EP0758373A1 - Fabric cleaning compositions containing subtilisin bpn' variants - Google Patents
Fabric cleaning compositions containing subtilisin bpn' variantsInfo
- Publication number
- EP0758373A1 EP0758373A1 EP95916426A EP95916426A EP0758373A1 EP 0758373 A1 EP0758373 A1 EP 0758373A1 EP 95916426 A EP95916426 A EP 95916426A EP 95916426 A EP95916426 A EP 95916426A EP 0758373 A1 EP0758373 A1 EP 0758373A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- glu
- amino acid
- asp
- gin
- asn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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
- C11D3/38609—Protease or amylase in solid compositions only
Definitions
- the present invention relates to fabric cleaning compositions comprising protease enzymes which are subtilisin variants.
- Enzymes make up the largest class of naturally occurring proteins.
- Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction.
- One class of enzymes known as proteases are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.
- protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
- the amino acid sequence of the protease determines the characteristics of the protease.
- a change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence.
- Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
- compositions comprising effective variants of proteases useful for cleaning fabric surfaces.
- Objects of the Present Invention It is an object of the present invention to provide fabric cleaning compositions comprising subtilisin enzyme variants.
- the BPN' variants useful in these compositions comprise at least one, two or three amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions, whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'.
- This invention relates to fabric cleaning compositions comprising a subtilisin enzyme, in particular BPN', that has been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme.
- BPN 1 variants The modified subtilisin enzymes (hereinafter, "BPN 1 variants") useful in the compositions of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. Certain of these BPN' variants are described in co-pending application U.S.S.N. 08/121 ,437, filed September 15, 1993 by Brode et al.
- subtilisin enzymes useful in the compositions of this invention belong to a class of enzymes known as proteases.
- a protease is a catalyst for the cleavage of peptide bonds.
- One type of protease is a serine protease.
- a serine protease is distinguished by the fact that there is an essential serine residue at the active site.
- this loop plays a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in this loop have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that this loop is important to the adsorption of the BPN' molecule for at least two reasons. First, the amino acids which comprise this exterior loop can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of this loop to the active-site and binding pocket of the BPN' molecule gives it a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).
- variant means an enzyme having an amino acid sequence which differs from that of wild-type.
- mutant BPN' gene means a gene coding for a BPN' variant.
- wild-type subtilisin BPN' refers to a subtilisin enzyme represented by SEQ ID NO:1.
- the amino acid sequence for subtilisin BPN' is further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell and E. Y. Chen, NUCLEIC ACIDS RESEARCH, Vol. II, 7911-7925 (1983), incorporated herein by reference.
- wild-type amino acid sequence encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 199-220.
- hydrophilicity table refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below.
- Table 1 lists amino acids in descending order of increasing hydrophilicity (see Hopp, T.P., and Woods, K.R., "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981 , incorporated herein by reference).
- Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9).
- the positively charged amino acids are Arg and Lys
- the negatively charged amino acids are Glu and Asp
- the remaining amino acids are neutral.
- the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).
- the statement “substitute Gin with an equally or more hydrophilic amino acid which is neutral or has a negative charge” means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin.
- the statement “substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge” means Pro would be substituted with Gin, Asn, Ser, Glu or Asp.
- the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence at one or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 218, 219 or 220 is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'.
- the positions having a substituted amino acid are 199, 200, 201 , 202, 205, 207, 208, 209, 210, 211 , 212 or 215; more preferably, 200, 201 , 202, 205 or 207.
- the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 201 is Gly, Gin,
- the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 203 is Met, Cys, His, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 204 is Glu.
- the substituting amino acid for position 205 is Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 206 Pro, Asn or Ser is selected from the group consisting of: Asn or Ser.
- the substituting amino acid for position 207 is Asp or Glu.
- the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 210 is Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 212 is Gin, Ser,
- the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu.
- the substituting amino acid for position 215 is Thr, Pro,
- the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 218 is Glu.
- the substituting amino acid for position 219 is Pro, Gin,
- the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Asp or Glu.
- the substituting amino acid for any of positions 199, 200, 201 , 202, 203, 205, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216,
- 219 and 220 is, with reference to Table 1 , is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'.
- the substituting amino acid for any of positions 199, 200, 201 , 202, 203, 205, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 219 and 220 is Asp, or Glu; and the substituting amino acid for positions 204 or 218 is Glu.
