WO1998017790A1 - Lipase, procede de production de cette lipase, micro-organisme produisant cette derniere et utilisation de celle-ci - Google Patents

Lipase, procede de production de cette lipase, micro-organisme produisant cette derniere et utilisation de celle-ci Download PDF

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Publication number
WO1998017790A1
WO1998017790A1 PCT/JP1996/003012 JP9603012W WO9817790A1 WO 1998017790 A1 WO1998017790 A1 WO 1998017790A1 JP 9603012 W JP9603012 W JP 9603012W WO 9817790 A1 WO9817790 A1 WO 9817790A1
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Prior art keywords
lipase
acinetobacter
strain
detergent
ferm
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PCT/JP1996/003012
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English (en)
Japanese (ja)
Inventor
Takashi Kotsuka
Masahiro Suzuki
Yuka Aoki
Junko Suzuki
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU73331/96A priority Critical patent/AU7333196A/en
Priority to PCT/JP1996/003012 priority patent/WO1998017790A1/fr
Publication of WO1998017790A1 publication Critical patent/WO1998017790A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase

Definitions

  • the present invention relates to a lipase having an effective activity in a detergent solution containing a bleaching agent, a detergent composition containing the lipase, a method for producing the same, and a bacterium producing the lipase.
  • lipase can be added to a detergent to decompose and remove lipids adhered to an object to be washed, thereby improving washing efficiency.
  • This use is described in a report entitled “Lipase as detergent components” by H. Andree et al. (Journal of Applied Biocnemistry 2 218-229 (1980)). It is described in.
  • Lipases which are desirable for detergent formulations are those which have sufficient lipase activity in detergent solutions under laundry conditions. Under normal washing conditions, the pH of the washing liquid is in the region on the liquid side, so a lipase that functions with the liquid pH is required. In general, lipid stains are relatively easy to remove under high-temperature and high-alkali conditions, but it is known that cleaning at low temperatures (60 ° C or less) is not sufficient. The washing temperature tends to decrease in the United States and Europe as well as in Japan, where low-temperature washing has been performed, and lipases that function well even at low temperatures are desirable as detergent-containing lipases.
  • Lipases produced by microorganisms include the genus Pseudomonas, the genus Alcaligenes, the genus Achromobacter, the genus Achromobacter, the genus Mucor, the genus Candida, and the genus Humicola. It is known to be derived from the genus Acinetobacter. However, most lipases obtained from these strains do not function adequately in alkaline detergent solutions because their optimal pH is neutral to slightly alkaline, Is also less stable. Furthermore, lipases of the genera Achromobacter, Mucor, Candida, and Humicola strongly increase their activity in the presence of surfactant. Be inhibited.
  • an object of the present invention is to provide a lipase which functions sufficiently in a detergent solution containing a bleaching agent, particularly, an anion-based detergent solution, and a method for producing the same.
  • Another object of the present invention is to provide a novel microorganism that produces the lipase. Disclosure of the invention
  • the present inventors isolated and cultured a large number of microorganisms in order to obtain a lipase functioning well in an anion-based detergent solution containing a bleaching agent, and as a result, SD706, SD707, Strains belonging to the genera Acinetobacter, Acinetobacter baumarmi and Pseudomonas C Pseudomonas) represented by the strains SD708 and SD710 produce new lipases satisfying the above conditions. They found that they produced it, and completed the present invention.
  • the present invention provides the following.
  • triolein emulsion When triolein emulsion is used as a substrate and the amount of oleic acid generated without adding detergent is 100%, anionic surfactant is 200 to 400 ppm, sodium perborate
  • anionic surfactant is 200 to 400 ppm
  • sodium perborate A detergent composition comprising a lipase and a surfactant, wherein the amount of oleic acid produced is at least 10% when measured in a detergent solution containing 1,000 ppm and 100 ppm of tetraacetylethylenediamine.
  • the detergent composition according to 1) or 2) which is a lipase derived from Acinetobacter bauman ni or Acinetobacter haemol yticus.
  • the action pH in the range of pH 4 to 12 using triolein emulsion as a substrate is 4 to 12, and the optimal pH is in the range of pH 9.5 to 11.5.
  • the working temperature is 30 to 80 ° C when measured in the range of 30 to 80 ° C using triolein emulsion as a substrate, and the optimum temperature is in the range of 55 to 70 ° C. In the enclosure.
