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
• • 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)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

• the present invention generally relates to enzyme technology, and more particularly relates to proteases, and to the manufacture thereof, whose amino acid sequence has been modified in particular with regard to use in washing and cleaning agents; to all sufficiently similar proteases having a corresponding modification; and to nucleic acids coding for them.
• the invention further relates to methods and uses of these proteases and to agents, in particular washing and cleaning agents, containing them.
• proteases are among the technically most important of all enzymes. For washing and cleaning agents they are the longest-established enzymes, contained in practically all modern high-performance washing and cleaning agents. They cause the breakdown of protein-containing stains on the material to be cleaned.
• proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62), which are categorized among the serine proteases because of the catalytically effective amino acids, are particularly important. They act as nonspecific endopeptidases and hydrolyze any acid amide bonds that are located within peptides or proteins. Their optimum pH is usually in the markedly alkaline range.
• subtilisin-like proteases by R. Siezen, in “Subtilisin enzymes” pp. 75-95, edited by R. Bott and C. Betzel, New York, 1996.
• Subtilases are formed naturally by microorganisms; among them, the subtilisins formed and secreted by Bacillus species are to be mentioned in particular as the most significant group within the subtilases.
• proteases of the subtilisin type used with preference in washing and cleaning agents are the subtilisins BPN' and Carlsberg, protease PB92, subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY, and the enzymes (to be classified, however, as subtilases and no longer as subtilisins in the strict sense) thermitase, proteinase K, and the proteases TW3 and TW7, as well as variants of the aforesaid proteases that comprise an amino acid sequence modified as compared with the initial protease.
• Proteases are modified in controlled or random fashion using methods known from the existing art, and are thereby optimized, for example, for use in washing and cleaning agents. These include point mutagenesis, deletion or insertion mutagenesis, or fusion with other proteins or protein parts. Correspondingly optimized variants are thus known for most proteases known from the existing art.
• the international patent applications WO 95/23221 and WO 92/21760 disclose variants of the alkaline protease from Bacillus lentus DSM 5483 that are suitable for use in washing or cleaning agents.
• the international patent application WO 2011/032988 furthermore discloses washing and cleaning agents that likewise contain variants of the alkaline protease from Bacillus lentus DSM 5483.
• the protease variants disclosed in these documents can be modified (in addition to further positions) at positions 3, 4, 99, and 199 in the count of the alkaline protease from Bacillus lentus DSM 5483, and can comprise at the aforesaid positions, for example, the amino acids 3T, 4I, 99D, 99E, or 199I. Combinations of these modifications as described hereinafter are, however, not evident from these documents.
• protease of the type of the alkaline protease from Bacillus lentus DSM 5483 or a protease sufficiently similar thereto (based on sequence identity), which comprises several of these modifications in combination, is particularly suitable for use in washing or cleaning agents and is advantageously improved in particular with regard to washing performance and/or stability.
• the subject matter of the invention is a protease comprising an amino acid sequence that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length, and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.
• a further subject of the invention is a method for manufacturing a protease, comprising the introduction of an amino acid substitution R99E or R99D, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, in the count in accordance with SEQ ID NO. 1, into an initial protease that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length.
• a “protease” for purposes of the present patent application therefore encompasses both the protease as such and a protease manufactured with a method according to the present invention. All statements with regard to the protease therefore refer both to the protease as a substance and to the corresponding method, in particular method for manufacturing the protease.
• a protease comprising an amino acid sequence that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.
• a method for manufacturing a protease comprising the introduction of an amino acid substitution R99E or R99D, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, in the count according to SEQ ID NO. 1, into a starting protease that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length.
• proteases according to the present invention Associated with the proteases according to the present invention respectively the manufacturing methods for proteases according to the present invention, as further subjects of the invention, are nucleic acids coding for said proteases, proteases or nucleic acids according to the present invention containing non-human host cells, as well as agents, in particular washing and cleaning agents, washing and cleaning methods, and uses defined by way of proteases according to the present invention, comprising proteases according to the present invention.
• Proteases according to the present invention consequently make possible improved removal of at least one, preferably of several protease-sensitive stains on textiles and/or on hard surfaces, for example dishes.
• Preferred embodiments of proteases according to the present invention exhibit particularly advantageous cleaning performance on blood-containing stains, for example on the following stains:
• Preferred embodiments of the present invention consequently make available stain-specific proteases whose cleaning performance is advantageous specifically with regard to one stain or to several stains.
• the stain focus of preferred embodiments of proteases according to the present invention with regard to blood-containing stains is consequently improved.
• proteases according to the present invention already achieve such advantageous cleaning performance effects even at low temperatures between 10° C. and 60° C., between 15° C. and 50° C., and between 20° C. and 40° C. Further preferred embodiments of proteases according to the present invention achieve improved cleaning performance of this kind over a broad temperature range, for example between 15° C. and 90° C., preferably between 20° C. and 60° C.
• proteases according to the present invention possess particular stability in washing or cleaning agents, for example with respect to surfactants and/or bleaching agents and/or with respect to temperature influences, in particular with respect to high or low temperatures, for example between 50 and 65° C., in particular 60° C., and/or with respect to acidic or alkaline conditions and/or with respect to changes in pH and/or with respect to denaturing or oxidizing agents and/or with respect to proteolytic breakdown and/or with respect to a change in redox conditions.
• protease variants that have improved performance and/or are more temperature-stable are therefore made available.
