[0039]

EXAMPLES The present invention will now be described with reference to examples, but the present invention is not limited to the following examples. In addition,
In the following description,% is based on weight unless otherwise specified.

Example 1: Alkaline lipase producing bacterium (SD
702) cultured soy flour 2%, glucose 2%, diammonium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate
2 ml of liquid medium containing 0.5% magnesium sulfate heptahydrate 0.1% and sodium carbonate 0.3% was placed in a 18 mm diameter test tube and sterilized by high pressure steam at 121 ° C for 20 minutes, and then Pseudomonas mendocina .
1 platinum loop inoculation of SD702 strain, 24 hours at 35 ℃,
Culture was performed at 130 rpm. After culturing, the bacterial cells were removed by centrifugation to obtain an alkaline lipase solution. The lipase activity of this solution was 5 U / ml.

Example 2: Alkaline lipase producing bacterium (SD
702) culture and acquisition of alkaline lipase Soybean flour 2%, glucose 2%, diammonium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate heptahydrate 0.1%, sodium carbonate 0.3 %, Tw
een 85 2 liters of liquid medium having a concentration of 1.0% was placed in a 5 liter culture tank, sterilized under high pressure steam at 121 ° C. for 20 minutes, and inoculated with Pseudomonas mendocina SD702 strain that had been cultivated in advance. Aeration-agitation culture was carried out at 1000 rpm for 20 hours at 35 ° C. After culturing, bacterial cells were removed by a centrifuge to obtain an alkaline lipase solution. The lipase activity of this solution was 20 U / ml. A 30-50% fraction precipitate was obtained by the acetone precipitation method from the thus obtained alkaline lipase solution, dissolved in distilled water, and then 20-20 by the ammonium sulfate precipitation method.
A 40% fraction precipitate was obtained. This precipitate was dissolved in 10 mM Tris-hydrochloric acid buffer (pH 7), desalted, and freeze-dried to obtain a bulk alkaline lipase powder.

Example 3: Purification of alkaline lipase The alkaline lipase bulk powder obtained in Example 2 was dissolved in 10 mM Tris-hydrochloric acid buffer (pH 7) and 10 mM Tris-hydrochloric acid buffer (pH 7) containing 10% ammonium sulfate. After dialysis with, the mixture was subjected to hydrophobic chromatography with Butyl-Toyopearl (trade name of Tosoh Corporation), and the active fraction was further fractionated by the same hydrophobic chromatography to obtain an active fraction. This active fraction was dialyzed with 10 mM Tris-hydrochloric acid buffer (pH 6) containing 1 mM calcium chloride, and then ion-exchanged with DEAE-Cellulofine A-800 (Seikagaku Corporation), which was equilibrated with the same buffer. It was adsorbed on a chromatographic resin and eluted with a NaCl solution to obtain an active fraction. This was desalted and then freeze-dried to obtain a purified enzyme. This lyophilized product was confirmed to be a single product by polyacrylamide gel electrophoresis.

Examples 4 to 6: NCIMB10541 strain, NCIMB10542
Strain, NCIMB10543 strain culture and acquisition of alkaline lipase Soybean flour 2%, glucose 2%, diammonium hydrogen phosphate 0.1%, dihydrogen potassium phosphate 0.5%, magnesium sulfate heptahydrate 0.1%, sodium carbonate 0.3%, Tw
een 85 2 liters of 1.0% liquid medium was placed in a 5 liter culture tank, sterilized under high pressure steam at 121 ° C for 20 minutes, and pre-cultured in these Pseudomonas mendocina NCIMB10541 strain, NC
The IMB10542 strain or the NCIMB10543 strain was inoculated, and aeration stirring culture was performed at 35 ° C. for 20 hours at 1000 rpm. After culturing, cells were removed from the culture solution by centrifugation to obtain an alkaline lipase solution. In this way, NCIMB10541 strain, NCIMB105
The lipase activities of the alkaline lipase solutions obtained from the culture solutions of 42 strains and NCIMB10543 strains were 16 U / ml and 12 U / ml, respectively.
ml, 16 U / ml. From each of the alkaline lipase solutions thus obtained, 30 to 5 by acetone precipitation method.
A 0% fraction precipitate was obtained, dissolved in distilled water, and then a 20-40% fraction precipitate was obtained by the ammonium sulfate precipitation method. The precipitate was dissolved in 10 mM Tris-HCl buffer (pH 7),
This was desalted and then freeze-dried to obtain a bulk alkaline lipase powder.