  • the molecular weight measured by SDS-polyacrylamide gel electrophoresis is in the range of 29,000 to 35,000.
  • triolein emulsion When triolein emulsion is used as a substrate and the amount of oleic acid formed when no detergent is added is set to 100%, the anionic surfactant is 200 to 400 ppm, and the excess The amount of oleic acid formed is 10% or more when measured in a detergent solution containing 1,000 ppm of sodium borate and 100 ppm of tetrathylethylenediamine.
  • a lipase-containing culture is obtained by culturing a bacterium belonging to the genus Acinetobacter or a genus Pseudomonas, and separating the lipase.
  • FIG. 1 is a graph showing the relationship between the reaction pH of lipase produced by the strain SD706 and the relative activity.
  • FIG. 2 is a graph showing the relationship between the reaction temperature and the relative activity of the lipase produced by the strain SD706.
  • FIG. 3 is a graph showing the residual activity of the lipase produced by the strain SD706 at pH 7 for 1 hour at various temperatures.
  • Figure 4 shows the lipase produced by the strain SD706 at different pHs. 4 is a graph showing residual activity after holding at C for 1 hour.
  • FIG. 5 is a graph showing the relationship between the reaction pH and the relative activity of the lipase produced by the strain SD707.
  • FIG. 6 is a graph showing the relationship between the reaction temperature and the relative activity of the lipase produced by the SD707 strain.
  • FIG. 7 is a graph showing the residual activity of the lipase produced by the strain SD707 after being kept at pH 7 at various temperatures for 1 hour.
  • FIG. 8 is a graph showing the residual activity of the lipase produced by the strain SD707 at 37 ° C. for 1 hour at various pHs.
  • FIG. 9 is a graph showing the relationship between the reaction pH and the relative activity of the lipase produced by the strain SD708.
  • FIG. 10 is a graph showing the relationship between the reaction temperature of the lipase produced by the strain SD708 and the relative activity.
  • FIG. 11 is a graph showing the residual activity of the lipase produced by the strain SD708 after being kept at pH 7 at various temperatures for 1 hour.
  • FIG. 12 is a graph showing the residual activity of lipase produced by the strain SD708 at 37 ° C. for 1 hour at various pHs.
  • FIG. 13 is a graph showing the relationship between the reaction pH and the relative activity of the lipase produced by the strain SD710.
  • FIG. 14 is a graph showing the relationship between the reaction temperature and the relative activity of the lipase produced by the strain SD710.
  • FIG. 15 is a graph showing the residual activity of the lipase produced by the SD710 strain at pH 7 for 1 hour at various temperatures.
  • FIG. 16 is a graph showing the residual activity of the lipase produced by the strain SD710 at 37 ° C for 1 hour at various pHs. Detailed description of the invention
  • the microorganism used for producing the lipase of the present invention is not particularly limited. Such microorganisms can be selected from among the stored microorganism strains or from microorganisms newly classified from nature. Natural or artificial mutants of these microorganisms that produce the lipase of the present invention are naturally included as long as they have the ability to produce lipase having the properties described below. Examples of strains belonging to the genus Acinetobacter that produce the lipase of the present invention include SD 706 and SD 707 strains isolated from the soil in Kanagawa Prefecture, and SD strains isolated from the soil in Nagano Prefecture by the present inventors. 7 08 shares.
  • 0 means decomposition by oxidation
  • 1 means no decomposition.
  • Table 1 morphological observations, physiological properties tests, and quinone-based and intracellular DNA GC content identification revealed that SD 706 and SD 707 strains were ⁇ Acinetobacter baumanni SD 708 was identified as belonging to the species Acinetobacter haemolyticus.
  • the SD706, SD707 and SD708 strains have been deposited with the National Institute of Advanced Industrial Science and Technology as FERM P-1488U FERM P-14882 and FERM P-14883, respectively.
  • Examples of the strain belonging to the genus Pseudomonas that produces the lipase of the present invention include the SD710 strain isolated by the present inventors from soil under Tokyo. Regarding the Dutch characteristics of this SD710 strain, Pseudomonas fluorescens (Pseudomonas fluorescens) similar to this bacterium with reference to (2) and (3) above, Pseudomonas puti da The results are shown in Table 2 in comparison with.