• protease variants that have improved performance and are more temperature-stable are made available. These advantageous embodiments of proteases according to the present invention consequently make possible improved washing results on protease-sensitive stains over a broad temperature range.
• the present invention is therefore a particularly advantageous selection of combinations of sequence modifications, the result of which is to obtain a particularly high-performance and/or temperature-stable protease variant for washing or cleaning agents.
• “Cleaning performance” is understood in the context of the invention as lightening performance on one or more stains, in particular on laundry or dishes.
• both the washing or cleaning agent that comprises the protease particularly the washing or cleaning bath constituted by said agent, and the protease itself have a respective cleaning performance.
• the cleaning performance of the enzyme thus contributes to the cleaning performance of the agent or of the washing or cleaning bath constituted by the agent.
• the cleaning performance is preferably ascertained as indicated below.
• a protease according to the present invention exhibits a proteolytic activity, i.e. it is capable of hydrolyzing peptide bonds of a polypeptide or protein, in particular in a washing or cleaning agent.
• a protease according to the present invention is therefore an enzyme that catalyzes the hydrolysis of peptide bonds and is thereby capable of cleaving peptides or proteins.
• a protease according to the present invention is furthermore preferably a mature protease, i.e. the catalytically active molecule having no signal peptide(s) and/or propeptide(s). Unless otherwise indicated, the sequences indicated also refer in each case to mature enzymes.
• the protease comprises an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.
• the protease comprises an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V 199I.
• Particularly preferred proteases according to the present invention are:
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E in combination with the amino acid substitutions S3T and V4I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, and R99E.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E in combination with the amino acid substitutions S3T and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, R99E, and V199I.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E in combination with the amino acid substitutions V4I and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions V4I, R99E, and V199I.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99D in combination with the amino acid substitutions S3T and V4I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, and R99D.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99D in combination with the amino acid substitutions S3T and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, R99D, and V 199I.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99D in combination with the amino acid substitutions V41 and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions V4I, R99D, and V199I.
• proteases according to the present invention are notable for the fact that they comprise the amino acid substitution R99E or R99D in combination with the three further amino acid substitutions S3T, V4I, and V199I.
• the following proteases in particular are very particularly preferred in this regard:
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO.
• amino acid substitution R99E in combination with the amino acid substitutions S3T, V4I, and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, R99E, and V199I.
• a protease of this kind is indicated in SEQ ID NO. 2.
• a protease comprising an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, and 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO.
• amino acid substitution R99D in combination with the amino acid substitutions S3T, V4I, and V199I, in particular a protease in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, R99D, and V199I.
• a protease of this kind is indicated in SEQ ID NO. 3.
• proteases as described above that furthermore comprise the amino acid leucine (L) at position 211 in the count in accordance with SEQ ID NO. 1.
• sequence comparison is based on the BLAST algorithm that is established in the existing art and usually used (cf. for example Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
• a comparison of this kind also allows a statement as to the similarity to one another of the sequences that are being compared. This is usually indicated as a percentage identity, i.e. the proportion of identical nucleotides or amino acid residues at the same positions, or at positions corresponding to one another in an alignment.
• the more broadly construed term “homology” also, in the context of amino acid sequences, incorporates consideration of the conserved amino acid exchanges, i.e. amino acids having a similar chemical activity, since these usually perform similar chemical activities within the protein.
• the similarity of the compared sequences can therefore also be indicated as a “percentage homology” or “percentage similarity.” Indications of identity and/or homology can be encountered over entire polypeptides or genes, or only over individual regions. Homologous or identical regions of various nucleic acid sequences or amino acid sequences are therefore defined by way of matches in the sequences. Such regions often have identical functions. They can be small, and can comprise only a few nucleotides or amino acids. Small regions of this kind often perform functions that are essential to the overall activity of the protein. It may therefore be useful to refer sequence matches only to individual, and optionally small, regions. Unless otherwise indicated, however, indications of identity or homology in the present application refer to the full length of the respectively indicated nucleic acid sequence or amino acid sequence.
• the protease is characterized in that its cleaning performance corresponds at least to that of a protease that comprises an amino acid sequence that corresponds to the amino acid sequence indicated in SEQ ID NO. 2, and/or at least to that of a protease that comprises an amino acid sequence that corresponds to the amino acid sequence indicated in SEQ ID NO. 3, the cleaning performance being determined in a washing system that contains a washing agent at a dosing ratio of between 4.5 and 7.0 grams per liter of washing bath as well as the protease, the proteases to be compared being used at identical concentration (based on active protein), and the cleaning performance being determined with respect to a blood stain on cotton, in particular with respect to the blood on cotton stain, product no.
• the concentration of protease in the washing agent stipulated for this washing system is from 0.001 to 0.1 wt %, preferably 0.01 to 0.06 wt %, based on active protein.
• a preferred liquid washing agent for a washing system of this kind has the following composition (all indications in percentage by weight): 0.3 to 0.5% xanthan, 0.2 to 0.4% antifoaming agent, 6 to 7% glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAEOS (fatty alcohol ether sulfate), 24 to 28% nonionic surfactants, 1% boric acid, 1 to 2% sodium citrate (dihydrate), 2 to 4% soda, 14 to 16% coconut fatty acids, 0.5% HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP (polyvinylpyrrolidone), 0 to 0.05% optical brightener, 0 to 0.001% dye, remainder deionized water.
