Classifications

• • 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)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
  • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/816—Enzyme separation or purification by solubility
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
  • Y10S435/836—Bacillus licheniformis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
  • Y10S435/839—Bacillus subtilis

Definitions

• the present invention relates to alkaline protease obtained as an elaboration product of the growth of the microorganism Bacillus licheniformis A.T.C.C. No. 21424 in a nutrient medium, to a method of producing the alkaline protease, and to detergent compositions containing the protease.
• Alkaline protease is a term used to describe a proteolytic enzyme which exhibits optimum activity in an alkaline medium.
• Proteolytic enzymes of this nature are useful as an active ingredient in present day enzyme detergents and laundry presoak compositions. The enzyme is used for its ability to attack and remove the common soils and stains of clothing that have a significant protein content such as blood and grass stains.
• alkaline protease A number of sources of alkaline protease are presently known. Some of the more important processes now in commercial use rely upon the fermentation of Bacillus subtilis and Bacillus licheniformis or some of the molds, such as the Aspergilli.
• the enzyme provided by the present invention provides a basis for supplying protease to detergent formulations at a much lower cost per unit of enzyme activity.
• the enzyme of this invention has a higher optimum pH for protoeolytic activity; viz., pH 9.0-1l.0 as compared with an optimum pH range of pH 8.5-9.5 for a commercially available enzyme prepared from Bacillus subtilis.
• the enzyme of the present invention removes stains at least as well as, and in some instances, better than, the enzymes presently available on the market, and it has a greater solution stability in the pH range of 9.5-11.0.
• the yield of alkaline protease obtained according to this invention is at least twice as high as is obtained from a hitherto high-yielding strain of Bacillus subtilis.
• Bacillus licheniformis A.T.C.C. No. 21424 of this invention is characterized by the following reactions:
• the media contained 0.2% of either sodium citrate, sodium acetate or sodium propionate.
• the media contained 1% NaCl, 0.1% (N I-I HPO 0.05 KH PO 0.02% MgSO .7H O, 1.5% agar and 0.0008% phenol red.
• the medium was adjusted to pH 7.0. At the end of the incubation period, growth was evaluated by changes in the color indicator from yellow to red.
• B. licheniformic metabolizes all three organic salts while B. subtilis utilizes only citrate. Utilization of the anions by the organism released Na which in turn raised the pH of the medium and changed the indicator from yellow to red. Thus, the B. subtilis displayed red coloration on the citrate slants but no change on the acetate and propionate media. The other two organisms exhibited red coloration on all of the slants.
• Control organisms used were B. subtilis A.T.C.C. No. 15841 and B. licheniformis A.T.C.C. NO. 12759. 'Both of these organisms as well as applicants B. licheniformz's A.T.C.C. No. 21424 were maintained on Trypticase Soy Agar slants (B.B.L. Labs) at 4 C. prior to use. All inoculations made in the tests were made directly from the slants.
• the culture medium used consists of from 1% to 4% protein, from 1% to 12% glucose, and such mineral supplements as are necessary to insure proper growth of the microorganism.
• Soybean meal is a preferred protein raw material.
• Other protein substrates such as casein, Pharmamedia, a cottonseed-derived protein material, and Proflo, a partially defatted cooked cottonseed flour, both sold by Traders Protein Div. of Traders Oil Mill Co., Fort Worth, Tex.
• the mineral salts found useful with the preferred culture medium for use according to this invention include MnCl MgCl CaCO CaCl NaH2PO4, Na HPO and ZnCl Maximum enzyme production is obtained when the medium is initially buffered at pH- 7.6. Yields became progressively less as the pH is varied in either direction from this value, and at pH values below 7 or above 8, the yield becomes unacceptably low.
• a calcium salt such as calcium acetate
• Insoluble calcium phosphates are formed which, in turn, absorb a gel-like material and both are removed by the centrifugation.