  • d is positive from 11 to 893 ⁇ 45 of strains belonging to the relevant species
  • the strain SD710 is a non-fermentative gram-negative bacillus with polar flagella and its quinone system is Q-9, and it was identified as a bacterium belonging to the genus Pseudoonas. Was.
  • SD710 strain has several flagella, produces fluorescent dye, and has a positive arginine dihydrolase test, which indicates that Pseudomonas fluorescens or Pseudomonas fluorescens can be used.
  • ⁇ Possibility of Pseudomonas putida was considered.
  • strain SD710 was determined to be Pseudomonas fluorescens or a closely related species of Pseudomonas putida. Deposited with the Institute of Biotechnology, Industrial Technology Research Institute as FERM P-14884.
  • mutants that produce the lipase having the following properties produced by the above-described strains can be obtained by natural or induced mutation using the above-mentioned strains as the original strains. It can be used as a fungus.
  • Conventional methods for preparing these mutants include, for example, irradiation of the original strain with ultraviolet light or artificial mutagenesis with an agent such as N-methyl-N'-nitro-N-nitro-soguanidine (NTG). And spread on an agar medium containing oil such as olive oil. The clear zone formed around the colonies from the growing strains selects larger colonies, and is used as the lipase production medium. And culturing the cells to select strains with excellent productivity.
  • the lipase of the present invention can be obtained from a culture obtained by culturing bacteria.
  • the genus Acinetobacter or pseudomona It can be obtained from a culture obtained by culturing the present lipase-producing bacterium belonging to the genus Pseudomonas.
  • the carbon source may be any assimilable carbon compound or a compound containing it.
  • the nitrogen source any assimilable nitrogen compound or a compound containing the same may be used.
  • ammonium salt, nitrate, soybean flour, meat extract, corn steep liquor, pharma media and the like are used.
  • salts such as phosphates such as ammonium hydrogen phosphate and potassium hydrogen phosphate, magnesium salts, calcium salts, and manganese salts can be appropriately used.
  • the cultivation conditions vary somewhat depending on the medium composition, but select conditions that allow production of the target lipase.
  • the culture temperature is between 10 and 40 ° C,
  • a range of 20 to 37 ° C is preferred.
  • the culturing time is suitably from about 8 hours to about 100 hours. Usually, the culturing is terminated when the production of the present lipase reaches the maximum.
  • the pH of the medium may be in the range of 5 to 9, and pH 6.5 to 7.5 is particularly suitable for the production of the present lipase.
  • the present lipase can be collected from the culture solution obtained in this manner by separation and purification according to a conventional method for collecting lipase.
  • the supernatant or the filtrate obtained by removing the bacterial cells and medium solids from the culture solution by a known appropriate method such as filtration or centrifugation is concentrated or concentrated without removing the soluble salts. Salting out method to precipitate enzymes by adding water, organic solvent precipitation to add enzymes and contaminants by adding hydrophilic organic solvent
  • the present lipase can be obtained by using a combination of two or more.
  • the lipase activity was measured using a measurement method using triolein-polyvinyl alcohol (PVA) emulsion.
  • PVA triolein-polyvinyl alcohol
  • the method for measuring the titer according to the present method was specifically performed by the following method.
  • Enzyme solution A mixed solution consisting of 0.4 ml of a buffer solution (pH 9.0) prepared by adjusting pH of 0.1 lm 1 and 200 mM Tris / HC1 with sodium hydroxide, and 0.5 ml of triolein emulsion was heated at 37 ° C. for 10 minutes in a test tube with a stopper, and the reaction was stopped using 0.2 ml of 1 N hydrochloric acid as a reaction stopping solution.
  • a buffer solution pH 9.0
  • triolein emulsion polyvinyl alcohol (PVA) 2% aqueous solution
  • the unit of activity was 1 unit (1 U) of the enzyme that produced 1 micromol of oleic acid per minute.
  • a glyceride substrate use is made of a glyceride-do-arabia gum emulsion prepared by adding 10 g of gum arabic and 100 g of distilled water to 10 g of each glyceride and homogenizing at 18000 rpm for 10 minutes.
  • the reaction rate of fatty acids at the time of reaction at 30 ° C. and pH 10 is determined by a pH-stat titration method using a 0.05N aqueous sodium hydroxide solution.
  • the fatty acid production rate is defined as the decomposition power of each substrate.
  • the decomposition power of triolein is 100
  • triptyline shows a relative activity in the range of 115-125
  • olive oil shows a relative activity in the range of 50-80.