• composition all indications in percentage by weight: 0.3 to 0.5% xanthan, 0.2 to 0.4% antifoaming agent, 6 to 7% glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAE
• the dosing ratio of the liquid washing agent is preferably between 4.5 and 6.0 grams per liter of washing bath, for example 4.7, 4.9, or 5.9 grams per liter of washing bath. Washing preferably occurs in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.
• a preferred powdered washing agent for a washing system of this kind has the following composition (all indications in percentage by weight): 10% linear alkylbenzenesulfonate (sodium salt), 1.5% C12 to C18 fatty alcohol sulfate (sodium salt), 2.0% C12 to C18 fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium hydrogen carbonate, 4.0% amorphous sodium disilicate, 17% sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1.0% carboxymethyl cellulose, 1.0% phosphonate, 27% sodium sulfate; remainder: foam inhibitors, optical brighteners, scents.
• the dosing ratio of the powdered washing agent is preferably between 4.5 and 7.0 grams per liter of washing bath, for example and particularly preferably 4.7 grams per liter of washing bath, or 5.5, 5.9, or 6.7 grams per liter of washing bath. Washing preferably occurs in a pH range between pH 9 and pH 11.
• Determination of the cleaning performance at 40° C. is performed in the context of the invention using a solid washing agent as indicated above, the washing operation occurring preferably for 70 minutes.
• the whiteness i.e. the lightening of the stains
• the whiteness is determined as an indication of washing performance, preferably using optical measurement methods, preferably photometrically.
• An instrument suitable for this is, for example, the Minolta CM508d spectrometer.
• the instruments used for measurement are usually calibrated beforehand using a white standard, preferably a white standard provided with the unit.
• protease activity can be determined quantitatively by way of the release of para-nitroaniline (pNA) chromophore from the suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide substrate (AAPF).
• pNA para-nitroaniline
• the protease cleaves the substrate and releases pNA.
• the release of pNA causes an increase in extinction at 410 nm, the change in which over time is an indication of enzymatic activity (see Del Mar et al., 1979).
• Measurement is performed at a temperature of 25° C., at pH 8.6, and a wavelength of 410 nm.
• the measurement time is 5 min, and the measurement interval 20 s to 60 s.
• the protease activity is usually indicated in protease units (PU). Suitable protease activities, for example, are 2.25, 5 or 10 PU per ml of washing bath. The protease activity is not, however, equal to zero.
• the protein concentration can be determined with the aid of known methods, for example the BCA method (bichinchoninic acid; 2,2′-biquinolyl-4,4′-dicarboxylic acid) or the biuret method (A. G. Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948), pp. 751-766).
• the active protein concentration can be determined, in this regard, by titrating the active centers using a suitable irreversible inhibitor (for proteases, for example, phenylmethylsulfonyl fluoride (PMSF)), and determining the residual activity (cf. M. Bender et al., J. Am. Chem. Soc. 88, 24 (1966), pp. 5890-5913).
• a suitable irreversible inhibitor for proteases, for example, phenylmethylsulfonyl fluoride (PMSF)
• proteins By reaction with an antiserum or a specific antibody, proteins can be combined into groups of immunologically related proteins.
• the members of such a group are notable for the fact that they comprise the same antigenic determinants that are recognized by an antibody. They are therefore structurally so similar to one another that they are detected by an antiserum or by specific antibodies.
• a further subject of the invention is therefore constituted by proteases which are characterized in that they comprise at least one and increasingly preferably two, three, or four antigenic determinants matching a protease according to the present invention. Because of their immunological matches, such proteases are structurally so similar to the proteases according to the present invention that a similar function is also be assumed.
• proteases according to the present invention can comprise further amino acid modifications, in particular amino acid substitutions, insertions, or deletions.
• Such proteases are, for example, further developed by targeted genetic modification, i.e. by way of mutagenesis methods, and optimized for specific purposes or with regard to special properties (for example, with regard to their catalytic activity, stability, etc.).
• nucleic acids according to the present invention can be introduced into recombination formulations and thereby used to generate entirely novel proteases or other polypeptides.
• the objective is to introduce targeted mutations, such as substitutions, insertions, or deletions, into the known molecules in order, for example, to improve the cleaning performance of enzymes according to the present invention.
• targeted mutations such as substitutions, insertions, or deletions
• the surface charges and/or isoelectric point of the molecules, and thereby their interactions with the substrate can be modified.
• the net charge of the enzymes can be modified in order thereby to influence substrate bonding, in particular for use in washing and cleaning agents.
• the stability of the protease can be enhanced by way of one or more corresponding mutations, and its cleaning performance thereby improved.
• Advantageous properties of individual mutations, e.g. individual substitutions can supplement one another.
• a protease already optimized with regard to specific properties, for example with regard to its stability in terms of surfactants and/or bleaching agents and/or other components, can therefore be additionally refined in the context of the invention.
• amino acid exchanges amino acid exchanges: Firstly the amino acid that is naturally present is designated in the form of the internationally usual single-letter code; this is followed by the relevant sequence position, and lastly by the inserted amino acid. Multiple exchanges within the same polypeptide chain are separated from one another by slashes. For insertions, additional amino acids are named after the sequence position. For deletions, the missing amino acid is replaced by a symbol, for example an asterisk or a dash.
