US3655570A - Detergent containing alkali protease - Google Patents

Detergent containing alkali protease Download PDF

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Publication number
US3655570A
US3655570A US795951A US3655570DA US3655570A US 3655570 A US3655570 A US 3655570A US 795951 A US795951 A US 795951A US 3655570D A US3655570D A US 3655570DA US 3655570 A US3655570 A US 3655570A
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percent
enzyme
weight
alkyl
medium
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Masao Isono
Katsumi Tomoda
Kouichi Miyata
Kazutaka Maejima
Keisuke Tsubaki
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • 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

Definitions

  • the present invention relates to a novel alkali protease and to a process for producing the protease as well as to detergents and other cleansers containing the enzyme.
  • alkali protease a process established forjproducing a potent al 'kali protease on a'cornmercial'scale and manufactured detergents and other clean'sers containing' the enzymes'lhe enzyme is referred toherein as alkali protease.
  • the principal object of the'present'invention is therefore-to provide an alkali proteasewhich exhibitspotent activityin the p'l-l r'arlge'from 8:0 to 1210, particularly, from 10.0 to 1 1 .5.
  • Another object is toprovidedetergents and other cleansers containing' thealkaliprotease.
  • conidiophores develop from aerial hyphae, unbranched, 10 to along, 1.0 to 1.5 uwide, hyaline.
  • Conidia are produced apically on conidiophore and grouped in a slimy cluster, ovate or kidnay-shaped, one to two-celled, rarely three-celled, 5 to 8 I by l to 2 p), hyaline.
  • -No sexual state develops. Chlamydospore usually absent, sometimes present. 3.
  • Physiological characteristics 1. Condition for growth:
  • Hydrogen ion'concentration Preferably grows in alkaline media,optimal at between pH 8.0 to 9.0 Temperature; Optimal at about 24 C. Allow to grow'between -l5 and 28 C.,-although better growth is observed around at 15 C. rather than at 28 C. Oxygen requirement: Aerobic. 2. Gelatin: liquefaction:
  • i-Eusarium is divided into Section 1fifl 2 follows:
  • a rmicroconidia develop, usually one-celled.
  • the organism Fusarium sp.-S-l9--5,' produces microconidia --dominantly and can be included in th Elegans Group of SectionA.
  • the Group Elegans'isitypified by F usarium oxysporum and includes 36 species which are all plant pathogens producing macroconidia in abundance.
  • the medium may be usedin either liquid or solid form.
  • the culture maybe effected under stationary conditions, however,
  • the-like, and, as nitrogen sources such materials as peptone,
  • soybean cake rice'bran, wheat bran, potato extract, casein,
  • gluten casein hydrolysate, corn steep liquor, urea, ammonium salts, nitrates and other organic or inorganic nitrogenous compounds.
  • inorganic salts various phosphates, sulfates and hydrochlorides, for example, may be incorporated.
  • various vitamins, amino acids, nucleic acids and their related compounds, etc. may be added.
  • a natural or synthetic defoarning agent e.g., soybean oil or silicone oil, may be effective to increase accumulation of the enzyme.
  • ln cultivating the microorganisms it is preferable to prepare a small scale preculture which is, in turn, inoculated into a main culture medium.
  • Culture conditions such as incubation temperature and time, pH of the medium, aeration rate, etc., should vary with the microorganism and medium compositions to be used.
  • the conditions should be selected and controlled so that the accumulation of the alkali protease is maximal.
  • the preferred conditions include the incubation temperature of from 20 to 30Co., an incubation time from 2 to 7 days, a medium pH of near 7, and an aeration rate of 0.5 to 1.5 liters per minute per liter of the medium.
  • the microogranism accumulates a large amount of the alkali protease in the culture.
  • the object enzyme occurs mostly in the liquid phase of the culture. Therefore, it is preferable to follow the steps of removing mycelia by filtration or centrifugation and, then, of recovering and isolating the enzyme from the filtrate or the supernatant fluid as the case may be.
  • the culture When a solid medium is employed, it is usually preferable to subject the culture to extraction with water or an aqueous solution of an inorganic salt and, then, to recover the enzyme from the resulting extract.
  • any of per se conventional isolation and purification means may be employed. These include salting-out of enzyme with an inorganic salt such as sodium sulfate, ammonium sulfate or sodium chloride as well as fractional precipitation of enzyme by adding a suitable hydrophilic organic solvent such as methanol, ethanol or acetone.
  • an enzyme solution may be concentrated under reduced pressure and/or demineralized by dialysis. It is also possible to employ means such as adsorption and desorption on calcium phosphate gel, alumina, bentonite, ion exchange resin, etc., chromatography using a cellulose derivative, e.g. diethylaminoethyl cellulose, gel-filtration, precipitation, electrodialysis, electrophoresis and removal of impurities as heavy metal complex.
