CN1151757A - Fabric cleaning composition containing subtilisin BPN' variants - Google Patents
Fabric cleaning composition containing subtilisin BPN' variants Download PDFInfo
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- CN1151757A CN1151757A CN95193811A CN95193811A CN1151757A CN 1151757 A CN1151757 A CN 1151757A CN 95193811 A CN95193811 A CN 95193811A CN 95193811 A CN95193811 A CN 95193811A CN 1151757 A CN1151757 A CN 1151757A
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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
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- 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
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- 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
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Abstract
The present invention relates to fabric cleaning compositions comprising subtilisin BPN' variants, wherein the BPN' variant comprises one or more 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 wild-type subtilisin BPN'.
Description
Technical field
The present invention relates to comprise the fabric cleaning composition of proteolytic enzyme, proteolytic enzyme wherein is the mutation of subtilysin.
Technical background
Enzyme is the maximum naturally occurring protein of a class.The different types of chemical reaction of the general catalysis of every fermentoid (promotion is reacted but can be consumed) itself.Known class of enzymes is a proteolytic enzyme, and other proteinic ability of their hydrolysis (compound decomposition is become two or more simple compounds, and these compounds have kept H and the OH part at the water molecules of the chemical bond either side that disconnects) is known.By naturally occurring proteolytic enzyme and the proteinic proteolytic enzyme of artificial generation are added in the detergent for washing clothes goods as additive, the ability of their protein hydrolysates has obtained utilization.Many spots on clothing are dirts of protein, and the wide proteolytic enzyme of characteristic can improve the removal to these spots basically.
Regrettably being in these virtuous protein natural, the bacterium environment usually can not convert to and be suitable for relative non-natural wash environment.Specifically, use outside the natural surroundings of proteolytic enzyme, the characteristic of the characteristic of proteolytic enzyme such as thermostability, pH stability, oxidative stability and matrix is not required the best.
The aminoacid sequence of proteolytic enzyme has determined the characteristic of proteolytic enzyme.The variation of proteolytic enzyme aminoacid sequence can change the character of enzyme, changes the grade of enzyme or even can make enzyme deactivation, position, character and/or quantity that this depends in aminoacid sequence to be changed.The aminoacid sequence that has adopted several schemes variation mild proteolytic enzyme is to attempt to improve their character, and purpose is to strengthen the effectiveness of proteolytic enzyme in wash environment.These schemes comprise that the change aminoacid sequence is to strengthen the oxidative stability under multiple condition in thermostability under the multiple condition and improvement.
Although described multiple scheme in the prior art, but still continuous demand is used for the composition that comprises the effective mutation of proteolytic enzyme on cleaning fabric surface.
The object of the invention
The object of the invention provides the fabric cleaning composition that comprises the subtilysin enzyme variant.
General introduction
The present invention relates to comprise the composition that is used for the cleaning fabric surface of subtilysin BPN ' mutation.BPN ' the mutation that is used for these compositions than mild subtilysin BPN ' at least one, there is different aminoacids (being surrogate) at two or three amino acid position places that clearly determine, this BPN ' mutation is compared with mild subtilysin BPN ' and has been reduced for the absorption of insoluble matrix and strengthened hydrolysis to insoluble matrix thus.
Detailed description 1. is used for the subtilisin BPN ' variants of fabric cleaning composition
The present invention relates to comprise the subtilysin enzyme, the fabric cleaning composition of BPN ' especially, described enzyme are that the aminoacid sequence that the various nucleotide sequences by the sudden change codase have changed enzyme thus is modified.The subtilysin enzyme (hereinafter claim " BPN ' mutation ") that is used for the modification of the present composition is compared with the subtilysin of mild and has been reduced the absorption of insoluble matrix and strengthened hydrolysis to insoluble matrix.In these BPN ' mutation some is described in the common pending application U.S.S.N.08/121 of people such as Brode in application on September 15th, 1993, in 437.
The subtilysin enzyme that is used for the present composition belongs to the class of enzymes that is known as proteolytic enzyme.Proteolytic enzyme is the catalyzer of division peptide bond.One type proteolytic enzyme is serine protease.The characteristics of serine protease are to have the residue that is mainly Serine at reactive site.
The speed that has many data proofs to observe the enzymic hydrolysis dissolvable matrix increases with enzyme concn.For for example for the matrix thing that is combined in the surface that is run into during many cleanings are used, hydrolysis rate increases and increases along with surface concn, and this is seemingly rational.Existing illustrated for this situation.(Brode, P.F.III and D.S.Rauch, LANGMUIR, " subtilysin enzyme BPN ': the activity on motionless matrix (Subtilisin BPN ': Activity on anImmobilized Substrate) ", 8 volumes, pp.1325-1329 (1992).In fact when the surface concn of enzyme changes, between the hydrolysis rate of discovery insoluble matrix thing and the surface concn linear relationship is arranged.(Rubingh, D.N. with " in of the catalysis (Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface.) of protein-solution interface place proteolytic enzyme " in POLYMER SOLUTIONS of M.D.Bauer to hydrolysis, BLENDS AND INTERFACES, publish by I.Noda and D.N.Rubingh, Elsevier, p, 464 (1992)).Be that we do not find that the enzyme that adsorbs more matrix thing can provide preferable performance when attempting to use this principle and seek the proteolytic enzyme mutation that provides better cleaning fabric performance unexpectedly.In fact we unexpectedly have been measured to opposite situation: reduce enzyme adsorbs the hydrolysis that has but caused the matrix thing to the matrix thing enhancing (that is, producing cleaning performance preferably).
Do not wish to accept keeping within bounds of opinion, but be sure of when the time a kind of mutation and other comparing, this improved performance is the result of the following fact, promptly adsorb also combined not too firm of few enzyme, so it has than high workability on the surface of the insoluble protein matrix thing that will be removed.Under the concentration of comparable enzyme solution, the flowability of this increase with discharge the higher concentration enzyme to the surface and compare and be enough to surpass any advantage.
Mutagenicity described herein refers to the variation (promptly reduce) of enzyme to the absorption of bonding dirt from the teeth outwards.In BPN ', form one than the imperial palace ring at the enzyme molecule from the amino acid of position, 199 position to 220.Found that this ring is adsorbed with remarkably influenced for playing remarkable effect and the specific change in this ring aspect the absorption that is combined in lip-deep peptide for this at the enzyme molecule.Do not wish to accept opinion and keep within bounds, believe that this ring has at least two reasons for the importance of BPN ' molecular adsorption effect.Amino acid in the first this interior ring can have close the contact with any surface that molecule is exposed.The binding site of the approaching and BPN ' molecule of second this ring and activity site gives enzyme certain effect at the enzyme that catalysis produces aspect the absorption that is combined in lip-deep matrix thing (peptide/albumen dirt).
" mutation " used herein meaning is the enzyme with the aminoacid sequence that is different from mild.
" mutant BPN ' gene " used herein meaning is the gene of coding BPN ' mutation.
" mild subtilysin BPN " used herein refers to the subtilysin enzyme of being represented by SEQ ID NO:1.The aminoacid sequence of subtilysin BPN ' is in addition by Wells, J.A., E.Ferrari, D.J.Henner, D.A.Estell and E.Y.Chen are described in nucleic acids research (NUCLEIC ACIDS RESEARCH), the II volume, among the 7911-7925 (1993), it is cited for referencial use at this paper.
Term used herein " mild aminoacid sequence " comprises SEQ ID NO:1 and locate vicissitudinous SEQ ID NO:1 beyond in the 199-220 position in aminoacid sequence.
" easy hydrophilic amino acid " used herein refers to any other amino acid that has bigger hydrophilic power with reference to the sample amino acid of the hydrophilic power in the following table than having.Be that the amino acid put by the descending order row of the hydrophilic power that increases is (referring to Hopp in the following table (table 1), T.P., and Woods, K.R., " Prediction of Protein Antigenic Determinants from AminoAcid Sequences ", PROCEEDINGS OF THE NATIONALACADEMY OF SCIENCE USA, 78 volumes, pp.3824-3828,1981, the document is quoted for referencial use at this paper).
Table 1
| Amino acid | Hydrophilic power value |
| ????Trp | ????-3.4 |
| ????Phe | ????-2.5 |
| ????Tyr | ????-2.3 |
| ????Leu，Ile | ????-1.8 |
| ????Val | ????-1.5 |
| ????Met | ????-1.3 |
| ????Cys | ????-1.0 |
| ????Ala，His | ????-0.5 |
| ????Thr | ????-0.4 |
| ????Pro，Gly | ????-0.0 |
| ????Gln，Asn | ????0.2 |
| ????Ser | ????0.3 |
| ??Arg +，Lys +，Glu -，Asp - | ????3.0 |
Table 1 has illustrated that also amino acid can have electric charge (it is about 8-9 that this character is based on pH).Positively charged amino acid is Arg and Lys, and electronegative amino acid is Glu and Asp, and all the other amino acid are neutral.In the preferred embodiment of the invention, substituted amino acid is neutral or electronegative, more preferably is electronegative (being Glu and Asp).