Examples 7 to 24: Washing evaluation-1 A washing test was carried out using the alkaline lipase bulk powder obtained in Example 2. The soiled cloth is defatted cotton cloth (15 cm x 1
5 cm) was soaked with 70 mg of triolein dissolved in benzene and dried, and the washing device was Terg-O-Tom.
eter was used. Various commercially available detergents were dissolved at standard working concentrations in 1 liter of distilled water in which calcium chloride was added to have a predetermined calcium concentration, and alkaline lipase was further added so as to have a predetermined concentration. In some cases, protease API-21 (Japanese Examined Patent Publication No. 60-55118) was further added so as to have a predetermined concentration. 4 to 6 per liter of washing liquid
A sheet of the above-mentioned triolein soiled cloth was put therein, and washed at a washing temperature of 20 ° C to 35 ° C for 30 minutes at 120 rpm. After washing, it was rinsed twice with 1 liter of the above calcium-containing distilled water and then dried. The amount of triolein of the unwashed and washed soiled cloth was the same as the above TLC after extraction with n-hexane.
-Quantification was carried out by the FID method, and the washing efficiency and the enzyme addition effect were calculated by the following formulas. Cleaning efficiency (%) = [(amount of triolein of uncleaned contaminated cloth-
(Amount of triolein in the contaminated cloth after washing) / Amount of triolein in the uncleaned contaminated cloth] x 100 Effect of enzyme addition (%) = cleaning efficiency with enzyme added (%)-cleaning efficiency with no enzyme added (detergent only) (%)

The results are shown in Table 3.

[0046]

[Table 3]

From Table 3, it is clear that the cleaning composition containing the present alkaline lipase enhances the cleaning effect on lipid stains.

Examples 25 to 27: Laundry evaluation-2 A washing test was conducted in the same manner as in Example 11 using the alkaline lipase bulk powder obtained in Example 4. The washing efficiency and the enzyme addition effect were calculated by the same formulas as those shown in Examples 7 to 24. The results are shown in Table 4.

[0049]

[Table 4]

It is clear from Table 4 that the cleaning effect against lipid stains is enhanced also in the cleaning composition containing the lipase obtained from several strains of Pseudomonas mendocina.

Comparative Examples 1-3: Washing Evaluation of Other Lipases In place of the alkaline lipase, lipases derived from Humicola lanuginosa described in JP-A-48-62990, JP-A-61-280274. Pseudomonas fragi 22-39B-derived lipase or achromobacter ( Achromoba
cter sp. )-derived lipase (Seikagaku) was added,
Using the commercially available detergents B and F, washing was performed in the same manner as in Example 11 or Example 17 to determine the enzyme addition effect. The results are shown in Table 5.

[0052]

[Table 5]

From Table 5, the difference from the cleaning composition containing the present alkaline lipase is clear.

From these results, in the case of adding the alkaline lipase according to the present invention, it was confirmed that the cleaning rate was improved by 10% or more as compared with the case of not adding.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the reaction pH and the relative activity of alkaline lipase produced by the SD702 strain.

FIG. 2 is a graph showing the relationship between the reaction temperature and the relative activity of alkaline lipase produced by the SD702 strain.

FIG. 3 is a graph showing the residual activity when alkaline lipase produced by the SD702 strain was treated at pH 8 for 1 hour at various temperatures.

FIG. 4 is a graph showing the residual activity of alkaline lipase produced by strain SD702 after being kept at various pHs at 37 ° C. for 1 hour.

[Procedure amendment]

[Submission date] July 22, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0002

[Correction method] Change

[Correction content]

[0002]

2. Description of the Related Art Lipase is used for food processing enzymes for flavoring dairy products, medical enzymes as digestive agents, diagnostic enzymes for measuring blood lipids, and hydrolysis and modification of fats and oils. It is widely used as an industrial enzyme, etc. Recently it is further noted lipase applications as a detergent formulation composition. It has been conventionally known that a protease is added to a detergent composition to decompose and remove protein and other dirt attached to an object to be washed. In addition, the addition of cellulase to remove the dirt attached to the cellulose fiber to be washed, or the addition of a glycolytic enzyme such as amylase to decompose and remove the sugar and other dirt attached to the washed material. Is also known. Further, in recent years, it has been known that lipase can be blended to decompose and remove lipids adhering to an object to be cleaned, thereby improving cleaning efficiency. This application is based on "Lipase as det" by H. Andree et al.
Journal of Applied Bioch
EMISTRY, 2 , 218-229 (1980)) and the like.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

(4) Range of operating temperature and optimum temperature Using triolein emulsion as a substrate , the temperature is 30 to 80 ° C.
Except that the reaction was carried out in the temperature range, the TLC-FI described above was used.
The relationship between the reaction temperature and the relative activity measured by the same method as the titer measuring method by the D method is as shown in FIG. 2, the working temperature is 30 to 80 ° C., and the optimum temperature is 55 to 65 ° C.
At 40 ° C. and 70 ° C., a relative activity of about 50% of the activity at the optimum temperature is shown.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0040

[Correction method] Change

[Correction content]

