Use of mutated subtilisin protease in cosmetic products

Details Use of mutated subtilisin protease in cosmetic products Use of mutated subtilisin protease in cosmetic products

1998-04-23

2003-01-21

Achutamurthy, Ponnathapu (Department: 1622)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Cosmetic, antiperspirant, dentifrice

C435S069100, C435S221000, C435S252310, C435S320100, C435S471000, C536S023200

Reexamination Certificate

active

06509021

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the use of mutated proteolytic enzymes with a low skin irritation potential, namely mutated subtilisin proteases, in cosmetic products, more particularly body-cleansing and body-care formulations and oral hygiene formulations.
Enzymes, such as proteases, lipases, amylases and cellulases, have long been used in detergents and cleaners—essentially to support their washing and cleaning performance. Among these enzymes, proteases occupy a position of paramount importance.
2. Discussion of the Related Art
Proteases are enzymes which catalyze the hydrolysis of peptide bonds in protein and peptide substrates and of ester bonds in certain terminal esters. Subtilisins are a family of bacterial extracellular proteases with molecular weights of about 20,000 to 45,000 dalton which can be obtained from soil bacteria, for example Bacillus amyloliquefaciens. Subtilisins belong to the group of serine proteases which initiate the nucleophilic attack on the peptide (ester) bond through a serine residue at the active site. They are physically and chemically well characterized enzymes. The three-dimensional structure of certain subtilisins was elucidated in detail by X-ray diffractograms (C. Betzel, G. P. Pal and W Saenger, (1988) Eur. J. Biochem. 178, 155-171; R. Bott, M. Ultsch, A. Kossiakoff, T. Graycar, B. Kartz and S. Power, (1988) J. Biol Chem. 263, 7895-7906; D. W. Goddette, C. Paech, S. S. Yang, J. R. Mielenz, C. Bystroff, M. Wilke and R. J. Fletterick, (1992) J. Mol. Biol. 228, 580-595; D. W. Heinz, J. P. Priestle, J. Rahuel, K. S. Wilson and M. G. Grütter (1991) J. Mol. Biol. 217, 353-371; J. Kraut (1977) Ann. Rev. Biochem. 46, 331-358; D. J. Neidhart and G. A. Petsko (1988) Protein Eng. 2, 271-276; A. V. Teplyakov, I. P. Kuranova, E. H. Harutyunyan, B. K Vainshtein, C. Frömmel, W.-E. Höhne und K. S. Wilson (1990) J. Mol. Biol. 214, 261-279).
Subtilisins are widely used in commercial products, for example in laundry and dishwashing detergents and in contact lens cleaners, and—above all for research purposes—in synthetic organic chemistry. One member of the subtilisin family, namely a highly alkaline protease which can be used in surfactant-containing formulations, is described in International patent application WO 91/02792. This alkaline protease from
Bacillus lentus
(
Bacillus lentus
alkaline protease; BLAP) can be obtained in commercially useful quantities from the strain
Bacillus licheniformis
ATCC 53926 which carries an expression plasmid that expresses the BLAP gene under the control of the promoter of the alkaline protease of
Bacillus licheniformis
ATCC 53926. The crystal structure of BLAP has been determined (D. W. Goddette et al. (1992) J. Mol. Biol. 228, 580-595; WO 92/21760) and the coordinates were deposited at the Brookhaven Protein Data Bank. If an optimal sequence homology of BLAP (269 amino acids) is aligned with that of subtilisin BPN′ (275 amino acids), the following pattern is obtained: the BLAP positions 1 to 35, 36 to 54, 55 to 160 and 161 to 269 correspond to positions 1 to 35, 37 to 55, 57 to 162 and 167 to 275, respectively, in subtilisin BPN′. Unless otherwise indicated, the numbering of the amino acids used in the present specification corresponds to that of BLAP.
The following nomenclature is used to describe the protease variants employed in the present invention: [original amino acid; position of the N terminus of the ripe enzyme; substituted amino acid]. For example, the replacement of valine by isoleucine in position 4 in BLAP is designated V4I. The list of the standard abbreviations for the typical amino acids is given in Table 1.