Therefore, for example this statement " with of equal value or easier hydrophilic aminoacid replacement Gln neutral or that the have negative charge " meaning is that Gln will be by Asn (it has the hydrophilic power that equates with Gln) or Ser, Glu or Asp (they are than the easier hydrophilic power of Gln) replacement; They are neutral or have negative charge, and than Gln bigger hydrophilic power value are arranged.Similar this statement " with easier to be hydrophilic aminoacid replacement Pro neutral or that the have negative charge " meaning is that Pro will be replaced by Gln, Asn, Ser, Glu or Asp.A. comprise the mutation of at least one place aminoacid replacement
In one embodiment of this invention, BPN ' mutation comprises the mild aminoacid sequence, and wherein a place or the many places of this mild aminoacid sequence in 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,218,219 or 220 positions are substituted; This BPN ' mutation is compared with mild subtilysin BPN ', has reduced the absorption of insoluble matrix thing and has strengthened hydrolysis to the insoluble matrix thing.Preferred substituted amino acid position is 199,200,201,202,205,207,208,209,210,211,212 or 215; More preferably 200,201,202,205 or 207.
The preferred amino acid that replaces 199 positions is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 200 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 201 positions is Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 202 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 203 positions is Met, Cys, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 204 positions is Glu.
The preferred amino acid that replaces 205 positions is Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 206 positions is Pro, Asn or Ser.
The preferred amino acid that replaces 207 positions is Asp or Glu.
The preferred amino acid that replaces 208 positions is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 209 positions is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 210 positions is Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 211 positions is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 212 positions is Gln, Ser, Asp or Glu.
The preferred amino acid that replaces 213 positions is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 214 positions is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu.
The preferred amino acid that replaces 215 positions is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 216 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 218 positions is Glu.
The preferred amino acid that replaces 219 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 220 positions is Pro, Gly, Gln, Asn, Asp or Glu.
The amino acid that more preferably replaces optional position 199,200,201,202,203,205,207,208,209,210,211,212,213,214,215,216,219 and 220 is neutrality or electronegative with reference to table 1, than the amino acid on the corresponding position of mild subtilysin BPN ' suitable or easier hydrophilic power are arranged, preferably easier to be hydrophilic.
The preferred amino acid that replaces optional position 199,200,201,202,203,205,207,208,209,210,211,212,213,214,215,216,219 and 220 in addition is Asp or Glu; The amino acid that replaces 204 or 218 positions is G1u.B. comprise the mutation of at least two place's aminoacid replacement
In another embodiment of the present invention, BPN ' mutation comprises the mild aminoacid sequence, and wherein two places or the many places of this mild aminoacid sequence in 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 or 220 positions are substituted; This BPN ' mutation is compared with mild subtilysin BPN ', has reduced the absorption of insoluble matrix thing and has strengthened hydrolysis to the insoluble matrix thing.The position that preferably has substituted amino acid is 199,200,201,202,205,207,208,209,210,211,212 or 215; More desired position is 200,201,202,205 or 207.
The preferred amino acid that replaces 199 positions is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 200 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 201 positions is Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 202 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 203 positions is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 204 positions is Asp or Glu.
The preferred amino acid that replaces 205 positions is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 206 positions is Pro, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 207 positions is Asp or Glu.
The preferred amino acid that replaces 208 positions is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 209 positions is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 210 positions is Ala, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 211 positions is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 212 positions is Gln, Ser, Asp or Glu.
The preferred amino acid that replaces 213 positions is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser or Asp.
The preferred amino acid that replaces 214 positions is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 215 positions is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 216 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 217 positions is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 218 positions is Gln, Ser, Asp or Glu.
The preferred amino acid that replaces 219 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 220 positions is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The amino acid that more preferably replaces optional position 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 or 220 is neutrality or electronegative with reference to table 1, than the amino acid on the corresponding position of mild subtilysin BPN ' suitable or easier hydrophilic power are arranged, preferably easier to be hydrophilic.
The preferred amino acid that replaces optional position 199,200,201,202,203,204,205,206,207,208,209,210,211,212,214,215,216,218,219 or 220 in addition is Asp or Glu; That replace 217 positions is Leu, Asp or Glu; That replace 213 positions is Asp.C. comprise the mutation of at least three place's aminoacid replacement
In another embodiment of the present invention, BPN ' mutation comprises the mild aminoacid sequence, and wherein three places or the many places of this mild aminoacid sequence in 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 and 220 positions are substituted; This BPN ' mutation is compared with mild subtilysin BPN ', has reduced the absorption of insoluble matrix thing and has strengthened hydrolysis to the insoluble matrix thing.The position that preferably has substituted amino acid is 199,200,201,202,205,207,208,209,210,211,212 or 215; More desired position is 200,201,202,205 or 207.
The preferred amino acid that replaces 199 positions is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 200 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 201 positions is Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 202 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 203 positions is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 204 positions is selected from Asp or Glu.
The preferred amino acid that replaces 205 positions is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 206 positions is Pro, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 207 positions is Asp or Glu.
The preferred amino acid that replaces 208 positions is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 209 positions is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 210 positions is Ala, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 211 positions is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 212 positions is Gln, Ser, Asp or Glu.
The preferred amino acid that replaces 213 positions is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 214 positions is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 215 positions is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 216 positions is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 217 positions is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 218 positions is Gln, Ser, Asp or Glu.
The preferred amino acid that replaces 219 positions is Pro, Gln, Asn, Ser, Asp or Glu.
The preferred amino acid that replaces 220 positions is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
The amino acid that more preferably replaces optional position 199,200,201,202,203,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 or 220 is neutrality or electronegative with reference to table 1, than the amino acid on the corresponding position of mild subtilysin BPN ' suitable or easier hydrophilic power are arranged, preferably easier to be hydrophilic.
The preferred amino acid that replaces optional position 199,200,201,202,203,204,205,206,207,208,209,210,211,212,214,215,216,218,219 or 220 in addition is Asp or Glu; That replace 217 positions is Leu, Asp or Glu; That replace 213 positions is Asp.D. the preparation of enzyme variant
Embodiment 1
Mutant BPN ' gene
The medium phage (phagemid) that will contain mild subtilysin BPN ' gene is (Mitchinson (pSS-5), C. and J.A.Wells (1989), " ProteinEngineering of Disulfide Bonds in Subtilisin BPN '; BIOCHEMISTRY; 28 volumes; 4807-4815 page or leaf) change into bacillus coli ung-bacterial strain CJ236, and use VCSM
13The helper phage preparation contains the dna profiling (Kunkel of strand uridylic, T.A., J.D.Roberts and R.A.Zakour, " Rapid and efficientsite-specific mutagenesis without phenotypic selection ", METHODS IN ENZYMOLOGY, 154 volumes, pp.367-382 (1987); By Yuckenberg, P.D., F.Witney, J.Geisselsoder and J.McClary revise, " Site-directed in vitro mutagenesis using uracil-containingDNA and phagemid vectors ", DIRECTED MUTAGENESIS-APRACTICAL APPROACH is published by M.J.McPherson, pp.27-48 (1991); These two pieces of documents are quoted for referencial use at this paper).Use the Zoller of direct single primer point mutagenesis and the modification method (Zoller of Smith, M.J., and M.Smith, " Oligonucleotide-directed mutagenesis using M13-derivedvectors:an efficient and general procedure for the productionof point mutations in any fragment of DNA " NUCLEICACIDS RESEARCH, 10 volumes, pp.6487-6500 (1982), the document quotes for referencial use at this paper) prepare all mutant (representing 1991 basically by people such as above Yuckenberg).Use Applied Biosystem Inc.380B DNA synthesizer to prepare oligonucleotide.The mutagenesis reaction product is converted to bacillus coli bacterial strain MM 294 (American Type Culture Collection E.Coli.33625).All mutant are determined by DNA sequence, separated DNA is converted into subtilis expression strain BG 2036 (Yang, M.Y., E.Ferrari and D.J.Henner (1984), " Cloning of theNeutral Protease Gene of Bacillus Subtillis and the Use ofthe Cloned Gene to Create an in Vitro-derived DeletionMutation ", JOURNAL OF BACTERIOLOGY, 160 volumes, pp.15-21).The pSS-5 mutant that has some modifications of the codon mutation that stops frameshit at amino acid 217 places is used to prepare the uridylic template.Make the normal read sign indicating number of oligonucleotide recovery in 217 positions, and also to be proofreaied and correct the sudden change that stops frameshit and generation official energy enzyme by random substituent coding (for all the three kinds of matrix under these codons, being the mixture that waits mole and/or change of all four kinds of Nucleotide) in 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 and 220 positions be by the ability evaluation of their casein digestions.Random substituent is determined by DNA sequence.