  • the decomposition power for the ester is determined by the colorimetric (A405) of p-nitrophenol generated by the hydrolysis reaction at pH 8.0 and 30 ° C using p-nitrophenyl fatty acid ester as a substrate. Ask.
  • p NPP p-ditrophenyl palmitate
  • p NPL p-ditropenyl laurate
  • p N PV p-Nitrofenyl valerate
  • the working temperature is 30 when measured by the same method as the titration method described above, except that the reaction is performed at a different reaction temperature in the range of 30 to 80 ° C using triolein emulsion as a substrate. ⁇ 80 ° C, optimal temperature is in the range of 55 ⁇ 70 ° C.
  • the residual activity after a 1 hour incubation at different temperatures in the temperature range of 20 ° C. to 70 ° C. at pH 7 was determined by the titration method described above. Has more than 80% activity at 60 ° C.
  • the buffer used for the treatment was 50 mM ⁇ -aminocaproic acid, 50 mM pistris (Bis (2-hydroxyethyl) imi notris (hydroxymethyl) methanone) ⁇ 50 mM TAPS (, -Tris (hyd roxymethyl) (Methyl-3-aminopropanesulfonic acid) Use a mixed buffer solution adjusted to pH 7 with hydrochloric acid.
  • the residual activity after treatment for 1 hour at 37 ° C with different pH in the range of pH 4 to l2 was determined to be 50% or less at pH 4 to 10 when measured by the above titration method. Has the above residual activity.
  • the buffer at the time of treatment contains 0.5 mM calcium chloride, 50 mM ⁇ -aminocaproic acid, 50 mM pistris (Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methanone), 50 mM TAPS ( Adjust pH of mixed buffer consisting of N-Tris (nydroxymethyl) methyl-3-aminopropanesulfonic acid) with hydrochloric acid or sodium hydroxide Use what was done.
  • the molecular weight obtained by SDS-polyacrylamide gel electrophoresis is in the range of 29,000-35,000.
  • JIS standard detergent (non-phosphorus) base 1,100 ppm, sodium linear alkylbenzene sulfonic acid (LAS) 400 ppm, sodium perborate l'OOOp pm ⁇ In a detergent solution containing 10 AMP (TA ED), the reaction PH was 10 and the reaction temperature was 35 ° C, except for using triolein emulsion as a substrate.
  • the activity is in the range of 10 to 50% as measured by a method similar to the above titration method.
  • the JIS standard detergent (free of phosphorus) base is 20 wt% zeolite, 69 wt% sodium sulfate anhydride, 4 wt% sodium carbonate, 6 wt% sodium silicate.
  • the composition of carboxymethylcellulose is 1 wt%.
  • the decomposing power of pNPP (P-nitrophenol palmitate) was set at 100
  • reaction pH The relationship between the reaction pH and the relative activity is as shown in Fig. 1, and when measured in the range of pH 4 to 12, the action PH is 4 to 12, and the optimal pH is 10.5 to 11.5. .
  • reaction temperature The relationship between the reaction temperature and the relative activity is shown in Fig. 2, and the working temperature is 30 to 80 ° C when measured in the range of 30 to 80 ° C, and the optimal temperature is 60 to 65. ° C.
  • Relationship treatment temperature and the residual activity is as shown in FIG. 3, it has a 6 0 D C of 80% or more active in the process.
  • the relationship between the treatment pH and the residual activity is as shown in FIG. 4, and the pH has a residual activity of 50% or more at pH 4 to 10.
  • the molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 31,000 2,000.
  • JIS standard detergent (no phosphorus) base 1 100 ppm of LAS, 400 ppm of LAS, 1,000 ppm of sodium perborate, 50% activity when measured in detergent solution containing TAEDIOOPP m is there.
  • p NPP p-nitrophenyl palmitate
  • p NPL p-nitrophenyl laurate
  • p NPV p —Nitrophenylvalerate
  • reaction PH The relationship between the reaction PH and the relative activity is as shown in Fig. 5, and the action pH is 4 to 12 when measured in the range of pH 4 to 12, and the optimal pH is 10.5 to 11.5. .
  • reaction temperature The relationship between the reaction temperature and the relative activity is as shown in Fig. 6, and the working temperature is 30 to 80 ° C when measured in the range of 30 to 80 ° C, and the optimal temperature is 60 to 80 ° C. 65 ° C.