• A95G describes the substitution of alanine at position 95 with glycine
• A95AG describes the insertion of glycine after the amino acid alanine at position 95
• A95* describes the deletion of alanine at position 95. This nomenclature is known to one skilled in the art of enzyme technology.
• a further subject of the invention is therefore a protease which is characterized in that it is obtainable from a protease as described above as an initial molecule by single or multiple conservative amino acid substitution, the protease still comprising, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, as described above.
• conservative amino acid substitution means the exchange (substitution) of one amino acid residue for another amino acid residue, where such exchange does not lead to a change in the polarity or charge at the position of the exchanged amino acid, e.g.
• the protease is characterized in that it is obtainable from a protease according to the present invention as an initial molecule by fragmentation or by deletion mutagenesis, insertion mutagenesis, or substitution mutagenesis, and comprises an amino acid sequence that matches the initial molecule over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, or 266 continuously connected amino acids, the amino acid substitution R99E or R99D contained in the initial molecule, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, still being present, as described above.
• the allergenicity of relevant enzymes can also be decreased by way of such fragmentation or deletion mutagenesis, insertion mutagenesis, or substitution mutagenesis, thus improving its overall usability.
• the enzymes retain their proteolytic activity even after mutagenesis, i.e. their proteolytic activity corresponds at least to that of the initial enzyme. Substitutions, too, can exhibit advantageous effects. Both individual and multiple continuously connected amino acids can be exchanged for other amino acids.
• the protease is characterized in that it is obtainable from a protease according to the present invention as an initial molecule by way of one or more amino acid substitutions in positions that are associated in an alignment with the positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255, and 268 of the protease from Bacillus lentus in accordance with SEQ ID NO. 1, where the protease still comprises, in the count in accordance with SEQ ID NO.
• the amino acid substitution R99E or R99D according to the present invention in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, as described above.
• the further amino acid positions are defined here by an alignment of the amino acid sequence of a protease according to the present invention with the amino acid sequence of the protease from Bacillus lentus as indicated in SEQ ID NO. 1.
• the association of the positions is furthermore directed toward the mature protein. This association is also to be utilized, in particular, when the amino acid sequence of a protease according to the present invention comprises a greater number of amino acid residues than the protease from Bacillus lentus in accordance with SEQ ID NO. 1. Proceeding from the aforesaid positions in the amino acid sequence of the protease from Bacillus lentus, the modification positions in a protease according to the present invention are those that are in fact associated with those positions in an alignment.
• amino acid residues located in the aforesaid positions in the wild type molecule of the protease from Bacillus lentus are the following: S36, N42, A47, T56, G61, T69, E87, A96, A101,I102, S104, N114, H118, A120, S130, S139, T141, S142, S154, S157, A188, V193, G205, L211, A224, K229, S236, N237, N242, H243, N255, respectively T268.
• Substitutions 61A, 154D, 154E, A188P, or V193M, for example, are particularly advantageous, to the extent the correspondingly homologous positions in a protease according to the present invention are not already naturally occupied by one of these preferred amino acids.
• a further confirmation of a correct association of the amino acids to be modified i.e. in particular their functional correspondence, can be supplied by comparison experiments in which the two positions associated with one another on the basis of an alignment are modified in the same way in both of the proteases being compared with each other, and an observation is made as to whether the enzymatic activity of the two is modified in the same way. For example, if an amino acid exchange at a specific position of the protease from Bacillus lentus in accordance with SEQ ID NO.
• an enzymatic parameter for example an elevation of the K M value
• a corresponding modification of the enzymatic parameter for example therefore likewise an elevation of the K M value
• a method according to the present invention therefore further comprises one or more of the following method steps:
• the protease respectively the protease manufactured with a method according to the present invention is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 98.8% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length.
• the protease respectively the protease manufactured with a method according to the present invention comprises the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.
• a further subject of the invention is a protease described above that is additionally stabilized, in particular by means of one or more mutations, for example substitutions, or by coupling to a polymer.
• a protease described above that is additionally stabilized, in particular by means of one or more mutations, for example substitutions, or by coupling to a polymer.
• All stabilization possibilities that are described in the existing art and/or are appropriate are suitable in principle. Those stabilization results which are achieved by mutations of the enzyme itself are preferred, since such stabilization requires no further working steps subsequent to recovery of the enzyme. Examples of sequence modifications suitable for this are recited above. Further suitable sequence modifications are known from the existing art.
• proteases can also be stabilized by exchanging one or more tyrosine residues for other amino acids.
• Preferred embodiments are those in which the enzyme is stabilized in several ways, since multiple stabilizing mutations have an additive or synergistic effect.
• a further subject of the invention is a protease as described above which is characterized in that it comprises at least one chemical modification.
• a protease having such a modification is referred to as a derivative, i.e. the protease is derivatized.
• “derivatives” are accordingly understood as those proteins whose pure amino acid chain has been chemically modified. Such derivatization operations can be performed, for example, in vivo by the host cell that expresses the protein. Linkages of low-molecular-weight compounds, such as of lipids or oligosaccharides, are particularly to be emphasized in this context. Derivatizations can also, however, be carried out in vitro, e.g. by chemical conversion of a side chain of an amino acid or by covalent bonding of a different compound onto the protein. Linkage of amines to carboxyl groups of an enzyme in order to modify the isoelectric point is, for example, possible.
• One such other compound can also be a further protein that is bound, for example, via bifunctional chemical compounds to a protein according to the present invention.