Embodiment 2
Fermentation
The useful subtilysin mutant that contains bacillus subtilis mycetocyte (BE 2036) is grown to medium-Log phase in 1 liter LB-dextrose bouillon culture, and to be inoculated into cumulative volume be 10 liters Biostat ED fermentor tank (B.Braun Biotech, Inc.Allentown, Pennsylvania) in.Fermentation media contains yeast extract, starch, kilfoam, buffer reagent and trace minerals (referring to FERMENTATION:A PRACTICAL APPROACH, publishing 1990 by B.McNeil and L.M.Harvey).To be maintained at pH constant be 7.0 to meat soup during the fermentation.Adding paraxin is used for the microbiotic of the plasmid of mutagenic treatment is selected.Cell is about 60 37 ℃ of following grow overnight to A600, and is collected.
Embodiment 3
Refining
The meat soup of fermentation is obtained pure enzyme through following steps.This meat soup goes out the bacillus subtilis mycetocyte clearly through centrifugal, and removes fine particle with the 100K cut film and make its clarification.Then on the 10K cut film, concentrate and reduce ionic strength, adjust pH to 5.5 with 0.025M MES buffer reagent (2-(N-morpholino) ethane sulfonic acid) through the via flow dialysis.Pack into this enzyme in cation-exchange chromatography post or the affinity adsorption chromatography post and this enzyme of wash-out from post is further made with extra care (referring to Scopes enzyme with NaCl or propylene glycol gradient, R.K.PROTEIN PURIFICATIONPRINCIPLES AND PRACTICE, Springer-Verlag, New York (1984), the document is quoted for referencial use at this paper).
Use pNA test (DelMar, E.G., C.Largman, J.W.Brodrick and M.C.Geokas, ANAL.BIOCHEM., Vol.99, pp.316-320 (1979), the document is quoted for referencial use at this paper) be determined at the organized enzyme concentration in the cut of collecting in the gradient elution process.This test determination enzymic hydrolysis synthetic dissolvable matrix thing: the speed of the p-Nitroaniline of succinyl--Ala-Ala-proline(Pro)-phenylalanine-p-nitroanilide (sAAPF-pNA) is discharged.On spectrophotometer, measure the xanchromatic speed that produces by hydrolysis reaction at the 410nm place, itself and organized enzyme concentration are proportional.In addition, measure absorption at the 280nm place to determine total protein concentration.Obtain enzyme purity by organized enzyme/gross protein ratio, and identify collected stoste cut with this ratio.
For fear of enzyme self-dissolving in storage process, can with etc. the propylene glycol of weight add by chromatographic column and collect in the cut that obtains.When this purification step is finished, test the purity of Zhengyuan enzyme solution with SDS-PAGE (SDS-PAGE), with use 11-T type trypsin inhibitor: (st.Louis, Missouri) the turkey eqq white of Gou Maiing passes through avtive spot titration measuring enzyme absolute concentration by Sigma Chemical Company.The transformation factor of being measured will show that variation that in enzyme molecule different positions place produces has obtained having than the enzyme of the mild enzyme variant for dissolvable matrix thing pNA enhanced activity.
When preparing to use, proenzyme liquid is removed propylene glycol and exchange buffering agent through Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion post wash-out.MES buffer reagent in proenzyme liquid is replaced by 0.1M Tris buffer reagent, and (three (methylol-aminomethanes), this buffer reagent contains 0.01M CaCl
2, pH is adjusted to 8.6 with HCl.All tests are to be 8.6 at pH, and under the Tris buffer reagent, constant temperature carries out under 25 ℃.E. the characteristic of enzyme variant
Embodiment 4
The surface preparation model
Use the controlled glass with hole of aminopropyl (CPG) bought by CPG Inc. (Fairfield, New Jersey) as with by Bachem, Inc (Torrence, California) the covalently bound carrier of sAAPF-pNA matrix thing of Gou Maiing.This is reflected in the dimethyl sulfoxide (DMSO) and carries out, use (1-ethyl-3-[3-(dimethylamino) propyl group] the carbodiimide hydrochloride) (EDC) as sequestrant.(survey) when reaction is finished, remove excessive solvent,, sweep oven dry with the N2 air-blowing down at about 70 ℃ then with dimethyl sulfoxide (DMSO) (DMSO) and second distillation water rinse CPG:sAAPF-pNA by pNA test mirror.The preparation of this reaction scheme and immobilization matrix is undertaken by following document description: Brode, P.F.III, with D.S.Rauch " Subtilisin BPN ': Activity on an Immobilized Substrate; " LANGMUIR, Vol.8, p.1325-1329 (1992), the document is quoted for referencial use at this paper.
The CPG surface has 62,000 ± 7,000 pNA molecule/μ m
2For the CPG that receives, surface-area will keep the 50.0m by CPG Inc. report
2/ g value is constant.This has pointed out and has used the step that adds sAAPF-pNA on CPG not destroy pore structure (mean diameter is 486A).
Embodiment 5
The surface hydrolysis test
Use CPG:SAAPF-pNA in a test, can measure the absorption of enzyme variant and in conjunction with the hydrolysis of CPG peptide.A spot of enzyme variant original solution is added in the degased flask that contains Tris buffer reagent and CPG:SAAPF-pNA.Flask is placed on the pendulum ring type transmission wobbler shook 90 minutes, this wobbler pauses at the different time interval (for example at initial adsorption of hydrolyzation in the stage therebetween, for example begin 20 minutes, paused once in per 2 minutes, back to reaction finishes to pause once in per 10 minutes).CPG:s AAPF-pNA is fixed, and this solution is as sample.This testing sequence and be by people such as above Brode to the absorption and the calculating of hydrolysis, 1992 descriptions are carried out.
Monitor the stability of the anti-self-dissolving of all enzymes, showing does not have tangible self-dissolving loss in this process of the test.Therefore, can measure the absorption of enzyme by measuring solution loss.Enzyme variant initial concentration and the difference between the concentration that each different time points is measured are the amount of the enzyme variant that is adsorbed.Get aliquots containig and calculate pNA amount by surface hydrolysis at the absorbancy reading at 410nm place.PNA total amount with remaining amount addition calculation hydrolysis in sample size and the flask.By deducting when not having enzyme to exist, be 8.6 at pH, the pNA that is hydrolyzed under the Tris buffer reagent measures and proofreaies and correct above numerical value.This alkaline hydrolysis is the 7-29% of total hydrolysis, and this depends on the effectiveness of enzyme.
Embodiment 6
The dissolvable matrix dynamic analysis
Monitor the hydrolysis rate of dissolvable matrix sAAPF-pNA by the absorption value of on the DU-70 spectrophotometer, measuring increase in time in the 410nm place.Constant and preparation simultaneously for each kinetic determination, changes matrix substrate concentration from 90-700 μ M sAAPF-pNA in the enzyme concn of 6-10 nmole scope.Get an absorption data p.s., through 900 seconds, these data were indicated on LOTUS
TMArticulation statement (Lotus Development Corporation, Cambridge, Massachusetts) on.With standard Lineweaver Burk analytical method kinetic parameter is analyzed, wherein the operation initial portion (generally being first minute) data adapting in the linear regression curve, obtain V thus
0With the standard handovers method with V
0And S
0Drawing obtains K
MAnd Kcat.F.BPN ' mutation example
BPN ' mutation of the present invention has been described in following table 2, and it has reduced the absorption that is combined in lip-deep matrix thing and has increased being combined in the hydrolysis of lip-deep matrix thing.In describing this specific sudden change, what at first provide is the amino acid of the existence of beginning most of gentle type, secondly is the Position Number at place, is the amino acid that replaces once more.
Table 2
BPN ' mutation example
-single sudden change-
Ala?216?Glu
Ala?216?Asp
Ala?216?Gly
Val?203?Glu
-two sudden changes-
Ile?205?Leu+Ala?216?Glu
Ile?205?Leu+Ala?216?Asp
Pro?210?Ala+Gly?215?Thr
Tyr?214?Phe+Tyr?217?Asn
Gln?206?Glu+Ala?216?Glu
Ala?216?Glu+Try?217?Leu
Gln?206?Glu+Tyr?217?Leu
The sudden change of-three places-
Gln?206?Glu+Ala?216?Glu+Tyr?217?Leu
Gln?206?Pro+Gly?211?Ala+Ala?216?Glu
-sudden change everywhere-
Val?203?Glu+Gln?206?Glu+Ala?216?Glu+Tyr?217?Leu
Val?203?Glu+Pro?210?Ala+Ala?216?Glu+Tyr?217?Leu
-five sudden change-Val of place 203 Glu+Gln 206 Glu+Gly 215 Thr+Ala 216 Glu+Tyr 217 Leu Val 203 Glu+Pro 210 Ala+Gly 215 Thr+Ala 216 Glu+Tyr 217 LeuII. fabric cleaning composition materials
Except containing above-described BPN ' mutation, fabric cleaning composition of the present invention also can comprise one or more cleaning combination materials compatible with proteolytic enzyme.Term used herein " cleaning combination material " meaning is by the cleaning combination of required specific type and the selected any liquid of product form (for example liquid, particle, bar), solid or gaseous substance, and used BPN ' mutation also is compatible in these materials and the composition.Consider the fabric that is cleaned and in use can easily carry out specific selection the cleaning combination material to the composition (for example using detergent washing) of cleaning condition desired form.The term of Shi Yonging " compatible " meaning is that can not make the proteolytic activity of BPN ' mutation required in normal environment for use reduce to proteolytic enzyme be invalid degree to the cleaning combination material in this article.Describe specific cleaning combination material hereinafter in detail.