• the clear supernatant liquid in this case, contains 98% of the total enzymatic activity in the culture medium.
• the enzyme may be recovered from solution by adding one-third volume of acetone at 2 C. to the clear supernatant. The resulting precipitate is removed by centrifugation and discarded. Additional acetone is added until 75% saturation of the fluid is obtained. The suspension is stored at 20 C. until precipitation is complete (about three hours). The precipitate is collected and dried to yield a powder.
• the alkaline protease of the present invention is used as an active ingredient in enzyme detergent compositions.
• the enzyme may be in powder or liquid form, and may be diluted with other ingredients. It is customary to dilute enzyme powder, as obtained by recovery from fermentation processes, with a compatable diluent such as sodium sulfate.
• Alkaline protease of this invention is used in an enzyme detergent composition at a level varying from 0.02% to 0.1% of the powder. When the enzyme powder is diluted approximately fivefold with sodium sulfate, it has an activity of about 1300 proteolytic units/ mg.
• detergents typically contain detergent actives.
• Various surface active agents known to the art can be used as detergent actives according to this invention, including anionic, nonionic, zwitterionic, ampholytic detergent compounds and mixtures thereof.
• Anionic detergent compositions which can be used in the compositions of this invention include both soap and nonsoap detergent compounds.
• suitable soaps are the sodium, potassium, ammonium and alkylolammo nium salts of higher fatty acids (C -C
• anionic organic non-soap detergent compounds are the water soluble salts, alkali metal salts, of organic sulfuric reaction products having in their molecular structure an alkyl radical taining [from about 8 to about 22 carbon atoms and a radical selected from the group consisting of sulfonic acid and sulfuric acid ester radicals. (Included in the term alkyl is the alkyl portion of higher acyl radicals.)
• Examples of the synthetic detergents which form a part of the compositions of the present invention are the sodium or potassium alkyl sulfates especially those obtained by sulfating the higher alcohols (Cy-C1 carbon atoms) produced by reducing the glycerides of tallow or coconut oil; sodium or potassium alkyl benzenesulfonates, such as are described in United States Letters Patents No. 2,220,099 and No.
• alkyl group contains from about 9 to about 15 carbon atoms
• alkali metal alkylbenzene sulfonates are those in which the alkyl radical is a straight chain aliphatic radical containing from about 10 to about 20 carbon atoms for instance, Z-phenyl-dodecanesulfonate and 3-phenyl-dodecanesulfonate
• sodium alkyl glyceryl ether sulfonates especially those ethers of the higher alcohols derived from tallow and coconut oil
• Nonionic synthetic detergents may be broadly defined as compounds aliphatic or alkylaromatic in nature which do not ionize in water solution.
• a well known class of nonionic synthetic detergents is made available on the market under the trade name of Pluronic. These compounds are formed by condensing ethylene oxide with an hydrophobic base formed by the condensation of propylene oxide with propylene glycol.
• the hydrophobic portion of the molecule which, of course, exhibits Water insolubility has a molecular weight of from about 1,500 to 1,800.
• the addition of polyoxyethylene radicals to this hydrophobic portion tends to increase the water solubility of the molecule as a whole and the liquid character of the product is retained up to the point where polyoxyethylene content is about 50% of the total weight of the condensation product.
• nonionic synthetic detergents include:
• the polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to 12 carbon atoms in either a straight chain or branched chain configuration with ethylene oxide, the said ethylene oxide being present in amounts equal to 10 to 25 moles of ethylene oxide per mole of alkyl phenol.
• the alkyl substituent in such compounds may be derived from polyrnerized propylene, diisobutylene, octane or nonene, for example.
• R R R N+O Long chain tertiary amine oxides corresponding to the following general formula, R R R N+O, wherein R is an alkyl radical of from about 8 to 18 carbon atoms, and R and R are each methyl or ethyl radicals.