• “Derivatization” is likewise to be understood as covalent bonding to a macromolecular carrier, or also as a non-covalent inclusion into suitable macromolecular cage structures. Derivatizations can, for example, influence the substrate specificity or strength of bonding to the substrate, or can bring about a temporary blockage of enzymatic activity if the linked-on substance is an inhibitor. This can be useful, for example, for the period of storage. Modifications of this kind can furthermore influence stability or enzymatic activity.
• linkages to macromolecular compounds for example polyethylene glycol, can improve the protein with regard to stability and/or skin compatibility.
• “Derivatives” of a protein according to the present invention can also be understood in the broadest sense as preparations of said proteins.
• a protein can be brought into association with a variety of other substances, for example from the culture of the producing microorganisms.
• a protein can also have had other substances deliberately added to it, for example in order to enhance its shelf stability. All preparations of a protein according to the present invention are therefore also in accordance with the present invention. This is also irrespective of whether or not it actually displays this enzymatic activity in a specific preparation. This is because it may be desirable for it to possess little or no activity during storage, and to perform its enzymatic function only at the time of use. This can be controlled, for example, by way of corresponding accompanying substances.
• the preparation of proteases together with protease inhibitors is a particular possibility in this regard.
• proteases or protease variants and/or derivatives described above those whose activity corresponds at least to that of the protease in accordance with SEQ ID NO. 2 and/or SEQ ID NO. 3, and/or whose cleaning performance corresponds at least to that of the protease in accordance with SEQ ID NO. 2 and/or SEQ ID NO. 3, are particularly preferred in the context of the present invention, the cleaning performance being determined in a washing system as described above.
• a further subject of the present invention is a nucleic acid that codes for a protease according to the present invention, as well as a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
• DNA molecules or RNA molecules can exist as an individual strand, as an individual strand complementary to said individual strand, or as a double strand.
• sequences of both complementary strands in all three possible reading frames are to be considered in each case.
• different codons i.e. base triplets
• can code for the same amino acids so that a specific amino acid sequence can be coded by multiple different nucleic acids.
• all nucleic acid sequences that can encode one of the above-described proteases are included in this subject of the invention.
• nucleic acids according to the present invention one or more codons can be replaced by synonymous codons.
• This aspect refers in particular to heterologous expression of the enzymes according to the present invention. For example, every organism, e.g. a host cell of a production strain, possesses a specific codon usage. “Codon usage” is understood as the translation of the genetic code into amino acids by the respective organism.
• Bottlenecks in protein biosynthesis can occur if the codons located on the nucleic acid are confronted, in the organism, with a comparatively small number of loaded tRNA molecules. Although it codes for the same amino acid, the result is that a codon becomes translated in the organism less efficiently than a synonymous codon that codes for the same amino acid. Because of the presence of a larger number of tRNA molecules for the synonymous codon, the latter can be translated more efficiently in the organism.
• Vectors are understood for purposes of the present invention as elements, made up of nucleic acids, that contain a nucleic acid according to the present invention as a characterizing nucleic acid region. They enable said nucleic acid to be established as a stable genetic element in a species or a cell line over multiple generations or cell divisions.
• vectors are special plasmids, i.e. circular genetic elements.
• a nucleic acid according to the present invention is cloned into a vector. Included among the vectors are, for example, those whose origins are bacterial plasmids, viruses, or bacteriophages, or predominantly synthetic vectors or plasmids having elements of widely differing origins.
• vectors are capable of establishing themselves as stable units in the relevant host cells over multiple generations. They can be present extrachromosomally as separate units, or can be integrated into a chromosome or into chromosomal DNA.
• Expression vectors comprise nucleic acid sequences which are capable of replicating in the host cells, preferably microorganisms, particularly preferably bacteria, that contain them, and expressing therein a contained nucleic acid. Expression is influenced in particular by the promoter or promoters that regulate transcription. Expression can occur in principle by means of the natural promoter originally located in front of the nucleic acid to be expressed, but also by means of a host-cell promoter furnished on the expression vector or also by means of a modified, or entirely different, promoter of another organism or of another host cell. In the present case at least one promoter for expression of a nucleic acid according to the present invention is made available and used for expression thereof.
• Expression vectors can furthermore be regulatable, for example by way of a change in culture conditions or when the host cells containing them reach a specific cell density, or by the addition of specific substances, in particular activators of gene expression.
• a substance is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon).
• IPTG galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside
• lac operon lac operon
• a further subject of the invention is a non-human host cell that contains a nucleic acid according to the present invention or a vector according to the present invention, or that contains a protease according to the present invention, in particular one that secretes the protease into the medium surrounding the host cell.
• a nucleic acid according to the present invention or a vector according to the present invention is preferably transformed into a microorganism, which then represents a host cell according to the present invention.
• individual components i.e. nucleic acid parts or fragments of a nucleic acid according to the present invention, can be also be introduced into a host cell in such a way that the host cell which then results contains a nucleic acid according to the present invention or a vector according to the present invention.
• This procedure is suitable in particular when the host cell already contains one or more constituents of a nucleic acid according to the present invention or a vector according to the present invention, and the further constituents are then correspondingly supplemented.
• Methods for the transformation of cells are established in the existing art and are sufficiently known to the skilled artisan. All cells are in principle suitable as host cells, i.e. prokaryotic or eukaryotic cells. Those host cells that can be manipulated in genetically advantageous fashion, e.g. as regards transformation using the nucleic acid or vector and stable establishment thereof, are preferred, for example single-celled fungi or bacteria. In addition, preferred host cells are notable for being readily manipulated in microbiological and biotechnological terms.