" fabric cleaning composition " used herein refers to the form of ownership of the detergent composition that is used for cleaning fabric, includes but not limited to particle, liquid and stick form.Preferred fabric cleaning composition is a liquid form.
The enzyme that " enzyme variant of significant quantity " used herein refers to acquisition to be needed in specific cleaning combination is urged active required enzyme variant amount.This significant quantity determined by those skilled in the art easily, and this is based on many factors, for example employed specific enzymes mutation, clean use, the composition of specific cleaning composition, whether need liquid or drying (for example particle) composition, or the like.Cleaning combination of the present invention preferably comprises one or more enzyme variants of about 0.0001%-10%, more preferably about 0.001%-1%, and 0.01%-0.1% more preferably from about.At the following several examples that further go through the different cleaning combinations of the present invention.Except as otherwise noted, all umbers used herein, percentage ratio and ratio are all by weight.
Enzyme variant of the present invention can use laundering of textile fabrics composition fully to be prepared with the component of various routines.This based composition can be liquid, particle or the like.This based composition can be formulated as contain as many as 30%-60% (weight) tensio-active agent " concentrate " washing composition now.
Fabric cleaning composition of the present invention optionally and preferably contains tensio-active agents such as various negatively charged ion, nonionic, zwitter-ion.
The general content of these tensio-active agents is about 5% to about 35% of composition.
The example of the unrestriced tensio-active agent of Shi Yonging comprises conventional C in the present invention
11-C
18Alkylbenzene sulfonate and uncle and random alkyl-sulphate, formula is CH
3(CH
2)
x(CHOSO
3 -M
+) CH
3And CH
3(CH
2)
y(CHOSO
3 -M
+) CH
2CH
3C
10-C
18Secondary (2,3) alkyl-sulphate, x and (y+1) be integer wherein at least about 7, preferably at least about 9, M is a water lyotropy positively charged ion, particularly sodium, C
10-C
18Alkyl alkoxy sulfate (especially EO 1-5 ethoxy sulfate), C
10-C
18Alkyl alkoxy carboxylate salt (especially EO 1-5 ethoxy carboxylate), C
10-C
18Alkyl polyglycoside and the many glycosides of they corresponding sulfations, C
12-C
18α-sulfonated fatty acid ester, C
12-C
18Alkyl and alkyl phenolic alkoxy thing (especially ethoxylate and blended oxyethyl group/propoxy-), C
12-C
18Trimethyl-glycine and sultaine (" sultaines "), C
10-C
18Amine oxide or the like.Preferred alkyl alkoxy sulfate of the present invention (AES) and alkyl alkoxy carboxylate salt (AEC).(also preferably this class tensio-active agent and aforesaid amine oxide and/or trimethyl-glycine or sultaine tensio-active agent are used in combination, this depends on the teacher's that fills a prescription requirement), other conventional useful tensio-active agent is listed in the standard textbook.Useful especially tensio-active agent comprises the C in the U.S. patent 5,194,639 that is disclosed in the people such as Connor that authorized on March 16th, 1993
10-C
18The N-methyl glucose amide, the document is quoted for referencial use at this paper.
Multiple other component useful in fabric cleaning composition can be included in the present composition, comprise other active ingredient, carrier, hydrotropic agent, processing aid, dyestuff or pigment, be used for the solvent of liquid formulations etc.Additionally increase foaming power if desired, can in composition, mix general about 1% to about 10% suds booster, as C
10-C
16Alkylolamide.C
10-C
14Single ethanol amide and diglycollic amide are the typical classes of this suds booster.It also is useful that this suds booster is added with high foaming power auxilliary that tensio-active agent such as above-mentioned amine oxide, trimethyl-glycine and sultaine use.If necessary, solubility magnesium salts such as MgCl
2, MgSO
4Add so that extra foaming power to be provided Deng amount that generally can about 0.1%-2%.
Liquid fabric cleaning combination of the present invention can contain water and other solvent as carrier.Lower molecular weight uncle or secondary alcohol, its typical example is that methyl alcohol, ethanol, propyl alcohol and Virahol suit.Monohydroxy-alcohol is preferred solubilizing surfactant, also can use but polyvalent alcohol for example contains have an appointment 2-6 carbon atom and about 2-6 hydroxyl those (for example 1, ammediol, ethylene glycol, glycerine and 1,2-propylene glycol).Can contain the 5%-90% that has an appointment in the composition, this class carrier of generally about 10%-50%.
Fabric cleaning composition of the present invention preferably is configured to and is being used for aqueous washing operation, and washing water have the about 6.8-11.0 of pH.Finished product is generally prepared in this scope.Control pH comprises use buffer reagent, alkali, acid etc. for the technology of suggestion use value, and these are well known to a person skilled in the art.
When preparation fabric cleaning composition of the present invention, prescription Shi Xiwang uses the various washing assistants of content for about 5%-50% (weight).Typical washing assistant comprises 1-10 micron zeolite, polycarboxylate such as citric acid and oxygen di-succsinic acid, layered silicate, phosphoric acid salt etc.Other conventional washing assistant is listed in the standard recipe book.
In addition, prescription Shi Xiwang uses the various additional enzymes of general consumption for about 0.001%-1% (weight) in this composition, as cellulase, lipase, amylase and proteolytic enzyme.Various enzyme fabric cares are known in the detergent for washing clothes technology.
Various bleaching compounds can be used in this composition as percarbonate, perborate etc., and general consumption is about 1%-15% (weight).If necessary, this composition also can contain bleach-activating agent such as tetraacetyl ethylene diamine, nonanoly acyloxy benzene sulfonate etc., and they also are well-known in the art.Its consumption is generally about 1%-10% (weight).
Various dirt release agents, especially negatively charged ion oligomer ester type, various sequestrants, especially amino phosphonates do and ethylenediamine disuccinate, various clay soil remover, especially the whitening agent of the tetren of ethoxylation, various dispersion agent, especially polyacrylate and polyaspartic acid salts, various whitening agent, especially anionic property, various suds suppressor, especially polysiloxane and secondary alcohol, various fabric softeners, especially smectic clays etc. all can be used in this composition, and its amount ranges is about 1%-35% (weight).In standard recipe book and disclosed patent, these conventional substances there are many detailed descriptions.
Enzyme stabilizers also can be used in the cleaning combination of the present invention, and this kind of enzyme stablizer comprises propylene glycol (preferably about 1%-10%), sodium formiate (preferably about 0.1%-1%) and calcium formiate (preferably about 0.1%-1%).A. granular fabric cleaning composition
The granular fabric cleaning composition of the present invention contains one or more enzyme variants of the present invention of significant quantity, preferably contains the about 0.001%-10% for composition weight, more preferably about 0.005%-5%, and 0.01%-1% organized enzyme most preferably from about.Except one or more enzyme variants, granular fabric cleaning composition generally comprises at least a tensio-active agent, one or more washing assistants and contains SYNTHETIC OPTICAL WHITNER in some cases.
By following examples granular fabric cleaning composition of the invention process is described.
Embodiment 7-10
Granular fabric cleaning composition
The embodiment numbering
Component 789 10Ala 216 Glu 0.10 0.20 0.03 0.05Gln 206 Glu+Tyr 217 Leu--0.02 0.05C13 linear alkylbenzene sulfonate (LAS)s 22.00 22.00 22.00 22.00 phosphate (for sodium phosphate trimer) 23.00 23.00 23.00 23.00 sodium carbonate, 23.00 23.00 23.00 23.00 sodium metasilicate, 14.00 14.00 14.00 14.00 zeolites, 8.20 8.20 8.20 8.20 chelating agents (diethylene-triamine pentaacetic acid), 0.40 0.40 0.40 0.40 sodium sulphate, 5.50 5.50 5.50 5.50 water surplus add to 100%
In embodiment 7-8, replace Ala 216 Glu with the BPN ' mutation except Ala 216 Glu listed in the table 2, obtain similar substantially result.
In embodiment 9-10, replace Ala 216 Glu and Gln 206 Glu+Tyr 217 Leu with other listed in the table 2 any BPN ' mutation combination, obtain similar substantially result.