• the arrow in the formula is a conventional representation of a semi-polar bond.
• amine oxides suitable for use in this invention include dimethyldodecylamine oxide, dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltetradecylamine oxide, dimethylhexadecylamine oxide.
• RRR"P- O Long chain tertiary phosphine oxides corresponding to the following formula, RRR"P- O, wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging from to 18 carbon atoms in chain length and R and R" are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms.
• R and R" are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms.
• the arrow in the formula is a conventional representation of a semi-polar bond.
• suitable phosphine oxides are:
• dimethyldodecylphosphine oxide dimethyltetradecylphosphine oxide, ethylmethyltetradecylphosphine oxide, octyldimethylphosphine oxide, dimethylstearylphosphine oxide, cetylethylpropylphosphine oxide, diethyldodecylphosphine oxide, diethyltetradecylphosphine oxide, bis(hydroxymethyl)dodecylphosphine oxide, bis (Z-hydroxyethyl dodecylphosphine oxide, 2-hydroxypropylmethyltetradecylphosphine oxide, dimethyloleylphosphine oxide, and dimethyl-Z-hydroxydodecylphosphine oxide.
• RRS O Dialkyl snlfoxides corresponding to the following formula, RRS O, wherein R is an alkyl, alkenyl, betaor gamma-monohydroxyalkyl radical containing one or two other oxygen atoms in the chain, the R groups ranging from 10 to 18 carbon atoms in chain length, and wherein R' is methyl or ethyl.
• suitable sulfoxide compounds are:
• tetradecylmethyl sulfoxide 3-hydroxytridecylmethyl sulfoxide, 2-hydroxydodecylmethyl sulfoxide, 3hydroXy-4-decoxybutylmethyl sulfoxide, 3-hydroxy-4-dodecoxybutylmethyl sulfoxide, 2-hydroxy-3-decoxypropylmethyl sulfoxide, 2-hydroxy-3-dodecoxypropylmethyl sulfoxide, dodecylethyl sulfoxide, and Z-hydroxydodecylethyl sulfoxide.
• the 3-hydroxy-4-decoxybutyl methyl sulfoxide has been found to be an especially effective detergent surfactant.
• An outstanding detergent composition contains this sulfoxide compound in combination with the polymaleate builder compound of this invention.
• Ampholytic synthetic detergents can be broadly de scribed as derivatives of aliphatic secondary and tertiary one contains an anionic water solubilizing group.
• Examples of compounds falling within this definition are 3- (N,N-dimethyl N hexadecylammonio)-propane-1-sulfonate and 3-(1N, N-dimethyl N hexadecylammonio)-2- hydroxypropane 1- sulfonate which are especially preferred for their excellent cool water detergency characteristics.
• anionic, nonionic, ampholytic and zwitterionic detergent surfactants mentioned above can be used singly or in combination in the practice of the present invention.
• the above examples are merely specific illustrations of the numerous detergents which can find application within the scope of this invention.
• organic detergent surfactant compounds can be formulated into any of the several commercially desirable composition forms, for example, granular, flake, liquid and tablet forms.
• These detergents may also optionally contain either organic or inorganic detergency builders such as sodium tripolyphosphate, tetra sodium pyrophosphate, tetra potassium pyrophosphate, potassium tripolyphosphate, sodium hexametaphosphate, sodium nitrilotriacetate and the sodium salt of ethylene diamine tetraacetic acid.
• organic or inorganic detergency builders such as sodium tripolyphosphate, tetra sodium pyrophosphate, tetra potassium pyrophosphate, potassium tripolyphosphate, sodium hexametaphosphate, sodium nitrilotriacetate and the sodium salt of ethylene diamine tetraacetic acid.
• the detergents of this invention do not contain agents which adversely affect or are adversely affected by the enzyme composition.
• agents which adversely affect or are adversely affected by the enzyme composition For example, hypochloride bleaching agents must not be used, because they completely inactivate the enzyme. The so-called oxy bleaches may also exhibit an adverse effect.
• other protein and protein-like materials generally should not be added to these detergent compositions as they may be attacked by the alkaline protease of this invention.
• EXAMPLE 1 A medium comprising 6% glucose, 2% soybean meal, 0.04% CaCI .02% MgCl in 0.1 M