  • the alkali protease produced and prepared in the foregoing manner is active over a broad pH range between 8.0 and 12.0, particularly in the pH region of 10.0 to l 1.5.
  • the optimal temperature for enzyme activity lies somewhere between about 20 and about 60 C., particularly between about 40 and about 50 C. This temperature range for the activity is in quite good accord with actual laundry conditions. Moreover, the activity of the alkali protease is not inhibited by surfactants and/or chelating agents which are principal ingredients in detergent products or cleansers.
  • the alkali protease may be employed not only as a highly purified preparation but as a crude enzyme product and thus the product is obtained in a desired purity in accordance with the purpose; either pure or crude preparation of the enzyme may be used as an ingredient of detergent products or cleansers.
  • surfactants contained in the detergent products there may be mentioned for purpose of exemplification various compounds including anionic surfactants of the fatty acid salt type, sulfate type or sulfonate type, such as natural fatty acid soap (NS), alkylsulfate (DAE), olefine sulfate, n-a-olefine sulfonate (AOS), tetrapropylbenzene sulfonate (ABS) and n-alkylbenzene sulfonate (LAS); and non-ionic surfactants of the ether type or ester type, such as polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyhydric alcohol alkyl esters, polyoxyethylene alkyl esters, sugar esters and the like.
  • anionic surfactants of the fatty acid salt type such as natural fatty acid soap (NS), alkylsulfate (DAE), olefine sulfate, n-a-
  • the detergent products or cleansers may contain builders such as tri-polyphosphate, sulfates, carbonates, borates, as well as carboxymethyl cellulose, fluorescent dyes, scents, bleaching agents, e.g. perborates, chelating agents, e.g., N(CH COONa);;, skin-protective agents, e.g. dimethyllaurylaminoxide, disinfectants, e.g. tertiarily amine, and
  • the alkali protease is mixed with other components of the product which may be a powdery, granular or liquid form and,
  • the resulting mixture when the resulting mixture is a liquid, it may be dried to a powdery or granular product by conventional means such as p y y s- Since there is no international system of unit for this kind of enzyme, it is difficult to prescribe the portion of the enzyme in detergent products or cleansers in general.
  • activity of the alkali protease is assayed by the following modified Kunitz method (J. Gen. Physiol. 30, 291, 1947).
  • 1.0 ml. of a 2 percent auqeous solution of casein (I-Iammarsteins) and 0.5 ml. of an NaOI-I buffer, pH 11.0, are mixed with 0.5 of an enzyme solution.
  • 2.0 ml. of the resulting mixture is allowed to incubate at 37 C. for 20 minutes, and then the reaction is stopped by the addition of 3.0 ml. of a 5 percent aqueous solution of trichloracetic acid.
  • the mixture is allowed to stand at 37 C. for a further 30 minutes, whereby the undigested casein is thoroughly precipitated.
  • the precipitated casein is filtered off and the resulting filtrate is subjected to optical density determination at 275 mu, from which the amount of the digested casein is calculated as the amount of tyrosine.
  • One unit of protease activity (PU) is defined as the amount of the enzyme which dissolved an amount of casein equivalent to 1.0 pg. of tyrosine per minute under the assay conditions,.
  • the specific activity of an enzyme sample is expressed as PU/mg.
  • the portion of the alkali protease to be incorporated in detergent products will vary with the type of product. In the case of a detergent to be used for washing cloth, the preferred amount will generally range from 5 to 10,000 in terms of units per gram of the detergent and for practical purposes from 10 to 5,000 units per gram.
  • An alkali protease-containing detergent prepared in the foregoing manner shows exceedingly powerful cleansing action against proteinous stains atributable to sweat, blood, broth and milk, which are hard to wash away with a conventional detergent.
  • the detergent is employed in prewashing as well as in mainwashing.
  • soiled materials are usually soaked in a detergent solution at a room temperature for several hours, while the mainwashing is preferably carried out at an elevated temperature between 40 and 60 C. for 1 hour or so.
  • ml, mg., p.g,p., mu and C mean milliliter(s), milligram(s), microgram(s), micron(s), millimicron(s) and degree centigrade, respectively. Percentages are calculated on the weight per volume basis. In the following examples, parts by weight bear the same relation to parts by volume as do gram(s) to milliliter(s).
  • EXAMPLE I 500 parts by volume of a liquid medium, composed of 5 percent defatted soybean meal, 5 percent glucose, 2 percent sodium dihydrogen phosphate, and adjusted to pH 7 is dispensed in a fermenter (its capacity being 2,000 parts by volume), sterilized, inoculated with Fusarium sp. S-l9-5 (IFO 8884) (ATCC 20192) and incubated at 28 C. for 5 days under aeration and agitation to prepare a seed culture.