Embodiment 11-14
Granular fabric cleaning composition
Embodiment numbers component 11 12 13 14Gln 206 Glu+Ala 216 Glu+Tyr 217 Leu 0.10 0.20 0.03 0.05Pro 210 Ala+Gly 215 Thr--0.02 0.05C
12Sad 4.00 4.00 4.00 4.00C of alkylbenzene sulfonate 12.00 12.00 12.00 12.00 zeolite A (1-10 micron) 26.00 26.00 26.00 26.002-butyl
12-C
14Secondary (2,3) sodium alkyl sulfate 5.00 5.00 5.00 5.00 Trisodium Citrates, 5.00 5.00 5.00 5.00 white dyes 0.10 0.10 0.10 0.10 sodium sulfate, 17.00 17.00 17.00 17.00 water and trace ingredients surplus add to 100%
In embodiment 11-12, replace Gln 206Glu+Ala 216 Glu+Tyr 217 Leu with other listed in the table 2 BPN ' mutation, obtain similar substantially result.
In embodiment 13-14, replace Gln 206 Glu+Ala 216 Glu+Tyr, 217 Leu and Pro 210 Ala+Gly 215Thr with other listed in the table 2 any BPN ' mutation combination, obtain similar substantially result.
Embodiment 15 and 16
Granular fabric cleaning composition
Component embodiment numbering
15 16 linear alkylbenzene sulfonates, 11.4 10.70 tallow alkyl vitriol, 1.80 2.40 C
14-15The C of 3.00 3.10 7 times of ethoxylations of alkyl-sulphate
14-15Tallow alcohol 1.80 1.80 dispersants 0.07 0.1 polysiloxane fluid 0.80 0.80 trisodium citrate 14.00 15.00 citric acids 3.00 2.50 zeolites 32.50 32.10 maleic acid acrylic copolymers 5.00 5.00 diethylenetriamine pentamethylenophosphonic acids 1.00 0.20 Ala 216 Glu+Tyr 217 Leu 0.30 0.30 lipase 0.36 0.40 amylase 0.30 0.30 sodium metasilicate 2.00 2.50 sodium sulphate 3.50 5.20 polyvinylpyrrolidones 0.30 0.50 perborate 0.5 1 phenolsulfonates 0.1 0.2 peroxidases 0.1 0.1 micro constitutents to 100 of 4.00 4.00 11 times of ethoxylations of alcohol are to 100
Embodiment 17 and 18
Granular fabric cleaning composition
Component embodiment numbering
17 18 straight chain C
12Sodium alkyl benzene sulfonate 6.5 8.0 sodium sulphate 15.0 18.0 Wessalith CSs 26.0 22.0 sodium nitrilo triacetates 5.0 5.0 polyvinylpyrrolidones 0.5 0.7 tetraacetyl ethylene diamines 3.0 3.0 boric acid 4.0-perboric acids 0.5 1 phenolsulfonates 0.1 0.2 Ile 205 Leu+Ala 216 Glu 0.4 0.4 fillers (silicate for example; Carbonate; Spices; Water) to 100 to 100
Embodiment 19
Concentrate granular fabric cleaning composition
The C of 2.0 3 times and 7 times ethoxylations of composition weight % alkyl-sulphate 8.0 alkyl ethoxy sulfates
25And C
45Copolymer 0.1 PEG 2,000 0.2 Val 203 Glu+Gln 206 Glu+Ala 216 Glu+Tyr 217 Leu 0.5 lipase 0.2 cellulase 0.2 tetraacetyl ethylene diamine 6.0 percarbonate 22.0 EDDSs 0.3 foam inhibitor 3.5 4 of mixture 6.0 polyhydroxy fatty acid amides 2.5 zeolites 17.0 phyllosilicates of alcohol/citrate 16.0 carbonate 7.0 maleic acid acrylic copolymers 5.0 soil release polymers 0.4 carboxymethyl cellulose 0.4 P4VP-N-oxide 0.1 vinyl imidazole and vinyl pyrrolidone; 4 '-two (2-morpholino-4-anilino--s-0.25 three azines-6-base is amino) stilbene-2; 2 '-disulfonic acid disodium 4,4 '-two (2-sulfo group cinnamyl (styril)) biphenyl disodium 0.05 water, spices and micro constitutent to 100
Embodiment 20
Granular fabric cleaning composition
Composition weight % linear alkylbenzene sulfonate 7.6 C
16-C
18The C of 1.3 7 times of ethoxylations of alkyl-sulphate
14-15Alcohol 4.0 coconut oil alkyl dimethyl hydroxyethyl ammonium chlorides 1.4 dispersants 0.07 polysiloxane fluid 0.8 trisodium citrate 5.0 4A 15.0 maleic acid acrylic copolymers 4.0 diethylenetriamine pentamethylenophosphonic acids 0.4 perborate 15.0 tetraacetyl ethylene diamines, 5.0 smectic clays, 10.0 polyoxyethylene (MW 300,000) 0.3 Tyr 214 Phe+Tyr 217 Asn 0.4 lipase 0.2 amylase 0.3 cellulase 0.2 sodium metasilicate 3.0 sodium carbonate 10.0 carboxymethyl celluloses 0.2 brightening agent 0.2 water that boils, spices and micro constitutent to 100
Embodiment 21
Granular fabric cleaning composition
The C of 2.05 7 times of ethoxylations of composition weight % linear alkylbenzene sulfonate 6.92 tallow alkyl vitriol
14-15The C of 4.4 3 times of ethoxylations of alcohol
12-15Alkyl ethoxy sulfate 0.16 zeolite 20.2 citric acids 5.5 carbonate 15.4 silicate 3.0 maleic acid acrylic copolymers 4.0 carboxymethyl celluloses 0.31 soil release polymers 0.30 Val 203 Glu+Pro 210 Ala+Gly 215 Thr+Ala 216 Glu+ 0.2 Tyr 217 Leu lipase 0.36 cellulase 0.13 perborate tetrahydrate 11.64 perborate monohydrates 8.7 tetraacetyl ethylene diamines 5.0 DTPMPs 0.38 magnesium sulfate 0.40 brightening agent 0.19 spices, siloxanes, foam inhibitor 0.85 micro constitutent are to 100b. liquid fabric cleaning combination
Liquid fabric cleaning combination of the present invention contains one or more enzyme variants of the present invention of significant quantity, preferably contains about 0.005%-5% of promising composition weight, more preferably from about the 0.01%-1% organized enzyme.This liquid fabric cleaning combination generally also comprises anion surfactant, lipid acid, water soluble detergency promoter and water.
By following examples liquid fabric cleaning combination of the invention process is described.
Embodiment 22-26
The liquid fabric cleaning combination
The embodiment numbering
Component 22 23 24 25 26Gln 206 Glu+Ala 216 Glu+Tyr 217 Leu 0.05 0.03 0.30 0.03 0.10Pro 210 Ala+Gly 215 Thr---0.01 0.20C
12-C
14Sad 5.00 5.00 5.00 5.00 5.00 Trisodium Citrates of sodium alkyl sulfate 20.00 20.00 20.00 20.00 20.002-butyl 1.00 1.00 1.00 1.00 1.00C
10Alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 monoethanolamines 2.50 2.50 2.50 2.50 2.50 water/propylene glycol/ethanol (100: 1: 1) surpluses add to 100%
In embodiment 22-24, replace Gln 206Glu+Ala 216 Glu+Tyr 217 Leu with other listed in the table 2 BPN ' mutation, obtain similar substantially result.
In embodiment 25-26, replace Gln 206 Glu+Ala 216 Glu+Tyr, 217 Leu and Pro 210 Ala+Gly 215 Thr with other listed in the table 2 any BPN ' mutation combination, obtain basic analog result.
Embodiment 27-28
The liquid fabric cleaning combination
The embodiment numbering
Component 27 28 C
12-14Alkenyl succinic 3.0 8.0 citric acid monohydrate compounds 10.0 15.0 C
12-15The C of 8.0 8.0 2 times of ethoxylations of sodium alkyl sulfate
12-15The C of alcohol sodium sulfate-3.0 7 times ethoxylation
12-15The C of-8.0 5 times of ethoxylations of alcohol
12-15Alcohol 8.0-diethylene triamine penta(methylene phosphonic acid) 0.2-oleic acid 1.8-ethanol 4.0 4.0 propane diols 2.0 2.0 Ala, 216 Glu+Tyr 217 Leu, 0.2 0.2 polyvinylpyrrolidone 1.0 2.0 foam inhibitors, 0.15 0.15 NaOH to pH 7.5 perborate, 0.5 1 phenolsulfonate 0.1 0.2 peroxidases, 0.4 0.1 water and micro constitutent to 100 part
Among the embodiment 27 and 28 here, replace Ala 216 Glu+Tyr 217 Leu, obtain similar substantially result with other listed in the table 2 BPN ' mutation.