  • the relationship between the treatment temperature and the residual activity is as shown in FIG. 7, and the treatment at 60 ° C. has an activity of 80% or more.
  • the molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 31,000 2,000.
  • triolein Assuming that the decomposition power of triolein is 100, the relative activities of triptyline 115 and olive oil 65 were exhibited.
  • p NPP p-nitrophenyl palmitate
  • p NPL p-nitrophenolate
  • p NPV p-2-nitrovalerate
  • reaction PH The relationship between the reaction PH and the relative activity is as shown in FIG. 9, and the action pH when measured in the range of pH 4 to pH 12 is 4 to 12, and the optimal pH is It is 9.5 to 10.5.
  • the relationship between the reaction temperature and the relative activity is shown in Fig. 10.
  • the working temperature is 30 to 80 ° C when measured in the range of 30 to 80 ° C, and the optimal temperature is 55 to 6 ° C. 0 ° C.
  • the relationship between the treatment temperature and the residual activity is as shown in FIG. 11, and it has an activity of 80% or more at 60 ° C.
  • the relationship between the treatment pH and the residual activity is as shown in FIG. 12, where the pH is 4 to 10 and the residual activity is 50% or more.
  • the molecular weight obtained by SDS_polyacrylamide gel electrophoresis is 33,000 soil 2,000.
  • JIS standard detergent (free of phosphorus) Base 1 lOOp pm, LAS 400ppm, sodium perborate l, 000 ppm, activity measured in detergent solution containing TAED lOO ppm, 1 8%.
  • p NPP p-ditrophenyl palmitate
  • p NPL p-ditrophenyl laurate
  • p NPV p- It showed a relative activity of 29.
  • reaction pH The relationship between the reaction pH and the relative activity is as shown in FIG. 13, and the action pH when measured in the range of pH 4 to pH 12 is 4 to 12, and the optimal pH is 1 0 to 11.
  • reaction temperature The relationship between the reaction temperature and the relative activity is shown in Fig. 14, and the working temperature is 30 to 80 ° C when measured in the range of 30 to 80 ° C, and the optimal temperature is 60 to 7 0 ° C.
  • the relationship between the treatment temperature and the residual activity is as shown in FIG. 15, and the activity is 80% or more at 60 ° C.
  • the relationship between the treatment pH and the residual activity is as shown in FIG. 16, and the pH has a residual activity of 50% or more at pH 4 to 10.
  • the molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 31,500 soil 2,000.
  • JIS standard detergent (no phosphorus) Base 1 lOOp pm, LA S 400p pm, Its activity is 12% when measured in a detergent solution containing 1,000 ppm of sodium perborate and 100 ppm of TAED.
  • Lipase which is an essential component of the detergent composition of the present invention, has the following activities in a detergent solution.
  • JIS standard detergent (free of phosphorus) Base 1 lOOp pm, LAS 400p pm, sodium perborate l, 000 ppm, detergent containing 100 ppm of tetracetyl ethylenediamine (TA ED) 100 ppm
  • TA ED tetracetyl ethylenediamine
  • the JIS standard detergent (free of phosphorus) base is 20 wt% zeolite, 69 wt% anhydrous sodium sulfate, 4 wt% sodium carbonate, 6 wt% sodium silicate, and carboxymethylcellulose. Consists of 1 wt% composition.
  • the lipase of the present invention can be used as an essential component of the detergent composition of the present invention, and the present invention provides a detergent composition containing the lipase having the above properties.
  • the amount of the lipase to be added to the detergent composition of the present invention is not particularly limited, but is generally 50 to 20,000 units, preferably 100 to 10,000 units per gram of the detergent composition. Mix. If the amount is too small, a sufficient improvement in the cleaning effect cannot be obtained, and if it is too large, the improvement in the cleaning effect is small compared to the amount of the enzyme, which is not preferable in terms of economy.
  • the lipase can be incorporated into any conventionally known detergent composition without any change in the composition, and is added to the detergent composition of the present invention. There is no particular limitation on the components other than the lipase to be prepared.