  • a liquid medium composed of 5 percent defatted soybean meal, 5 percent glucose, 2 percent sodium dihydrogen phosphate, and adjusted to pH 7 is dispensed in a fermenter (its capacity being 2,000 parts by volume), sterilized, inoculated with Fusarium sp. S-l9-5 (IFO 8884) (ATCC 20192) and incubated at 28 C. for 5 days under aeration and agitation to prepare a seed culture.
  • the seed culture is inoculated to a fermenter (its capacity being 50,000 parts by volume) containing 30,000 parts by volume of the same liquid medium as above, and the fermenter is incubated at 25 C. for 144 hours with the aeration rate of 45,000 pars by volume per minute under agitation of 500 rev./min. During the incubation, foaming is suppressed by the addition of a suitable amount of soy bean oil from ime to im Changes of the pH value and the protease activity course of the cultivation are shown as follows:
  • EXAMPLE 2 Soluble in water and aqueous mineral salt solutions hav-
  • the culture obtained after 144 hours, as in Example 1, is $25235 s figfifi iogginzerlnsoluble 1n cooled to about C. and, then, passed through a filter press 9 pH activity (See FIG with the filter aid, Hyflo Super-Ce] (Johns-Manville Products optimal PH at about 11 Corp. U.S.A.), whereby the mycelia is removed.
  • To the result- Temperature activity s FIG ing 20,000 parts by volume of the filtrate is added 0.6-satu- Optimal temperature about 5 rated ammonium sulfate, and the salted-out precipitate is col- 1 1 pH Stability (See FIG.
  • EXAMPLE 3 1n 3,000 parts by volume of cold water is dissolved 30 parts by weight of the crude enzyme powder prepared in Example 2, and insoluble materials in the enzyme solution are filtered off with the filter aid to obtain a clear solution. 0.6-Saturated ammonium sulfate is added to the clear filtrate to give precipitate of the enzyme which is, then, re-dissolved in 1,500 parts by volume of cold water, decolorized with charcoal, dialyzed against cold water at 45 C. for 4 days and liphilized to a powder. The above procedure yields 4.5 parts by weight of a partially purified enzyme powder with a specific activity of 1,580 PU/mg.
  • Enzyme-containing detergent compositions are prepared by blending 0.05 g. of the crude enzyme powder obtained in Example 2 with 100 g. each of the above different detergents.
  • Soiled Cloth (5 cm X cm) The cleaning activity is evaluated by a panel monitation. Five pieces of the soiled cloth are assigned to the test of each detergent and, after the sequence of washing, rinsing and drying under the specified laundry test, the resulting washed cloths are scored by five judges according to the following standards of score.
  • the cultures are then centrifuged to give supernatant fluids which are used as enzyme solutions.
  • To 2,000 parts by volume each of the enzyme solutions are added 5 parts by weight of the LAS-detergent described in Example 4, and protease activity of the resulting solution is determined by the specified assay method.
  • the results are set forth in Table 5 indicating that the LAS-detergent does not inhibit any activity of the enalkali zymes produced by those protease-producing microogranisms.
  • EXAMPLE 7 A liquid detergent for kitchen use: In 55 parts by volume of hot water at 60 65 C. are dissolved 18 parts by weight of sodium tetrapropylbenzenesulfonate, 12 parts by weight of sodium n-C alkylphenolether-sulfate, 5 parts by weight of lauryldiethanolamide and 10 parts by weight of sodium xylenesulfonate. After allowing to cool, the solution is supplemented with 0.5 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a liquid detergent composition for kitchen use.
  • EXAMPLE 8 Hair shampoo In 64 parts by weight of hot water at 60-65 C. are dissolved 5 parts by weight of acetylated lanolin, 6 parts by weight of Alkylolarnine (American Alcolac Corp.) and 25 parts by weight of Duponol XL E. I. duPont de Nemours Company). After allowing to cool, the solution is supplemented with 0.2 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a hair shampoo.
  • a detergent or cleanser composition comprising at least one surfactant and alkali protease selected from the group consisting of Fusarium sp. S-19-5 (ATCC 20192), Fusarium oxysporum f. lini (IFO 5880) and Gibberella saubinetti (ATTC 20193), characterized by the following properties:
  • composition as in claim 1, wherein the amount of the said alkali protease is from about 5 to about 10,000 PU per gram of the final product by the modified Kunitz method.
  • composition as in claim 1, wherein the said surfactant is selected from an anionic surfactant and a nonionic surfactant.
  • composition as in claim 3 wherein the said anionic surfactant is from the group of a fatty acid salt, alkyl sulfate, olefine sulfate, alkyl sulfonate, olefine sulfonate, and alkylaryl sulfonate.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Health & Medical Sciences (AREA)
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  • Microbiology (AREA)
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  • Biotechnology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Detergent Compositions (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
US795951A 1968-02-08 1969-02-03 Detergent containing alkali protease Expired - Lifetime US3655570A (en)