Embodiment 29-31
The liquid fabric cleaning combination
The embodiment numbering
Component 29 30 31 citric acids 7.10 3.00 3.00 aliphatic acid-2.00 2.00 ethanol 1.93 3.20 3.20 boric acid 2.22 3.50 3.50 MEAs 0.71 1.09 1.09 1,2-PD 7.89 8.00 8.00 cumene sodium sulfonates 1.80 3.00 3.00 sodium formates 0.08 0.08 0.08 NaOH 6.70 3.80 3.80 polysiloxanes anti-foaming agents 1.16 1.18 1.18 Ala 216 Glu 0.0145--Ala 216 Glu+Tyr 217 Leu-0.0145-Gln 206 Glu+Ala 216 Glu+Tyr 217 Leu--0.0145 lipase .200 .200 .200 cellulases-7.50 7.50 soil release polymers 0.29 0.15 0.15 anti-foaming agent 0.06 0.085 0.085 brightening agent 36 0.095--brightening agent 3-0.05 0.05 C12Alkyl benzene sulphonate (ABS) 9.86--C
12-15Alkyl polyethoxye (2,5) vitriol 13.80 18.00 18.00 C
12Glucamide-5.00 5.00 C
12-13Alkyl polyethoxylated (9) 2.00 2.00 2.00 water, spices and trace ingredients surplus add to 100%C. stick fabric cleaning composition
Be suitable for hand-washing one or more enzyme variants of the present invention that the stick fabric cleaning composition of the present invention of being with the dirt fabric contains significant quantity, be preferably about 0.001%-10% of composition weight, more preferably about 0.01%-1%.
By following examples stick fabric cleaning composition of the invention process is described.
Embodiment 33-36
The stick fabric cleaning composition
The embodiment numbering
Component 33 34 35 36 Val 203 Glu 0.3-0.1 0.02 Tyr 214 Phe+Tyr 217 Asn-0.3 0.4 0.03 C
12-C
16Sodium alkyl sulfate 20.0 20.0 20.0 20.00 C
12-C
14Methyl glucose amide 5.0 5.0 5.0 5.00 C
11-C
13Sodium alkyl benzene sulfonate 10.0 10.0 10.0 10.00 sodium carbonate 25.0 25.0 25.0 25.00 sodium pyrophosphates, 7.0 7.0 7.0 7.00 sodium phosphate trimers, 7.0 7.0 7.0 7.00 Wessalith CSs (0.1-10 μ) 5.0 5.0 5.0 5.00 carboxymethyl celluloses, 0.2 0.2 0.2 0.20 polyacrylates (MW 1400) 0.2 0.2 0.2 0.20 coconut oil single ethanol amides 5.0 5.0 5.0 5.00 brightening agents, spices 0.2 0.2 0.2 0.20 CaSO41.0 1.0 1.0 1.00 MgSO
41.0 1.0 1.0 1.00 water, 4.0 4.0 4.0 4.00 fillers
*Surplus adds to 100%
*Can be selected from suitable material, for example CaCO
3, talcum, clay, silicate etc.
In embodiment 33, replace Val 203 Glu with other listed in the table 2 BPN ' mutation, obtain similar substantially result.
In embodiment 34, replace Tyr 214 Phe+Tyr 217 Asn with other listed in the table 2 BPN ' mutation, obtain similar substantially result.
In embodiment 35-36, replace Val 203 Glu and Tyr 214 Phe+Tyr 217 Asn by other listed in the table 2 any BPN ' mutation combination, obtain similar substantially result.
Specific embodiments of the present invention are disclosed, obviously can make variations and modifications for those skilled in the art to the present invention under the situation that does not depart from essence of the present invention and scope.This means and has covered this class modification of within the scope of the present invention all in the appended claims.
The general information of sequence information (1):
(i) applicant: BRODE, PHILIP F.
BARNETT，BOBBY?L.
RUBINGH，DONN?N.
GHOSH，CHANCHAL?K.
(ii) invention exercise question: the fabric cleaning composition that contains subtilisin BPN ' variants
(iii) sequence number: No. 1 information of 1 (2) serial ID:
(i) sequence signature:
(A) length: 275 amino acid
(B) type: amino acid
(C) topological framework: linearity
(ii) molecule type: protein
(xi) sequence description: serial ID NO:1:Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu1 5 10 15His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20??????????????????25??????????????????30Ser?Gly?Ile?Asp?Ser?Ser?His?Pro?Asp?Leu?Lys?Val?Ala?Gly?Gly?Ala
35??????????????????40??????????????????45Ser?Met?Val?Pro?Ser?Glu?Thr?Asn?Pro?Phe?Gln?Asp?Asn?Asn?Ser?His
50??????????????????55??????????????????60Gly?Thr?His?Val?Ala?Gly?Thr?Val?Ala?Ala?Leu?Asn?Asn?Ser?Ile?Gly65??????????????????70??????????????????75??????????????????80Val?Leu?Gly?Val?Ala?Pro?Ser?Ala?Ser?Leu?Tyr?Ala?Val?Lys?Val?Leu
85??????????????????90??????????????????95Gly?Ala?Asp?Gly?Ser?Gly?Gln?Tyr?Ser?Trp?Ile?Ile?Asn?Gly?Ile?Glu
100?????????????????105?????????????????110Trp?Ala?Ile?Ala?Asn?Asn?Met?Asp?Val?Ile?Asn?Met?Ser?Leu?Gly?Gly
115?????????????????120?????????????????125Pro?Ser?Gly?Ser?Ala?Ala?Leu?Lys?Ala?Ala?Val?Asp?Lys?Ala?Val?Ala
130?????????????????135?????????????????140Ser?Gly?Val?Val?Val?Val?Ala?Ala?Ala?Gly?Asn?Glu?Gly?Thr?Ser?Gly145?????????????????150?????????????????155?????????????????160Ser?Ser?Ser?Thr?Val?Gly?Tyr?Pro?Gly?Lys?Tyr?Pro?Ser?Val?Ile?Ala
165?????????????????170?????????????????175Val?Gly?Ala?Val?Asp?Ser?Ser?Asn?Gln?Arg?Ala?Ser?Phe?Ser?Ser?Val
180?????????????????185?????????????????190Gly?Pro?Glu?Leu?Asp?Val?Met?Ala?Pro?Gly?Val?Ser?Ile?Gln?Ser?Thr
195?????????????????200?????????????????205Leu?Pro?GIy?Asn?Lys?Tyr?Gly?Ala?Tyr?Asn?Gly?Thr?Ser?Met?Ala?Ser
210?????????????????215?????????????????220Pro?His?VaI?Ala?G1y?Ala?Ala?Ala?Leu?Ile?Leu?Ser?Lys?His?Pro?Asn225?????????????????230?????????????????235?????????????????240Trp?Thr?Asn?Thr?Gln?Val?Arg?Ser?Ser?Leu?Glu?Asn?Thr?Thr?Thr?Lys
245?????????????????250?????????????????255Leu?Gly?Asp?Ser?Phe?Tyr?Tyr?Gly?Lys?Gly?Leu?Ile?Asn?Val?Gln?Ala
260?????????????????265?????????????????270Ala?Ala?Gln
275
Claims (19)
1. fabric cleaning composition, comprise the BPN ' mutation that contains the mild aminoacid sequence of (a) significant quantity, wherein a place or the plurality of positions in the mild aminoacid sequence 199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,218,219 or 220 is substituted, and it is characterized in that:
When i occurred in 199 positions when replacement, the amino acid that replaces 199 positions was Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When ii occurred in 200 positions when replacement, the amino acid that replaces 200 positions was His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When iii occurred in 201 positions when replacement, the amino acid that replaces 201 positions was Gly, Gln, Asn, Ser, Asp or Glu;
When iv occurred in 202 positions when replacement, the amino acid that replaces 202 positions was Pro, Gln, Asn, Ser, Asp or Glu;
When v occurred in 203 positions when replacement, the amino acid that replaces 2 positions was Met, Cys, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When vi occurred in 204 positions when replacement, the amino acid that replaces 204 positions was Glu;
When vii occurred in 205 positions when replacement, the amino acid that replaces 205 positions was Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When viii occurred in 206 positions when replacement, the amino acid that replaces 206 positions was Pro, Asn or Ser;
When ix occurred in 207 positions when replacement, the amino acid that replaces 207 positions was Asp or Glu;
When x occurred in 208 positions when replacement, the amino acid that replaces 208 positions was Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xi occurred in 209 positions when replacement, the amino acid that replaces 209 positions was Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xii occurred in 210 positions when replacement, the amino acid that replaces 210 positions was Gly, Gln, Asn, Ser, Asp or Glu;
When xiii occurred in 211 positions when replacement, the amino acid that replaces 211 positions was Ala, Pro, Gln, Asn, Ser, Asp or Glu;
When xiv occurred in 212 positions when replacement, the amino acid that replaces 212 positions was Gln, Ser, Asp or Glu;
When xv occurred in 213 positions when replacement, the amino acid that replaces 213 positions was Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xvi occurred in 214 positions when replacement, the amino acid that replaces 214 positions was Phe, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
When xvii occurred in 215 positions when replacement, the amino acid that replaces 215 positions was Thr, Pro, Gln, Asn, Ser, Asp or Glu;
When xviii occurred in 216 positions when replacement, the amino acid that replaces 216 positions was His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xix occurred in 218 positions when replacement, the amino acid that replaces 218 positions was Glu.