  • detergent compositions include surfactants of 10 to 50% by weight, detergents of 0 to 50% by weight, and alkaline agents of 1 to 50% by weight, based on the weight of the detergent composition. Or at least one compound selected from the group consisting of an inorganic electrolyte, Q. 1 to 5% by weight of a recontamination inhibitor, an enzyme, a bleaching agent, a fluorescent dye, a caking inhibitor and an antioxidant. And a detergent composition comprising:
  • the surfactant for example, linear or branched alkyl or alkenyl sulfate, amide sulfate, having a linear or branched alkyl or alkenyl group, ethylene oxide, propylene oxide and Aliphatic sulphates such as alkyl or alkenyl ether sulphates to which one or more of the pentylene oxides have been added, alkyl sulphonates, amide sulphonates, dialkyl sulphosuccinates, ⁇ -olefins, vinylidene-type olefins Aliphatic sulfonates such as sulfonic acid salts of internal and internal olefins, aromatic sulfonates such as linear or branched alkylbenzene sulfonates, linear or branched alkyl groups or alkenyl groups Group, ethylene oxide, propylene oxide Or alkenyl ether carboxylate or amide, mono-sulfo
  • phosphates such as orthophosphate, pyrrolinate, tripolyphosphate, methacrylate, hexamethacrylate, phytate, etc. 1,1-diphosphonic acid and derivatives thereof, phosphonic acids such as ethanehydroxy-1,1,2-triphosphonic acid, ethane-1,2-dicarboxy-1,2,2-diphosphonic acid and methanehydroxyphosphonic acid Salts, phosphonocarboxylates such as 2-phosphonobutane-1,2, -dicarboxylic acid, 1-phosphonobutane-12,3,4-tricarboxylic acid, monomethylphosphonosuccinic acid, and amides such as aspartic acid and glutamic acid Aminopolyacetates such as phosphate, nitrate triacetate, ethylenediaminetetraacetate, and methyltriaminepentaacetate, polyacrylic acid, and polyitanate Polyelectrolytes such as con
  • Organic compounds such as urea and urea, and inorganic compounds such as sodium chloride and bentonite can be used. Furthermore, triethanolamine, diethanolamine, monoethanolamine as organic solvent agents can be used. And triisopropanol amine can be used.
  • the detergent composition of the present invention contains a surfactant, a lipase, and the like as constituent components.
  • an amphoteric surfactant such as a bleaching agent such as sodium percarbonate or sodium perborate is used.
  • Agents, pigments, builders, e.g., anti-recontamination agents such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, potassium oxymethylcellulose, anti-caking agents, antioxidants, and other lipases and proteases.
  • Other enzymes can be included as needed.
  • any method may be used to mix enzymes such as protease, cellulase, and lipase.Forming in the form of fine powder may cause dust during handling of the detergent. It is not preferable from the viewpoint of safety and hygiene of detergent users and workers in the detergent industry, and therefore, it is preferable to shape the solvent in a solution state or in a shape that suppresses dust in advance. This shaping may be performed by any of the commonly used malm granulation, extrusion granulation, fluidized granulation, centrifugal fluidized granulation and other methods, and the protease or cellulase to be added to the detergent composition of the present invention.
  • Example 2 Culture of lipase-producing bacteria (SD706 strain) and lipase 2 liters of the liquid medium having the concentration of Example 1 was placed in a 5 liter culture tank, and subjected to high-pressure steam sterilization at 121 ° C. for 20 minutes. Inoculated with E. coli (Acinetobacter_baumanni) SD 706 strain, and cultured with aeration and stirring at 10,000 rpm for 24 hours at 30 ° C. After the culture, the cells were removed by centrifugation, and the lipase solution was removed. The lipase activity of this solution was 600 U / m1.
  • Acinetob ⁇ ter bau manni SD707 strain was cultured in the same manner as in Example 1. After the culture, the cells were removed by centrifugation to obtain a lipase solution. The lipase activity of this solution was 50 U / ml.
  • Example 4 Culture of a lipase-producing bacterium (strain SD 707) and acquisition of lipase As in Example 2, the strain Acinetobacter bau manni SD 707 was cultured. After the culture, the cells were removed by centrifugation to obtain a lipase solution. The lipase activity of this solution was 500 U / ml. A lipase powder was obtained from the lipase solution thus obtained in the same manner as in Example 2.
  • Example 5 Culture of lipase-producing orchid (SD 708 strain)
  • Example 1 Acinetobacter haemolyticus SD708 strain was cultured. After the culture, the cells were removed by centrifugation to obtain a lipase solution. The lipase activity of this solution was 50 U / m 1.