• buffer at pH 7.6 was prepared.
• the glucose was provided as a 50% solution, autoclaved separately.
• the balance of the medium is also autoclaved separately and the two portions combined under aseptic conditions.
• An inoculum was prepared by adding 5 ml. of sterile distilled water to a 24-hour-old Trypticase Soy slant of Bacillus liclzeniformis, A.T.C.C. No. 21424, [Trypticase Soy Agar (BBL) has the following composition: Trypticase, a peptone derived from casein by pancreatic digestion, 15 grams; Phytone, a papian digest of soya meal, 5 grams; NaCl, 5 grams; and Agar, 15 grams, in 1 liter of water] that had been incubated for 24 hours at 37 C., and a cell suspension was prepared. The suspension was then introduced aseptically into a 250 ml.
• the fermentation flasks were shaken on a reciprocal shaker at 175 r.p.m. and 37 C. Maximum protease production occurred after 48-72 hours or at a terminal pH of 7.2-7.4.
• the strength of the enzyme preparations was assayed by the following assay based on digestion of casein.
• One proteolytic unit as the term is used herein and in the appended claims is arbitrarily defined as the amount of enzyme producing an increase in absorbancy (optical density) of 1.0 at 280 mu in ten minutes at 37 C. Eight hundred eighty-eight proteolytic units of this invention equals 1 Anson Unit.
• Soybean meal (percent) 1 1 Glucose (percent) 1 2 3 4 6 Mg. niltrogen/ml. medium: Soybean e 2. 60 Total reducing sugars as mg. glucose/ml. medium:
• the activity of the enzyme is expressed in glycine units per ml. or per mg.
• the enzyme was isolated from the culture broth after the indicated incubation time by cooling the liquid to 5 C., and centrifuging it at 17,300 g.350 ml./minute in a Sorvall cooled centrifuge type RC 2 B equipped with a Szent Gyorgiy continuous flow system. The broth was then mixed with 2 vol. acetone (20 C.) and after one hour the precipitate was centrifuged (6870 X g. 350 ml./ min). The precipitate was resuspended in about 300 ml. of a water-acetone mixture (1 vol./2 vol.). The slurry was filtered on a Buchner funnel and the precipitate was dried by adding more cold acetone.
• a method for producing an alkaline protease which comprises the steps of introducing a culture of Bacillus licheniformis A.T.C.C. No. 21424 into a culture medium, allowing fermentation to take place until the culture medium contains a high level of alkaline protease, and recovering the accumulated alkaline protease.
• a method for the production of alkaline protease which comprises the steps of growing Bacillus licheniformis A.T.C.C. No. 21424 under aerobic conditions in an aqueous medium having a pH between 7 and 8 and containing a carbon source, a protein and traces of min eral salts at a temperature within the range of 35 C. to 40 C. for a period of time from 48 to 96 hours, and thereafter recovering the enzyme from the culture medium.
• a method for producing an alkaline protease in accordance with claim 2, wherein the culture medium comprises 6% glucose, 2% soybean meal, 0.04% CaCl and 0.02% MgCl in a 0.1 molar Na HPO NaH PO aqueous buffer at a pH of 7.6, the percentages being by weight.
• alkaline protease is separated from the culture medium by (a) adding calcium acetate to a level of 0.15 M in the culture medium, (b) centrifuging at 4 C., and discarding the solids, (c) adding one-third volume of acetone to the supernatant at 2 C., (d) centrifuging and discarding the solids, (e) adding additional acetone until saturation is reached, (f) storing the suspension at -20 C. until precipitation is complete, and (g) recovering and drying the precipitate.

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Cited By (16)

Families Citing this family (5)

• 0
  • BE BE755886D patent/BE755886A/xx unknown
• 1969
  • 1969-09-08 US US00856182A patent/US3748233A/en not_active Expired - Lifetime
• 1970
  • 1970-09-03 AU AU19571/70A patent/AU1957170A/en not_active Expired
  • 1970-09-04 ZA ZA706071A patent/ZA706071B/xx unknown
  • 1970-09-05 DE DE19702044161 patent/DE2044161A1/de active Pending
  • 1970-09-07 FR FR7032477A patent/FR2061067A5/fr not_active Expired
  • 1970-09-07 GB GB4275970A patent/GB1306867A/en not_active Expired
  • 1970-09-07 CH CH1333870A patent/CH542279A/de not_active IP Right Cessation
  • 1970-09-08 NL NL7013286A patent/NL7013286A/xx unknown

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