When xx occurred in 219 positions when replacement, the amino acid that replaces 219 positions was Pro, Gln, Asn, Ser, Asp or Glu;
When xxi occurred in 220 positions when replacement, the amino acid that replaces 220 positions was Pro, Gly, Gln, Asn, Asp or Glu; Be characterised in that this BPN ' mutation compares with mild subtilysin BPN ', reduced the absorption of insoluble matrix thing and strengthened hydrolysis the insoluble matrix thing; (b) one or more cleaning combination materials compatible with BPN ' mutation.
2. the fabric cleaning composition of claim 1 is characterized in that a. when replacement occurs in 206 positions, and the amino acid that replaces 206 positions is Asn or Ser; B. when replacement occurred in 211 positions, the amino acid that replaces 211 positions was Pro, Gln, Asn, Ser, Asp or Glu; C. when replacement occurred in 214 positions, the amino acid that replaces 214 positions was Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; With d. when replacing when occurring in 215 positions, the amino acid that replaces 215 positions is Pro, Gln, Asn, Ser, Asp or Glu.
3. the fabric cleaning composition of claim 2 is characterized in that replacing the Ala of 216 positions with Gly when 216 positions are substituted.
4. the fabric cleaning composition of claim 2, it is characterized in that the amino acid that replaces position, any place in 199,200,201,202,203,205,207,208,209,210,211,212,213,214,215,216,219 or 220 is Asp or Glu when replacing the place occur in 199,200,201,202,203,205,207,208,209,210,211,212,213,214,215,216,219 or 220 or plurality of positions; When replacement occurred in a place in 204 or 208 or this position, two places, the amino acid that replaces 204 or 218 positions was Glu; Wherein replace and preferably occur in 199,200,201,202,205,207,208,209,210,211,212 or 215, in a place or plurality of positions, more preferably occur in a place or plurality of positions in 200,201,202,205 or 207.
5. the fabric cleaning composition of claim 1, it has place's aminoacid replacement, is characterised in that this replacement is:
A.Glu replaces the Ala of 216 positions;
B.Asp replaces the Ala of 216 positions;
C.Glu replaces the Val of 203 positions.
6. fabric cleaning composition, comprise: (a) contain the BPN ' mutation of mild aminoacid sequence, wherein two places or the plurality of positions in the mild aminoacid sequence 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, and is characterised in that:
When i occurred in 199 positions when replacement, the amino acid that replaces 199 positions was Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When ii occurred in 200 positions when replacement, the amino acid that replaces 200 positions was His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When iii occurred in 201 positions when replacement, the amino acid that replaces 201 positions was Gly, Gln, Asn, Ser, Asp or Glu;
When iv occurred in 202 positions when replacement, the amino acid that replaces 202 positions was Pro, Gln, Asn, Ser, Asp or Glu;
When v occurred in 203 positions when replacement, the amino acid that replaces 203 positions was Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When vi occurred in 204 positions when replacement, the amino acid that replaces 204 positions was Asp or Glu;
When vii occurred in 205 positions when replacement, the amino acid that replaces 205 positions was Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When viii occurred in 206 positions when replacement, the amino acid that replaces 206 positions was Pro, Asn, Ser, Asp or Glu;
When ix occurred in 207 positions when replacement, the amino acid that replaces 207 positions was Asp or Glu;
When x occurred in 208 positions when replacement, the amino acid that replaces 208 positions was Pro, Gly, Gln, Asn or Ser;
When xi occurred in 209 positions when replacement, the amino acid that replaces 209 positions was Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xii occurred in 210 positions when replacement, the amino acid that replaces 210 positions was Ala, Gly, Gln, Asn, Ser, Asp or Glu;
When xiii occurred in 211 positions when replacement, the amino acid that replaces 211 positions was Ala, Pro, Gln, Asn, Ser, Asp or Glu;
When xiv occurred in 212 positions when replacement, the amino acid that replaces 212 positions was Gln, Ser, Asp or Glu;
When xv occurred in 213 positions when replacement, the amino acid that replaces 213 positions was Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xvi occurred in 214 positions when replacement, the amino acid that replaces 214 positions was Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn or Ser;
When xvii occurred in 215 positions when replacement, the amino acid that replaces 215 positions was Thr, Pro, Gln, Asn, Ser, Asp or Glu;
When xviii occurred in 216 positions when replacement, the amino acid that replaces 216 positions was His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xix occurred in 217 positions when replacement, the amino acid that replaces 217 positions was Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
When xx occurred in 218 positions when replacement, the amino acid that replaces 218 positions was Gln, Ser, Asp or Glu.
When xxi occurred in 219 positions when replacement, the amino acid that replaces 219 positions was Pro, Gln, Asn, Ser, Asp or Glu; With
When xxii occurred in 220 positions when replacement, the amino acid that replaces 220 positions was Pro, Gly, Gln, Asn, Ser, Asp or Glu; Be characterised in that this BPN ' mutation compares with mild subtilysin BPN ', reduced the absorption of insoluble matrix thing and strengthened hydrolysis the insoluble matrix thing; (b) one or more cleaning combination materials compatible with BPN ' mutation.
7. the fabric cleaning composition of claim 6 is characterised in that this mild BPN ' is substituted in position, two places.
8. the fabric cleaning composition of claim 7 is characterised in that said two places replace and is: a.Ala replaces the Pro of 210 positions and the Gly that Thr replaces 215 positions; B.Phe replaces the Tyr of 2l4 position and the Tyr that Asn replaces 217 positions; C.Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions; D.Leu replaces the Ile of 205 positions and the Ala that Glu replaces 216 positions; E.Leu replaces the Ile of 205 positions and the Ala that Asp replaces 216 positions; F.Glu replaces the Gln of 206 positions and the Ala that Glu replaces 216 positions; G.Asp replaces the Ala of 216 positions and the Try that Leu replaces 217 positions; Or h.Glu replaces the Gln of 206 positions and the Try that Leu replaces 217 positions.
9. the fabric cleaning composition of claim 7 is characterized in that: a. is when replacing when occurring in 206 positions, and the amino acid that replaces 206 positions is Glu, Asn or Ser; B. when replacement occurred in 210 positions, the amino acid that replaces 210 positions was Gly, Gln, Asn, Ser, Asp or Glu; C. when replacement occurred in 211 positions, the amino acid that replaces 211 positions was Pro, Gln, Asn, Ser, Asp or Glu; D. when replacement occurred in 214 positions, the amino acid that replaces 214 positions was Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; With e. when replacing when occurring in 215 positions, the amino acid that replaces 215 positions is Pro, Gln, Asn, Ser, Asp or Glu.
10. the fabric cleaning composition of claim 6, it is characterized in that when replacement occurred in position, 199,200,201,202,203,204,205,206,207,208,209,210,211,212,214,215,216,217,218,219 or 220 place, the amino acid that replaces optional position in 199,200,201,202,203,204,205,206,207,208,209,210,211,212,214,215,216,217,218,219 or 220 was Asp or Glu; When replacement occurred in 213 positions, the amino acid that replaces 213 positions was Asp; Wherein replace two places or the plurality of positions that preferably occurs in 199,200,201,202,205,207,208,209,210,211,212 or 215, more preferably replace two places or the plurality of positions that occurs in 200,201,202,205 or 207.
11. the fabric cleaning composition of claim 6 is characterized in that Glu or Asp replace the Ala of 216 positions and the Tyr that Leu replaces 217 positions.
12. the fabric cleaning composition of claim 6 is characterized in that mild BPN ' is substituted in position, three places.
13. the fabric cleaning composition of claim 12, the replacement of wherein said three places is: a.Pro replaces the Gln of 206 positions, and Ala replaces the Gly of 211 positions and the Ala that Glu replaces 216 positions; B.Val replaces the Ile of 205 positions, and Ala replaces the Pro of 210 positions and the Lys that Glu replaces 213 positions; Or c.Glu replaces the Gln of 206 positions, and Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions.
14. the fabric cleaning composition of claim 12 is characterised in that: when a. occurred in 206 positions when replacement, the amino acid that replaces 206 positions was Asn or Ser; B. when replacement occurred in 210 positions, the amino acid that replaces 210 positions was Gly, Gln, Asn, Ser, Asp or Glu; C. when replacement occurred in 211 positions, the amino acid that replaces 211 positions was Pro, Gln, Asn, Ser, Asp or Glu; D. when replacement occurred in 214 positions, the amino acid that replaces 214 positions was Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; With e. when replacing when occurring in 215 positions, the amino acid that replaces 215 positions is Pro, Gln, Asn, Ser, Asp or Glu.