  • Example 6 Cultivation of lipase-producing bacterium (SD 708 strain) and acquisition of lipase
  • Acinetobacter haemolyticus SD 708 strain was cultured. After the culture, the cells were removed by centrifugation to obtain a lipase solution. The lipase activity of this solution was 200 UZm1.
  • a lipase powder was obtained from the lipase solution thus obtained in the same manner as in Example 2.
  • Example 7 Culture of lipase-producing bacteria (SD710 strain)
  • Example 8 Culture of lipase-producing bacteria (strain SD710) and acquisition of lipase Pseudomonas sp. Strain SD710 was cultured in the same manner as in Example 2. After the culture, the cells were removed by centrifugation to obtain a lipase solution. The lipase activity of this solution was 20 U Zm 1.
  • a lipase powder was obtained from the lipase solution thus obtained in the same manner as in Example 2.
  • Example 9 Lipase purification
  • Example 2 The lipase bulk powder obtained in Example 2, Example 4, Example 6, or Example 8 was dissolved in 10% saturated ammonium sulfate, and the mixture was dissolved in Butyl Toyopearl 65 M (Tosoichi). An active fraction was obtained by performing hydrophobic chromatography on a (trade name) Co., Ltd.
  • This active fraction was dialyzed against 10 mM Tris-HCl buffer (pH 8) containing 0.3 mM calcium chloride dihydrate, and then equilibrated with the same buffer to give DEAE-Cel lulofine A-
  • the product was adsorbed on an ion-exchange chromatographic resin under the trade name of 800 (Seikagaku Corporation) and eluted with a NaC1 concentration gradient to obtain an active fraction. This was desalted and freeze-dried to obtain a purified enzyme.
  • Example 2 The lipase bulk powder obtained in Example 2, Example 4, Example 6, and Example 8 was used. Chromopactor Piscosum
  • LAS sodium linear alkylbenzene sulfonate ( sodium linear alkylbenzenesulfonate)
  • P ⁇ EAE polyoxyethylene alkyl ethe (polyoxyethylene alkyl ethe) (Daiichi Kagaku Pharmaceutical Co., Ltd., Neugen ET-127)
  • TA ED is tetraacetyl 0 tyed with ethylenediamine (tetraacetyl ethylene diamine) ⁇ With bleed: 1,440 ppm,
  • Synthetic detergent 1 JIS standard detergent (no phosphorus) Base 1,100Ppm,
  • Synthetic detergent 2 JIS standard detergent (no phosphorus) Base 1, IOOP pm,
  • Synthetic detergent 3 JIS standard detergent (no phosphorus) base ⁇ , ⁇ pm,
  • Synthetic detergent JIS standard detergent (no phosphorus) Base l, 100 ppm, LAS 200 ppm
  • Table 3i shows the relative activities when the activity without adding the detergent 1 wt% was set to 100%.
  • SD706-SD710 is a lipase obtained from the so-called 6 SD710 strain
  • C. vise is Chromobacter p. Pipis cocosa samum, M M .. mmiieehh is lipase from Mum coco kor le Mie,
  • a commercially available bleach-containing detergent type, Wizpriichi (P & G brand name), was prepared using the lipase obtained in Examples 2, 4, 6, and 8.
  • a combined detergent composition was prepared.
  • the washing evaluation was performed as follows.
  • the stained cloth is degreased cotton cloth (15 cm x 15 cm), 250 mg of lipstick, 0.5 ml of lard, and 0.5 ml of olive oil 0.5 m 1 each, and then dried (75 ° C (After 0 minutes, at room temperature overnight).
  • the washing device used was Terg-0-Tometer.
  • the lipase-containing detergent composition or the commercially available bleach-containing detergent Tide With Bleach (P & G) was dissolved in 1 liter of distilled water to which calcium ion was added at a final concentration of 40 ppm, respectively, at standard concentrations.
  • Washing efficiency (%) ⁇ (Z value of soiled cloth after washing-Z value of unwashed soiled cloth) ⁇
  • the lipase of the present invention has high activity in detergent solutions containing bleach, Under the conditions, an excellent oil / fat stain removing effect can be provided, so that the detergent composition of the present invention can enhance the detergency during washing.
  • a lipase produced by Acinetobacter baumanni and Acinetobacter haemolyticus can be added to the detergent to provide a detergent composition having excellent washing properties.