- Variants comprising at least two amino acid substitutions comprising at least two amino acid substitutions
- the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence at two or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 or 220 is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'.
- the positions having a substituting amino acid are 199, 200, 201, 202, 205, 207, 208, 209, 210, 211 , 212, or 215; more preferably, positions 200, 201 , 202, 205 or 207.
- the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 200 is His, Thr,
- the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 204 is Asp or Glu.
- the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu.
- the substituting amino acid for position 207 is Asp or Glu.
- the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 210 is Ala, Gly,
- the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 212 is Gin, Ser, Asp or Glu.
- the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly. Gin, Asn, Ser, or Asp.
- the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 215 is Thr, Pro,
- the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 218 is Gin, Ser, Asp or Glu.
- the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 220 is Pro, Gly,
- the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 or 220 is, with reference to Table 1 , is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'. More preferably still, the substituting amino acid for any of positions
- the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence of three or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220, is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild- type subtilisin BPN'.
- the positions having a substituting amino acid are 199, 200, 201 , 202, 205, 207, 208, 209, 210, 211 , 212, or 215; more preferably positions 200, 201 , 202, 205 or 207.
- the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 202 is Pro, Gin,
- the substituting amino acid for position 203 Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 204 is selected from the group consisting of Asp or Glu.
- the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu.
- the substituting amino acid for position 207 is Asp or Glu.
- the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 209 is lie, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 210 is Ala, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 211 is Ala, Pro,
- the substituting amino acid for position 212 is Gin, Ser, Asp or Glu.
- the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 215 is Thr, Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 218 is Gin, Ser,
- the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu.
- the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Ser Asp or Glu.
- the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220 is, with reference to Table 1, is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'.
- the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 218, 219 or 220 is Asp or Glu; for position 217 is Leu, Asp, or Glu; and for position 213 is Asp. D. Preparation of enzyme variants
- Oligonucleotides are designed to restore the proper reading frame at position 217 and also encoded for random substitutions at positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.
- the fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION: A PRACTICAL APPROACH, Ed. B. McNeil and L. M. Harvey, 1990).
- the broth is kept at a constant pH of 7.0 during the fermentation run. Chloramphenical is added for antibiotic selection of mutagenized plasmid.
- the cells are grown overnight at 37°C to an AQOO °f about 60 and harvested.
- the fermentation broth is taken through the following steps to obtain pure enzyme.
- the broth is cleared of Bacillus subtilis cells by centrifugation, and clarified by removing fine particulates with a 100K cutoff membrane. This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5.5 using 0.025M MES buffer (2-( ⁇ /-morpholino)ethanesulfonic acid).
- the enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI or a propylene glycol gradient (See Scopes, R. K., PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Springer-Verlag, New York (1984), incorporated herein by reference).
- the pNA assay (DelMar, E.G., C. Largman, J.W. Brodrick and M.C. Geokas, ANAL BIOCHEM., Vol. 99, pp. 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution.
- This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (sAAPF-pNA).
- the rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration.
- absorbance measurements at 280 nm are used to determine the total protein
- SUBSTITUTE SHEET " (RULE 26) concentration The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution.
- the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer.
- the MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCl2 and pH adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C.
- Example 4 Model Surface Preparation Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, California). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3- (dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water.
- DMSO dimethyl sulfoxide
- the CPG surface will have 62,000 ⁇ 7,000 pNA molecules/ ⁇ m 2 .
- the surface area will remain unchanged from the value of 50.0 ⁇ .2/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).
- CPG:sAAPF-pNA Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment.
- a small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed.
- the flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e.g., the first 20 minutes - and every 10 minutes towards the end of the experiment).
- the CPG:sAAPF-pNA is allowed to settle and the solution is sampled. Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode er a/., 1992, above.
- enzyme adsorption can be determined by measuring solution depletion. The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed.
- the amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm.
- the total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask. This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme.
- Example 6 Soluble Substrate Kinetic Analysis The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer.
- the enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 ⁇ M sAAPF-pNA for each kinetic determination.
- An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUSTM spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts).
- BPN' variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Table 2, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third. TABLE 2 Example BPN' Variants :
- the fabric cleaning compositions of the present invention also comprise, in addition to the BPN' variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme.
- cleaning composition material means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid; granule; bar), which materials are also compatible with the BPN' variant used in the composition.
- the specific selection of cleaning composition materials are readily made by considering the fabric to be cleaned, and the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use).