15. the fabric cleaning composition of claim 6, be characterised in that mild BPN ' everywhere or position, five places be substituted.
16. the fabric cleaning composition of claim 15, wherein said replacement is: a.Glu replaces the Val of 203 positions, and Glu replaces the Gln of 206 positions, and Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions; B.Glu replaces the Val of 203 positions, and Ala replaces the Pro of 210 positions, and Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions; C.Glu replaces the Val of 203 positions, and Glu replaces the Gln of 206 positions, and Thr replaces the Gly of 215 positions, and Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions; Or d.Glu replaces the Val of 203 positions, and Ala replaces the Pro of 210 positions, and Thr replaces the Gly of 215 positions, and Glu replaces the Ala of 216 positions and the Tyr that Leu replaces 217 positions.
17. any fabric cleaning composition of claim 1-16 is characterised in that said composition is a liquid form.
18. any fabric cleaning composition of claim 1-16, wherein said composition be included as said composition weight at least about 5% tensio-active agent with at least about 5% washing assistant.
19. a method of cleaning fabrics, this method comprise that the fabric that needs are cleaned contacts with the arbitrary composition of right 1-18.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23793994A | 1994-05-02 | 1994-05-02 | |
| US08/237,939 | 1994-05-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1151757A true CN1151757A (en) | 1997-06-11 |
Family
ID=22895871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95193811A Pending CN1151757A (en) | 1994-05-02 | 1995-04-17 | Fabric cleaning composition 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) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899428B (en) * | 2002-11-06 | 2015-08-05 | 诺维信公司 | Subtilase variant |
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| US5858948A (en) * | 1996-05-03 | 1999-01-12 | Procter & Gamble Company | Liquid laundry detergent compositions comprising cotton soil release polymers and protease enzymes |
| ATE409743T1 (en) | 1996-11-04 | 2008-10-15 | Novozymes As | SUBTILATE VARIANTS AND COMPOUNDS |
| AU4772697A (en) | 1996-11-04 | 1998-05-29 | 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 |
| WO1999011769A1 (en) | 1997-08-29 | 1999-03-11 | Novo Nordisk A/S | Protease variants and compositions |
| CA2301851C (en) | 1997-08-29 | 2012-08-07 | Novo Nordisk A/S | Protease variants and compositions |
| US6773907B2 (en) | 1997-11-21 | 2004-08-10 | Peter Kamp Hansen | Subtilase enzymes |
| KR100762164B1 (en) | 1997-11-21 | 2007-10-01 | 노보자임스 에이/에스 | Protease variants and compositions |
| 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 |
| CN1301305A (en) | 1998-03-26 | 2001-06-27 | 宝洁公司 | Serine protease variants having amino acid substitutions |
| EP1115376A1 (en) * | 1998-09-22 | 2001-07-18 | The Procter & Gamble Company | 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 |
| ATE402996T1 (en) | 1999-05-20 | 2008-08-15 | Novozymes As | SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUBGROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID RESIDUE BETWEEN POSITIONS 125 AND 126 |
| DE60040287D1 (en) | 1999-05-20 | 2008-10-30 | Novozymes As | SUBTILASE ENZYMS OF I-S1 AND I-S2 SUB-GROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID BETWEEN POSITIONS 128 AND 129 |
| WO2000071689A1 (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 127 and 128 |
| 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 |
| WO2000071688A1 (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 126 and 127 |
| EP1183339B2 (en) | 1999-05-20 | 2013-03-13 | 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 |
| US6946128B1 (en) | 1999-07-22 | 2005-09-20 | The Procter & Gamble Company | Protease conjugates having sterically protected epitope regions |
| EP1210415A2 (en) | 1999-07-22 | 2002-06-05 | The Procter & Gamble Company | Subtilisin protease variants having amino acid substitutions in defined epitope regions |
| BR0012694A (en) | 1999-07-22 | 2002-04-09 | Procter & Gamble | Protease conjugate, cleaning composition and personal treatment composition |
| 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 |
| JP2003509537A (en) * | 1999-09-09 | 2003-03-11 | ザ、プロクター、エンド、ギャンブル、カンパニー | Detergent composition containing 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 |
| US7153820B2 (en) | 2001-08-13 | 2006-12-26 | Ecolab Inc. | Solid detergent composition and method for solidifying a detergent composition |
| CN102766545B (en) | 2003-05-07 | 2014-08-06 | 诺维信公司 | Variant subtilisin enzymes (subtilases) |
| JP4880469B2 (en) | 2003-10-23 | 2012-02-22 | ノボザイムス アクティーゼルスカブ | Protease with improved stability in detergents |
| EP1700904A1 (en) | 2005-03-11 | 2006-09-13 | Unilever N.V. | Liquid detergent composition |
| EP1700907A1 (en) | 2005-03-11 | 2006-09-13 | Unilever N.V. | Liquid bleaching composition |
| WO2007122175A1 (en) | 2006-04-20 | 2007-11-01 | 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 |
| DE102007044415A1 (en) | 2007-09-17 | 2009-03-19 | Henkel Ag & Co. Kgaa | Performance-enhanced proteases and detergents and cleaners containing these proteases |
| US8894898B2 (en) | 2007-10-18 | 2014-11-25 | Ecolab Usa Inc. | Pressed, waxy, solid cleaning compositions and methods of making them |
| US8728790B2 (en) * | 2009-12-09 | 2014-05-20 | Danisco Us Inc. | Compositions and methods comprising protease variants |
| US8758520B2 (en) | 2011-05-20 | 2014-06-24 | Ecolab Usa Inc. | Acid formulations for use in a system for warewashing |
| KR101890147B1 (en) | 2011-05-20 | 2018-08-21 | 에코랍 유에스에이 인코퍼레이티드 | 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 |
| EP3561034B1 (en) | 2013-09-09 | 2022-07-13 | Ecolab USA Inc. | Method of producing a detergent solid block having synergistic stain removal through novel chelator combination |
| BR112016005143B1 (en) | 2013-09-09 | 2021-10-19 | Ecolab Usa Inc | CONCENTRATED DETERGENT COMPOSITION, AQUEOUS SOLUTION AND USE OF A CONCENTRATED DETERGENT COMPOSITION |
| BR112016009177B1 (en) | 2013-10-24 | 2021-12-21 | Ecolab Usa Inc | METHOD FOR REMOVING DIRT FROM A SURFACE TO BE CLEANED |
| WO2021022045A1 (en) | 2019-07-31 | 2021-02-04 | Ecolab Usa Inc. | Personal protective equipment free delimer compositions |
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| US4990452A (en) * | 1986-02-12 | 1991-02-05 | Genex Corporation | Combining mutations for stabilization of subtilisin |
| US5013657A (en) * | 1988-04-12 | 1991-05-07 | Bryan Philip N | Subtilisin mutations |
| CA2034486A1 (en) * | 1989-06-26 | 1990-12-27 | Eric Casteleijn | Enzymatic detergent compositions |
| EP0563169B2 (en) * | 1990-12-21 | 2006-04-12 | Novozymes A/S | ENZYME MUTANTS HAVING A LOW DEGREE OF VARIATION OF THE MOLECULAR CHARGE OVER A pH RANGE |
| AU682314B2 (en) * | 1992-07-17 | 1997-10-02 | Genencor International, Inc. | High alkaline serine proteases |
| DE69434873T2 (en) * | 1993-09-15 | 2007-09-06 | The Procter & Gamble Company, Cincinnati | SUBTILISIN BPN 'VARIANTS WITH REDUCED ADSORPTION AND INCREASED HYDROLYSIS |
-
1995
- 1995-03-17 ZA ZA952220A patent/ZA952220B/en unknown
- 1995-04-17 BR BR9507593A patent/BR9507593A/en not_active Application Discontinuation
- 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 JP JP7528271A patent/JPH09512433A/en active Pending
- 1995-04-17 EP EP95916426A patent/EP0758373A1/en not_active Withdrawn
- 1995-04-17 MX MX9605359A patent/MX9605359A/en unknown
- 1995-04-17 CA CA002189428A patent/CA2189428A1/en not_active Abandoned
- 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
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899428B (en) * | 2002-11-06 | 2015-08-05 | 诺维信公司 | Subtilase variant |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0758373A1 (en) | 1997-02-19 |
| ZA952220B (en) | 1995-12-14 |
| MX9605359A (en) | 1997-12-31 |
| FI964411A (en) | 1996-11-01 |
| WO1995029979A1 (en) | 1995-11-09 |
| PE49595A1 (en) | 1996-02-05 |
| CA2189428A1 (en) | 1995-11-09 |
| JPH09512433A (en) | 1997-12-16 |
| BR9507593A (en) | 1997-09-23 |
| AU2293195A (en) | 1995-11-29 |
| FI964411A0 (en) | 1996-11-01 |
| NO964590D0 (en) | 1996-10-30 |
| NO964590L (en) | 1996-10-30 |
| PE12396A1 (en) | 1996-04-18 |
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