  • Acinetobacter baumanni (Acinetobacter baumanni) SD706, SD707, and Acinetobacter haemolyticus SD708, Pseudomonas sp. (Pseudomonas sp,) SD710 strain, strains that are bacteriologically equivalent to these four strains, and mutants of these four strains are useful for effectively producing the lipase of the present invention.
  • Information on deposited microorganisms Information on deposited microorganisms
  • Acinetobacter baumanni SD706 strain accesion No. FERM P-14881:
  • Acinetobacter haemolyticus SD 708 strain accesion No. FERM P-14883:

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Abstract

Cette invention concerne une lipase capable de produire de l'acide oléique dans une quantité de 10 % ou plus. Cette quantité est mesurée dans une solution détergente qui contient de 200 à 400 mg/l d'un tensioactif anionique, 1000 mg/l de perborate de sodium et 100 mg/l de tetraacétyléthylènediamine, ceci lorsque l'on utilise une émulsion de trioléine comme substrat, et lorsque l'on considère que la quantité d'acide oléique produite sans ajouter aucun détergent est de 100 %. Cette invention concerne également une composition détergente contenant cette lipase, ainsi qu'un procédé de production de cette composition détergente. Etant donné son activité élevée dans les détergents disponibles sur le marché et contenant des agents blanchissants, cette lipase peut être ajoutée auxdits détergents afin d'accroître le pouvoir détergent.
PCT/JP1996/003012 1996-10-17 1996-10-17 Lipase, procede de production de cette lipase, micro-organisme produisant cette derniere et utilisation de celle-ci WO1998017790A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU73331/96A AU7333196A (en) 1996-10-17 1996-10-17 Lipase, process for producing the same, microorganism producing the same, and use of the lipase
PCT/JP1996/003012 WO1998017790A1 (fr) 1996-10-17 1996-10-17 Lipase, procede de production de cette lipase, micro-organisme produisant cette derniere et utilisation de celle-ci

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PCT/JP1996/003012 WO1998017790A1 (fr) 1996-10-17 1996-10-17 Lipase, procede de production de cette lipase, micro-organisme produisant cette derniere et utilisation de celle-ci

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7282128B2 (en) 2001-07-19 2007-10-16 Applera Corporation Buffers for electrophoresis and use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63161087A (ja) * 1986-12-10 1988-07-04 ユニリーバー・ナームローゼ・ベンノートシヤープ 酵素含有漂白洗剤組成物
JPH02245100A (ja) * 1989-01-30 1990-09-28 Unilever Nv 粒状洗剤組成物及びその使用
JPH0638746A (ja) * 1992-05-27 1994-02-15 Showa Denko Kk アルカリリパーゼ、それを生産する微生物およびアルカリリパーゼ含有洗剤組成物
JPH06504311A (ja) * 1991-01-16 1994-05-19 ザ、プロクター、エンド、ギャンブル、カンパニー リパーゼと水溶性第四級アンモニウム化合物とを含有する洗剤組成物
JPH0767636A (ja) * 1993-08-30 1995-03-14 Showa Denko Kk リパーゼ及びそれを生産する微生物及びリパーゼ製造方法及びリパーゼ含有洗剤組成物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63161087A (ja) * 1986-12-10 1988-07-04 ユニリーバー・ナームローゼ・ベンノートシヤープ 酵素含有漂白洗剤組成物
JPH02245100A (ja) * 1989-01-30 1990-09-28 Unilever Nv 粒状洗剤組成物及びその使用
JPH06504311A (ja) * 1991-01-16 1994-05-19 ザ、プロクター、エンド、ギャンブル、カンパニー リパーゼと水溶性第四級アンモニウム化合物とを含有する洗剤組成物
JPH0638746A (ja) * 1992-05-27 1994-02-15 Showa Denko Kk アルカリリパーゼ、それを生産する微生物およびアルカリリパーゼ含有洗剤組成物
JPH0767636A (ja) * 1993-08-30 1995-03-14 Showa Denko Kk リパーゼ及びそれを生産する微生物及びリパーゼ製造方法及びリパーゼ含有洗剤組成物

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7282128B2 (en) 2001-07-19 2007-10-16 Applera Corporation Buffers for electrophoresis and use thereof
US8512537B2 (en) 2001-07-19 2013-08-20 Applied Biosystems, Llc Buffers for electrophoresis and use thereof

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