- compatible means the cleaning composition materials do not reduce the proteolytic activity of the BPN' variant to such an extent that the protease is not effective as desired during normal use situations.
- Specific cleaning composition materials are exemplified in detail hereinafter.
- fabric cleaning composition refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form.
- the cleaning compositions of the present invention comprise from about 0.0001% to about 10% of one or more enzyme variants, more preferably from about 0.001 % to about 1%, more preferably still from about 0.01% to about 0.1%.
- the enzyme variants of the present invention can be used with various conventional ingredients to provide fully-formulated fabric laundering compositions.
- Such compositions can be in the form of liquids, granules and the like.
- Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants.
- the fabric cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.
- Nonlimiting examples of surfactants useful herein include the conventional C-
- alkyl alkoxy sulfates AES
- alkyl alkoxy carboxylates AEC
- AES alkyl alkoxy sulfates
- AEC alkyl alkoxy carboxylates
- Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C18 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al., issued March 16, 1993, incorporated herein by reference.
- compositions herein A wide variety of other ingredients useful in fabric cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc.
- suds boosters such as the C1 -C 6 alkolamides can be inco ⁇ orated into the compositions, typically at about 1 % to about 10% levels.
- the C-10- 1 monoethanol and diethanol amides illustrate a typical class of such suds boosters.
- Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous.
- liquid fabric cleaning compositions herein can contain water and other solvents as carriers.
- Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable.
- Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1 ,3-propanediol, ethylene glycol, glycerine, and 1 ,2-propanediol) can also be used.
- the compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.
- the fabric cleaning compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
- the formulator may wish to employ various builders at levels from about 5% to about 50% by weight.
- Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like.
- Other conventional builders are listed in standard formularies.
- the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001 % to about 1% by weight.
- additional enzymes such as cellulases, lipases, amylases and proteases
- Various fabric care enzymes are well-known in the laundry detergent art.
- bleaching compounds such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight.
- such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.
- Various soil release agents especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials. Enzyme stabilizers may also be used in the cleaning compositions of the present invention.
- Such enzyme stabilizers include propylene glycol (preferably from about 1 % to about 10%), sodium formate (preferably from about 0.1 % to about 1 %) and calcium formate (preferably from about 0.1 % to about 1 %).
- the granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1% by weight of active enzyme of the composition.
- the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.
- the granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.
- Examples 7-8 the BPN' variants recited in Table 2, among others, are substituted for Ala216Glu, with substantially similar results.
- Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 26.00
- Fillers e.g., silicates; carbonates; perfumes; Up to 100 Up to 100 water
- Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition.
- Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water.
- the liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples.
- any combination of the BPN' variants recited in Table 2, among others, are substituted for Gln206Glu + Ala216Glu + Tyr217Leu and Pro210Ala + Gly215Thr, with substantially similar results.
- Citric acid monohydrate 10.0 15.0
- Citric Acid 7.10 3.00 3.00
- Silicon anti-foam agent 1.16 1.18 1.18
- Anti-foam ing agents 0.06 0.085 0.085
- Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01 % to about 1 % by weight of the composition.
- the bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples.
- Zeolite A (0.1 -.10 ⁇ ) 5.0 5.0 5.0 5.00
- Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20
- Example 33 the BPN' variants recited in Table 2, among others, are substituted for Val203Glu, with substantially similar results.
- Example 34 the BPN' variants recited in Table 2, among others, are substituted for Tyr214Phe + Tyr217Asn, with substantially similar results.
- Example 35-36 any combination of the BPN' variants recited in Table 2, among others, are substituted for Val203Glu and Tyr214Phe + Tyr217Asn, with substantially similar results.