Example 1: Alkaline lipase producing bacterium (SD
702) culture soybean flour 2%, glucose 2%, diammonium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate heptahydrate 0.1%, sodium carbonate 0.
2 ml of a liquid medium having a concentration of 3% was placed in a test tube having a diameter of 18 mm, and sterilized under high pressure steam at 121 ° C. for 20 minutes, and then Pseudomonas men ( Pseudomonas men
docina ) SD702 strain was inoculated with 1 platinum loop, and
It was cultured at 130 rpm for 24 hours at ℃. After culturing, the bacterial cells were removed by centrifugation to obtain an alkaline lipase solution. The lipase activity of this solution was 5 U / ml.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0044

[Correction method] Change

[Correction content]

Examples 7 to 24: Washing evaluation-1 A washing test was carried out using the alkaline lipase bulk powder obtained in Example 2. The soiled cloth is defatted cotton cloth (15 cm x
15 cm), 70 m of triolein dissolved in benzene
g was soaked and dried, and the cleaning device was Te
An rg-O-Tometer was used. Various commercially available detergents were dissolved at standard working concentrations in 1 liter of distilled water in which calcium chloride was added to have a predetermined calcium concentration, and alkaline lipase was further added so as to have a predetermined concentration. In some cases, further protease API-21 (Japanese Patent Publication No. 60
No. -55118) was added so as to have a predetermined concentration.
Add 4 to 6 sheets of the above triolein soil cloth per 1 liter of washing liquid, and wash at 120 ° C at a washing temperature of 20 ° C to 35 ° C.
It was washed with pm for 30 minutes. After washing, it was rinsed twice with 1 liter of the above calcium-containing distilled water and then dried. The amount of triolein of unwashed and washed was n-
After extraction with hexane, quantification by the TLC-FID method,
The washing efficiency and the enzyme addition effect were calculated by the following formulas. Cleaning efficiency (%) = [(amount of triolein of uncleaned contaminated cloth-
After washing contaminated triolein amount of cloth) / Not Application Benefits <br/> oleic amount of washing soiled cloth] × 100 enzyme addition effect (%) = cleaning efficiency during enzyme addition (%) - no enzyme added during (detergent only ) Cleaning efficiency (%)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location (C12N 1/20 C12R 1:38) (72) Inventor Shie Goto 2-24 Tamagawa, Ota-ku, Tokyo −25 Showa Denko KK Biochemistry Research Lab (72) Inventor Keimi Tanaka 2-24 Tama River, Ota-ku, Tokyo Showa Denko KK Biochemistry Research Lab (72) Inventor Michihiro Takama 1 Shibadaimon, Minato-ku, Tokyo −13-9 Showa Denko Co., Ltd. (72) Inventor Norio Moriya 5-1 Okawacho, Kawasaki-ku, Kawasaki-shi, Kanagawa Showa Denko Engineering Research Center

Claims (11)

[Claims] 1. An alkaline lipase having the following properties. (1) Working pH and optimum pH Working pH when using triolein emulsion as a substrate
Is 4 to 11.5, and the optimum pH is 7.0 to 9.5. (2) Working temperature and optimum temperature When the triolein emulsion is used as a substrate, the working temperature is 10 to 80 ° C and the optimum temperature is 55 to 65 ° C. (3) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 28000 ± 2000. (4) Isoelectric point The isoelectric point is 4.5 ± 1.5 as measured by isoelectric focusing polyacrylamide gel electrophoresis. (5) Inhibition of lipase activity by detergent component The activity inhibition by sodium linear alkylbenzene sulfonate is 50% or less. 2. The alkaline lipase according to claim 1, which is obtained from a culture of a bacterium belonging to the genus Pseudomonas . 3. Pseudom
The alkaline lipase according to claim 1, which is obtained from a culture of a bacterium belonging to onas mendocina ). 4. Pseudom
onas mendocina ) SD702 strain (Microtech Lab.
Alkaline lipase according to claim 1, which is obtained from the culture of No. 1). 5. A Pseudomonas mesobacterium ( Pseudomonas me) producing the alkaline lipase according to claim 1.
ndocina ) SD702 strain ( Ministry of Microbiology Research Institute No. 12944). 6. A Pseudom
An enzyme-containing detergent composition comprising the alkaline lipase according to claim 1 produced by onas mendocina ). 7. Pseudom
onas mendocina ) SD702 strain (Microtech Lab.
An enzyme-containing detergent composition comprising the alkaline lipase according to claim 1 produced by No.). 8. Pseudom
onas mendocina ) Bacteria NCIMB10541 strain, or NCIMB105
An enzyme-containing detergent composition comprising the alkaline lipase according to claim 1 obtained from a culture of 42 strains or NCIMB10543 strain. 9. The enzyme-containing detergent composition according to any one of claims 6 to 8, wherein the enzyme additive is provided as a dust-free granule. 10. The enzyme-containing detergent composition according to any one of claims 6 to 8, wherein the enzyme additive is provided as a liquid or a slurry. 11. The enzyme-containing detergent composition according to claim 6, which contains a protease in addition to the alkaline lipase.