TABLE 1
Abbreviations of the amino acids
A =
Ala =
Alanine
C =
Cys =
Cysteine
D =
Asp =
Aspartic acid
E =
Glu =
Glutamic acid
F =
Phe =
Phenyl alanine
G =
Gly =
Glycine
H =
His =
Histidine
I =
Ile =
Isoleucine
K =
Lys =
Lysine
L =
Leu =
Leucine
M =
Met =
Methionine
N =
Asn =
Asparagine
P =
Pro =
Proline
Q =
Gln =
Glutamine
R =
Arg =
Arginine
S =
Ser =
Serine
T =
Thr =
Threonine
V =
Val =
Valine
W =
Trp =
Tryptophan
Y =
Tyr =
Tyrosine
Where several mutations occur within the same protein molecule, this is characterized through the sum of the individual mutations, such as for example S3T+V4I+A188P+V193M+V199I.
Protection against thermal and chemical inactivation and improvement of washing and cleaning performance and dermatological compatibility are primary functions if new proteases are to be developed for industrial and institutional applications. Several enzymes, including proteases of the subtilisin type, have been developed by random mutagenesis or site-specific mutagenesis. They provide some indicators as to how improved thermal and chemical stability can be rationally achieved (D. A. Estell, T. P. Graycar and J. A. Wells (1985) J. Biol. Chem. 260, 6518-6521; M. Matsumura, W. J. Becktel, M. Levitt and B. W. Matthews (1989) Proc. Natl. Sci. US 86, 6562-6566; M. W. Pantoliano, M. Whitlow, J. F. Wood, M. L. Rollence, B. C. Finzel, G. L. Gilliland, T. L. Poulos and P. N. Bryan (1988) Biochemistry 27, 8311-8317; A. J. Russell and A. R. Fersht (1987) Nature 328, 496-500; R. J. Siezen, W. M. De Vos, J. A. M. Leunissen and B. W. Dijkstra (1991) Protein Eng. 4, 719-737; J. H. van Ee (1991) Chimicaoggi (7/8), 31-35; J. A. Wells and D. A. Estell (1988) Trends Biochem. Sci. 13, 291-297). By contrast, the modification of enzymatic activity, particularly improving or optimizing the activity rate for certain substrates, is a far more complex problem. EP 0 260 105 discloses the production of subtilisin-BPN′-mutants with modified ratios of transesterification rate to hydrolysis rate and nucleophilic specificities by modifying specific amino acid residues within 15 Å of the catalytic triad. A. J. Russell and A. R. Fersht (1987), J. Mol. Biol 193, 803-813, describe the isolation of a subtilisin-BPN′-mutant (DO99S) which has a modification to the surface charge at a distance of 14 to 15 Å from the active center. This substitution influences the pH dependence of the catalytic reaction of the subtilisin. None of these publications teaches whether the modifications to the amino acids also produce a change in the dermatological compatibility of the enzymes. EP 0 130 756, EP 0 247 647 and U.S. Pat. No. 4,760,025 disclose a saturation mutation process in which at least one mutation is inserted into the subtilisin BPN′ at the amino acid residues (BPN′ numbering) Asp32, Asn 155, Tyr104, Met222, Gly166, His64, Ser221, Gly169, Glu156, Ser33, Phe189, Tyr217 and/or Ala152. Mutated proteases which show improved oxidative stability, modified substrate specificity and/or modified pH activity are obtained using this procedure. The documents in question also teach that mutations in the vicinity of the active center of the protease have the most influence on activity. However, none of the documents in question discloses a process with which it is possible to predict whether and which changes in the amino acid sequence improve the dermatological compatibility of proteases.
Most of the information on the catalytic activity of subtilisins has been gathered in investigations into the hydrolysis of small well-defined peptide substrates. Hitherto, little has been known about interactions with large protein substrates. This applies in particular to information on the washing performance of proteases when their substrate is bound to a textile surface and the catalysis has to take place in the presence of substances which interact with the enzyme, such as bleaching agents, surfactants and builders. In addition, nothing is known of the interaction of proteases with the substances normally present in skin-care and hair-care products an

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