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Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US23793994A | 1994-05-02 | 1994-05-02 | |
US237939 | 1994-05-02 | ||
PCT/US1995/004691 WO1995029979A1 (en) | 1994-05-02 | 1995-04-17 | Fabric cleaning compositions containing subtilisin bpn' variants |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0758373A1 true EP0758373A1 (en) | 1997-02-19 |
Family
ID=22895871
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP95916426A Withdrawn EP0758373A1 (en) | 1994-05-02 | 1995-04-17 | Fabric cleaning compositions containing subtilisin bpn' variants |
Country Status (12)
Country | Link |
---|---|
EP (1) | EP0758373A1 (en) |
JP (1) | JPH09512433A (en) |
CN (1) | CN1151757A (en) |
AU (1) | AU2293195A (en) |
BR (1) | BR9507593A (en) |
CA (1) | CA2189428A1 (en) |
FI (1) | FI964411A0 (en) |
MX (1) | MX9605359A (en) |
NO (1) | NO964590L (en) |
PE (2) | PE12396A1 (en) |
WO (1) | WO1995029979A1 (en) |
ZA (1) | ZA952220B (en) |
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CA2270180C (en) | 1996-11-04 | 2011-01-11 | Novo Nordisk A/S | Subtilase variants and compositions |
US6284246B1 (en) | 1997-07-30 | 2001-09-04 | The Procter & Gamble Co. | Modified polypeptides with high activity and reduced allergenicity |
AU8798198A (en) | 1997-08-29 | 1999-03-22 | Novo Nordisk A/S | Protease variants and compositions |
EP1012251A1 (en) | 1997-08-29 | 2000-06-28 | Novo Nordisk A/S | Protease variants and compositions |
EP1032655B1 (en) | 1997-11-21 | 2005-06-29 | Novozymes A/S | Protease variants and compositions |
US6773907B2 (en) | 1997-11-21 | 2004-08-10 | Peter Kamp Hansen | Subtilase enzymes |
US6780629B2 (en) | 1997-11-21 | 2004-08-24 | Novozymes A/S | Subtilase enzymes |
US6495136B1 (en) | 1998-03-26 | 2002-12-17 | The Procter & Gamble Company | Proteases having modified amino acid sequences conjugated to addition moieties |
US6908757B1 (en) | 1998-03-26 | 2005-06-21 | The Procter & Gamble Company | Serine protease variants having amino acid deletions and substitutions |
CA2325568A1 (en) | 1998-03-26 | 1999-09-30 | The Procter & Gamble Company | Serine protease variants having amino acid substitutions |
AU743760B2 (en) * | 1998-09-22 | 2002-02-07 | Procter & Gamble Company, The | Personal care compositions containing subtilisin enzymes bound to water insoluble substrates |
EP1803817B1 (en) | 1998-12-18 | 2011-04-06 | Novozymes A/S | Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in an active site loop region |
AU4393100A (en) | 1999-05-20 | 2000-12-12 | Novozymes A/S | Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 125 and 126 |
EP1183340B1 (en) | 1999-05-20 | 2008-09-03 | Novozymes A/S | Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 126 and 127 |
ATE408680T1 (en) | 1999-05-20 | 2008-10-15 | Novozymes As | SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUBGROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID BETWEEN POSITIONS 128 AND 129 |
WO2000071687A1 (en) | 1999-05-20 | 2000-11-30 | Novozymes A/S | Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 129 and 130 |
ATE408676T1 (en) | 1999-05-20 | 2008-10-15 | Novozymes As | SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUBGROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID BETWEEN POSITIONS 132 AND 133 |
AU4392900A (en) | 1999-05-20 | 2000-12-12 | Novozymes A/S | Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 127 and 128 |
US6946128B1 (en) | 1999-07-22 | 2005-09-20 | The Procter & Gamble Company | Protease conjugates having sterically protected epitope regions |
MXPA02000840A (en) | 1999-07-22 | 2002-07-30 | Procter & Gamble | Subtilisin protease variants having amino acid substitutions in defined epitope regions. |
EP1206526A2 (en) | 1999-07-22 | 2002-05-22 | The Procter & Gamble Company | Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions |
KR20020021395A (en) | 1999-07-22 | 2002-03-20 | 데이비드 엠 모이어 | Protease conjugates having sterically protected clip sites |
CN1375000A (en) * | 1999-09-09 | 2002-10-16 | 宝洁公司 | A detergent composition containing a protease |
US6727085B2 (en) | 1999-12-15 | 2004-04-27 | Fanoe Tina Sejersgaard | Subtilase variants having an improved wash performance on egg stains |