• Preferred host cells according to the present invention secrete the (transgenically) expressed protein into the medium surrounding the host cells.
• the proteases can furthermore be modified, after their manufacture, by the cells producing them, for example by the addition of sugar molecules, formylation, amination, etc. Post-translation modifications of this kind can functionally influence the protease.
• Further preferred embodiments are represented by those host cells whose activity can be regulated on the basis of genetic regulation elements that are made available, for example, on the vector, but can also be present a priori in those cells. They can be stimulated to expression, for example, by controlled addition of chemical compounds that serve as activators, by modifying the culture conditions, or when a specific cell density is reached. This makes possible economical production of the proteins according to the present invention.
• One example of such a compound is IPTG, as described earlier.
• Preferred host cells are prokaryotic or bacterial cells.
• Bacteria are notable for short generation times and few demands in terms of culturing conditions. As a result, economical culturing methods or manufacturing methods can be established.
• the skilled artisan has a great wealth of experience with bacteria in fermentation technology.
• Gram-negative or Gram-positive bacteria may be suitable for a specific production instance, for a wide variety of reasons to be ascertained experimentally in the individual case, such as nutrient sources, product formation rate, time requirement, etc.
• Gram-negative bacteria such as, for example, Escherichia coli
• Gram-negative bacteria can furthermore also be configured so that they discharge the expressed proteins not only into the periplasmic space but into the medium surrounding the bacterium.
• Gram-positive bacteria such as e.g. bacilli or actinomycetes, or other representatives of the actinomycetals, possess no external membrane, so that secreted proteins are delivered immediately into the medium, as a rule the nutrient medium, surrounding the bacteria, from which medium the expressed proteins can be purified.
• Gram-positive bacteria are related or identical to most originating organisms for technically important enzymes, and usually themselves form comparable enzymes, so that they possess similar codon usage and their protein synthesis apparatus is of course correspondingly directed.
• Host cells according to the present invention can be modified in terms of their requirements for culture conditions, can comprise other or additional selection markers, or can also express other or additional proteins. They can, in particular, be those host cells that transgenically express multiple proteins or enzymes.
• the present invention is applicable in principle to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and its result is that proteins according to the present invention can be manufactured by the use of such microorganisms. Such microorganisms then represent host cells for purposes of the invention.
• the host cell is characterized in that it is a bacterium, preferably one that is selected from the group of the genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas, and Pseudomonas, more preferably one that is selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coel
• the host cell can, however, also be a eukaryotic cell, which is characterized in that it possesses a cell nucleus.
• a further subject of the invention is therefore represented by a host cell which is characterized in that it possesses a cell nucleus.
• eukaryotic cells are capable of post-translationally modifying the protein that is formed. Examples thereof are fungi such as Actinomycetes, or yeasts such as Saccharomyces or Kluyveromyces. This may be particularly advantageous, for example, when the proteins are intended to experience, in connection with their synthesis, specific modifications made possible by such systems.
• eukaryotic systems carry out in particular in conjunction with protein synthesis are, for example, the bonding of low-molecular-weight compounds such as membrane anchors or oligosaccharides. Oligosaccharide modifications of this kind can be desirable, for example, in order to decrease the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, for example cellulases or lipases, can also be advantageous.
• Thermophilic fungal expression systems for example, can furthermore be particularly suitable for the expression of temperature-resistant proteins or variants.
• the host cells according to the present invention are cultured and fermented in a usual manner, for example in discontinuous or continuous systems.
• a suitable nutrient medium is inoculated with the host cells, and the product is harvested from the medium after a period of time to be ascertained experimentally.
• Continuous fermentations are notable for the achievement of a flow equilibrium in which, over a comparatively long period of time, cells die off in part but are also in part renewed, and the protein formed can simultaneously be removed from the medium.
• Host cells according to the present invention are preferably used to manufacture proteases according to the present invention.
• a further subject of the invention is therefore a method for manufacturing a protease, comprising
• This subject of the invention preferably comprises fermentation methods. Fermentation methods are known from the existing art and represent the actual industrial-scale production step, generally followed by a suitable method for purifying the product that was manufactured, for example the protease according to the present invention. All fermentation methods that are based on a corresponding method for manufacturing a protease according to the present invention correspondingly represent embodiments of this subject of the invention.
• Fermentation methods which are characterized in that fermentation is carried out by way of an inflow strategy are particularly appropriate.
• the constituents of the medium that are consumed during continuous culturing are fed in.
• the fermentation operation can also be configured so that undesired metabolic products are filtered out, or are neutralized by the addition of a buffer or respectively suitable counterions.
• the protease that has been manufactured can be harvested from the fermentation medium.
• a fermentation method of this kind is preferred over isolation of the protease from the host cell, i.e. product preparation from the cell mass (dry mass), but requires that suitable host cells, or one or more suitable secretion markers respectively mechanisms and/or transport systems, be made available so that the host cells secrete the protease into the fermentation medium.
• suitable host cells i.e. product preparation from the cell mass (dry mass)
• suitable secretion markers respectively mechanisms and/or transport systems
• isolation of the protease from the host cell can occur, i.e. purification thereof from the cell mass, for example by precipitation using ammonium sulfate or ethanol, or by chromatographic purification.