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JP786368 1968-02-08
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DE (1) DE1906001C3 (en))
DK (1) DK130190B (en))
ES (2) ES363379A1 (en))
FI (1) FI47205C (en))
FR (1) FR2001575A1 (en))
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3006769A1 (de) * 1979-02-23 1980-08-28 Radiometer As Verfahren zum betreiben von vorrichtungen, die protein enthaltende biologische fluessigkeiten analysieren, und zubereitung fuer die verwendung in diesem verfahren
US4711739A (en) * 1986-12-18 1987-12-08 S. C. Johnson & Son, Inc. Enzyme prespotter composition stabilized with water insoluble polyester or polyether polyol
US5078898A (en) * 1987-11-02 1992-01-07 Novo Nordisk A/S Detergent compositions comprising pseudomonas lipase and a specific protease
US5156761A (en) * 1988-07-20 1992-10-20 Dorrit Aaslyng Method of stabilizing an enzymatic liquid detergent composition
US5543322A (en) * 1991-05-21 1996-08-06 Takeda Chemical Industries, Ltd. DNA coding for alkaline protease
US20070010416A1 (en) * 2004-10-22 2007-01-11 Novozymes A/S Protease with improved stability in detergents

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK563986A (da) * 1986-11-25 1988-06-17 Novo Industri As Fremstilling af en lav-temperatur-aktiv protease
DK564086A (da) * 1986-11-25 1988-06-17 Novo Industri As Enzymatisk detergent-additiv
FR3115901B1 (fr) 2020-11-05 2022-11-18 Commissariat Energie Atomique Système sur puce de sommation en arbre binaire de valeurs flottantes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3451935A (en) * 1966-04-25 1969-06-24 Procter & Gamble Granular enzyme-containing laundry composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3451935A (en) * 1966-04-25 1969-06-24 Procter & Gamble Granular enzyme-containing laundry composition

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3006769A1 (de) * 1979-02-23 1980-08-28 Radiometer As Verfahren zum betreiben von vorrichtungen, die protein enthaltende biologische fluessigkeiten analysieren, und zubereitung fuer die verwendung in diesem verfahren
US4867797A (en) * 1979-02-23 1989-09-19 Radiometer A/S Method for cleaning instruments used for analyzing protein-containing biological liquids
US4711739A (en) * 1986-12-18 1987-12-08 S. C. Johnson & Son, Inc. Enzyme prespotter composition stabilized with water insoluble polyester or polyether polyol
US5078898A (en) * 1987-11-02 1992-01-07 Novo Nordisk A/S Detergent compositions comprising pseudomonas lipase and a specific protease
US5156761A (en) * 1988-07-20 1992-10-20 Dorrit Aaslyng Method of stabilizing an enzymatic liquid detergent composition
US5543322A (en) * 1991-05-21 1996-08-06 Takeda Chemical Industries, Ltd. DNA coding for alkaline protease
US20070010416A1 (en) * 2004-10-22 2007-01-11 Novozymes A/S Protease with improved stability in detergents

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DE1906001C3 (de) 1978-12-07
NL157657B (nl) 1978-08-15
SE368028B (en)) 1974-06-17
FI47205B (en)) 1973-07-02
ES363379A1 (es) 1971-02-16
BE728027A (en)) 1969-07-16
NO129637B (en)) 1974-05-06
GB1231150A (en)) 1971-05-12
ES380768A1 (es) 1973-04-01
NL6901889A (en)) 1969-08-12
DE1906001B2 (de) 1978-04-06
SE409335B (sv) 1979-08-13
DK130190B (da) 1975-01-13
DK130190C (en)) 1975-06-30
DE1906001A1 (de) 1969-09-11
FR2001575A1 (en)) 1969-09-26
FI47205C (fi) 1973-10-10

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