US6777218B1 (en) | 2000-03-14 | 2004-08-17 | Novozymes A/S | Subtilase enzymes having an improved wash performance on egg stains |
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TWI319007B (en) * | 2002-11-06 | 2010-01-01 | Novozymes As | Subtilase variants |
ATE407202T1 (en) | 2003-05-07 | 2008-09-15 | Novozymes As | ENZYME VARIANTS OF SUBTILISIN (SUBTILASES) |
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EP2013339B1 (en) | 2006-04-20 | 2013-09-11 | Novozymes A/S | Savinase variants having an improved wash performance on egg stains |
US8093200B2 (en) | 2007-02-15 | 2012-01-10 | Ecolab Usa Inc. | Fast dissolving solid detergent |
US20100190682A1 (en) | 2007-04-30 | 2010-07-29 | Sang-Kyu Lee | Use of protein hydrolysates to stabilize metalloprotease detergent formulations |
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WO2009050684A2 (en) | 2007-10-18 | 2009-04-23 | Ecolab Inc. | Pressed, waxy, solid cleaning compositions and methods of making them |
BR112012014082B1 (en) | 2009-12-09 | 2020-12-15 | Danisco Us Inc | isolated protease variant and its production method, isolated nucleic acid, expression vector, recombinant host cell, composition and method for cleaning an item or surface in need of cleaning |
US8758520B2 (en) | 2011-05-20 | 2014-06-24 | Ecolab Usa Inc. | Acid formulations for use in a system for warewashing |
EP2902471A1 (en) | 2011-05-20 | 2015-08-05 | Ecolab USA Inc. | Non-phosphate detergents and non-phosphoric acids in an alternating alkali/acid system for warewashing |
CA2857277C (en) | 2011-12-13 | 2016-10-25 | Ecolab Usa Inc. | Concentrated warewashing compositions and methods |
US9745543B2 (en) | 2012-09-10 | 2017-08-29 | Ecolab Usa Inc. | Stable liquid manual dishwashing compositions containing enzymes |
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AU2013403675B2 (en) | 2013-10-24 | 2017-01-12 | Ecolab Usa Inc. | Compositions and methods for removing soils from surfaces |
WO2021022045A1 (en) | 2019-07-31 | 2021-02-04 | Ecolab Usa Inc. | Personal protective equipment free delimer compositions |
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US5013657A (en) * | 1988-04-12 | 1991-05-07 | Bryan Philip N | Subtilisin mutations |
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ES2110488T3 (en) * | 1990-12-21 | 1998-02-16 | Novo Nordisk As | ENZYMATIC MUTANTS WITH A LOW DEGREE OF VARIATION IN MOLECULAR LOAD IN A PH INTERVAL. |
PL178045B1 (en) * | 1992-07-17 | 2000-02-29 | Genencor Int | Highly alkaline serine proteases |
WO1995007991A2 (en) * | 1993-09-15 | 1995-03-23 | The Procter & Gamble Company | Subtilisin bpn' variants with decreased adsorption and increased hydrolysis |
-
1995
- 1995-03-17 ZA ZA952220A patent/ZA952220B/en unknown
- 1995-04-17 AU AU22931/95A patent/AU2293195A/en not_active Abandoned
- 1995-04-17 WO PCT/US1995/004691 patent/WO1995029979A1/en not_active Application Discontinuation
- 1995-04-17 CA CA002189428A patent/CA2189428A1/en not_active Abandoned
- 1995-04-17 MX MX9605359A patent/MX9605359A/en unknown
- 1995-04-17 EP EP95916426A patent/EP0758373A1/en not_active Withdrawn
- 1995-04-17 BR BR9507593A patent/BR9507593A/en not_active Application Discontinuation
- 1995-04-17 JP JP7528271A patent/JPH09512433A/en active Pending
- 1995-04-17 CN CN95193811A patent/CN1151757A/en active Pending
- 1995-04-24 PE PE1995267036A patent/PE12396A1/en not_active Application Discontinuation
- 1995-04-28 PE PE1995267454A patent/PE49595A1/en not_active Application Discontinuation
-
1996
- 1996-10-30 NO NO964590A patent/NO964590L/en unknown
- 1996-11-01 FI FI964411A patent/FI964411A0/en unknown
Non-Patent Citations (1)
Title |
---|
See references of WO9529979A1 * |
Also Published As
Publication number | Publication date |
---|---|
BR9507593A (en) | 1997-09-23 |
NO964590D0 (en) | 1996-10-30 |
AU2293195A (en) | 1995-11-29 |
CA2189428A1 (en) | 1995-11-09 |
ZA952220B (en) | 1995-12-14 |
CN1151757A (en) | 1997-06-11 |
PE49595A1 (en) | 1996-02-05 |
MX9605359A (en) | 1997-12-31 |
WO1995029979A1 (en) | 1995-11-09 |
NO964590L (en) | 1996-10-30 |
FI964411A (en) | 1996-11-01 |
FI964411A0 (en) | 1996-11-01 |
PE12396A1 (en) | 1996-04-18 |
JPH09512433A (en) | 1997-12-16 |
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