• a further subject of the invention is an agent which is characterized in that it contains a protease according to the present invention as described above.
• the agent is preferably a washing or cleaning agent. Because proteases according to the present invention exhibit advantageous cleaning performance effects in particular on blood-containing stains, the agents are suitable and advantageous in particular for removing such stains.
• washing or cleaning agents both concentrates and agents to be used undiluted, for use on a commercial scale, in washing machines, or for hand laundering or cleaning.
• washing agents for textiles, carpets, or natural fibers, for which the term “washing agent” is used.
• dishwashing agents for automatic dishwashers or manual dishwashing agents, or cleaners for hard surfaces such as metal, glass, porcelain, ceramic, tiles, stone, painted surfaces, plastics, wood, or leather, for which the term “cleaning agent” is used, i.e. in addition to manual and automatic dishwashing agents, for example also scouring agents, glass cleaners, toilet cleaners, etc.
• washing and cleaning agents are washing adjuvants that are dispensed into the actual washing agent in the context of manual or automatic textile laundering in order achieve a further effect.
• textile pre- and post-treatment agents i.e. those agents with which the laundered item is brought into contact before actual laundering, for example in order to loosen stubborn stains, as well as those agents that, in a step following actual textile laundering, impart to the washed item further desirable properties such as a pleasant feel, absence of creases, or low static charge.
• the fabric softeners are classified among the latter agents.
• the washing or cleaning agents according to the present invention which can be present as in particular powdered solids, in recompressed particle form, as homogeneous solutions or suspensions, can contain besides a protease according to the present invention all known ingredients usual in such agents, at least one further ingredient preferably being present in the agent.
• the agents according to the present invention can contain, in particular, surfactants, builders (detergency builders), peroxygen compounds, or bleach activators. They can further contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators, and/or further adjuvants such as optical brighteners, anti-gray agents, foam regulators, as well as dyes and scents, as well as combinations thereof.
• a combination of a protease according to the present invention with one or more further ingredient(s) of the agent is particularly advantageous, since in preferred configurations according to the present invention such an agent exhibits improved cleaning performance thanks to synergies that result.
• Such a synergy can be achieved in particular by combining a protease according to the present invention with a surfactant and/or a builder (detergency builder) and/or a peroxygen compound and/or a bleach activator.
• An agent according to the present invention contains the protease advantageously in a quantity from 2 ⁇ g to 20 mg, preferably from 5 ⁇ g to 17.5 mg, particularly preferably from 20 ⁇ g to 15 mg, and very particularly preferably from 50 ⁇ g to 10 mg per g of the agent.
• the protease contained in the agent, and/or further ingredients of the agent can be encased with a substance that is impermeable to the enzyme at room temperature or in the absence of water, which substance becomes permeable to the enzyme under utilization conditions of the agent.
• Such an embodiment of the invention is thus characterized in that the protease is encased with a substance that is impermeable to the protease at room temperature or in the absence of water.
• the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing operation.
• the agent is characterized in that it is
• inventions of the present invention encompass all solid, powdered, liquid, gelled, or pasty administration forms of agents according to the present invention, which optionally can also be made up of multiple phases and can be present in compressed or uncompressed form.
• the agent can be present as a pourable powder, in particular having a bulk weight from 300 g/l to 1200 g/l, in particular 500 g/l to 900 g/l, or 600 g/l to 850 g/l.
• Further included among the solid administration forms of the agent are extrudates, granulates, tablets, or pouches.
• the agent can also be liquid, gelled, or pasty, for example in the form of a nonaqueous liquid washing agent or a nonaqueous paste or in the form of an aqueous liquid washing agent or a hydrous paste.
• the agent can furthermore be present as a one-component system. Such agents are made up of one phase. Alternatively, an agent can also be made up of multiple phases. An agent of this kind is thus subdivided into multiple components.
• Washing or cleaning agents according to the present invention can contain exclusively a protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes, in a concentration useful for the effectiveness of the agent.
• a further embodiment of the invention is thus represented by agents that moreover comprise one or more further enzymes.
• All enzymes that can display catalytic activity in the agent according to the present invention are preferably usable as further enzymes, in particular a protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase, or a lipase, as well as mixtures thereof.
• Further enzymes are contained in the agent advantageously in a quantity in each case from 1 ⁇ 10 ⁇ 8 to 5 weight percent, based on active protein.
• each further enzyme is contained in agents according to the present invention in a quantity from 1 ⁇ 10 ⁇ 7 to 3 wt %, from 0.00001 to 1 wt %, from 0.00005 to 0.5 wt %, from 0.0001 to 0.1 wt %, and particularly preferably from 0.0001 to 0.05 wt %, based on active protein.
• the enzymes exhibit synergistic cleaning performance effects with respect to specific stains or spots, i.e. the enzymes contained in the agent composition mutually assist one another in their cleaning performance.
• a synergism of this kind exists between the protease contained according to the present invention and a further enzyme of an agent according to the present invention, thereamong in particular between the aforesaid protease and the amylase and/or a lipase and/or a mannanase and/or a cellulase and/or a pectinases.
• Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and further ingredients of the agent according to the present invention.
• a further subject of the invention is a method for cleaning textiles or hard surfaces which is characterized in that in at least one method step an agent according to the present invention is utilized; or that in at least one method step a protease according to the present invention becomes catalytically active, in particular in such a way that the protease is used in a quantity from 40 ⁇ g to 4 g, preferably from 50 ⁇ g to 3 g, particularly preferably from 100 ⁇ g to 2 g, and very particularly preferably from 200 ⁇ g to 1 g, per utilization.
• Methods for cleaning textiles are notable in general for the fact that, in multiple method steps, various substances having cleaning activity are applied onto the material to be cleaned and are washed out after the contact time, or that the material to be cleaned is treated in another fashion with a washing agent or a solution or dilution of said agent.
• washing or cleaning methods can be supplemented, in at least one of the method steps, by the utilization of a washing or cleaning agent according to the present invention or of a protease according to the present invention, and then represent embodiments of the present invention.
• an individual and/or the only step of such a method can consist in bringing a protease according to the present invention, if desired as a sole component having cleaning activity, into contact with the stain, preferably in a buffer solution or in water. This represents a further embodiment of this subject of the invention.
• Alternative embodiments of this subject of the invention are also represented by methods for treating textile raw materials or for textile care, in which a protease according to the present invention becomes active in at least one method step.
• Preferred thereamong are methods for textile raw materials, fibers, or textiles having natural constituents, and very particularly for those having wool or silk.
• a further subject of the invention is the use of an agent according to the present invention for the cleaning of textiles or hard surfaces, or of a protease according to the present invention for the cleaning of textiles or hard surfaces, in particular in such a way that the protease is used in a quantity from 40 ⁇ g to 4 g, preferably from 50 ⁇ g to 3 g, particularly preferably from 100 ⁇ g to 2 g, and very particularly preferably from 200 ⁇ g to 1 g.
• a protease variant according to the present invention was manufactured by site-directed mutagenesis in the nucleic acid coding for the protease, using the PHUSION Site-Directed Mutagenesis Kit (Finnzyme, F541).
• the codons for the amino acid positions indicated were modified so that, with reference to the amino acid sequence, an exchange of the amino acids occurred as indicated.
• Expression of the protease variant occurred in a manner usual in the art, by transforming Bacillus subtilis DB 104 (Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444) with a corresponding expression vector and subsequent culturing of the transformands expressing the protease variant.
• Protease variant 1 Protease having an amino acid sequence in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, R99E, V199I in the count in accordance with SEQ ID NO. 1 (SEQ ID NO. 2);
• Protease variant 2 Protease having an amino acid sequence in accordance with SEQ ID NO. 1 having the amino acid substitutions S3T, V4I, R99D, V199I in the count in accordance with SEQ ID NO. 1 (SEQ ID NO. 3).
• washing-agent formulations were investigated in terms of their cleaning performance. For this, the batches were washed for 70 minutes at a temperature of 20° C. or 40° C. The dosing ratio was 4.7 g of washing agent per liter of washing bath. Washing was performed with tap water having a hardness of 16 degrees of German hardness.
• a baseline washing-agent formulation of the following composition was used as a control washing agent (all indications in percent by weight): 0.3 to 0.5% xanthan, 0.2 to 0.4% antifoaming agent, 6 to 7% glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAEOS (fatty alcohol ether sulfate), 24 to 28% nonionic surfactants, 1% boric acid, 1 to 2% sodium citrate (dihydrate), 2 to 4% soda, 14 to 16% coconut fatty acid, 0.5% HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP (polyvinylpyrrolidone), 0 to 0.05% optical brightener, 0 to 0.001% dye, remainder deionized water.
• xanthan 0.2 to 0.4% antifoaming agent
• 6 to 7% glycerol 0.3 to 0.5% ethanol
• 4 to 7% FAEOS fatty alcohol ether sulfate
• the baseline washing-agent formulation had the following proteases added to it on an identical active-protein basis (0.03 wt % active substance), for the various series of experiments.
• Protease variant 1 (batch 1) from Example 1 was used as a protease according to the present invention.
• the reference used was a protease that is disclosed in FIG. 2 such as SEQ ID NO. 3 of the international patent application WO 03/057713 (batch 2). This reference protease exhibits very good cleaning performance in liquid washing and cleaning agents.
• protease according to the present invention exhibits improved cleaning performance, in particular on blood-containing stains.
• proteases indicated below were incubated at a concentration of 10 to 20 ⁇ g/ml in 0.1M glycine/NaOH buffer at 60° C. and pH 10.0. At regular intervals over a period of 60 minutes, samples were taken, held on ice, and measured using an activity test by determining residual proteolytic activity via the release of para-nitroaniline (pNA) chromophore from the substrate.
• the substrate is a suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide substrate (suc-AAPF-pNA). The protease cleaves the substrate and releases pNA.
• the release of pNA causes an increase in extinction at 410 nm, the time course of which is an indication of enzymatic activity (see Del Mar et al., 1979).
• the half life was calculated based on the residual activity values that were determined. The following half lives (t 1 ⁇ 2) were obtained:
• Proteases according to the present invention consequently exhibit improved cleaning performance and are advantageously temperature-stable.
• Example 4 Ascertaining the cleaning performance of proteases according to the present invention when used in a commercially usual liquid washing agent at a washing temperature of 60° C.
• Example 2 Using this test material, a variety of washing agent formulations were investigated in terms of their washing performance. The experiments were carried out as described in Example 2, except that washing occurred at a higher temperature, specifically 60° C.
• the protease variant 1 (hereinafter batch 1) from Example 1 was used as a protease according to the present invention.

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