Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-05-07
2001-03-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S069100, C435S221000, C435S252310, C435S320100, C536S023200, C536S023400
Reexamination Certificate
active
06197589
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel mutant proteolytic enzymes with improved properties relative to the wild type enzyme in cleaning and detergent formulations, to nucleotide sequences encoding the improved proteases, and to host organisms containing the nucleotide sequences encoding the novel proteases. This invention also includes within its scope new and improved detergent and cleansing compositions containing an effective cleansing amount of said enzymes.
2. Description of the Related Art
Subtilisins are a family of bacterial extracellular proteases with molecular masses of 20,000 to 45,000 daltons produced by a soil bacillus e.g.
Bacillus amyloliquefaciens
. Proteases are enzymes which catalyze the hydrolysis of peptide linkages in protein and peptide substrates and of ester bonds in some terminal esters. Subtilisins belong to the group of serine proteases which initiate the nucleophilic attack on the peptide (ester) bond by a serine residue at the active site. Subtilisins are physically and chemically well characterized enzymes.
The three-dimensional structure of several subtilisins has been elucidated in detail by X-ray diffraction studies (Betzel, C., Pal, G. P., and Saenger, W. (1988) Eur. J. Biochem. 178, 155-171; Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B., and Power, S. (1988) J. Biol. Chem. 263, 7895-7906; Goddette, D. W., Paech, C., Yang, S. S., Mielenz, J. R., Bystroff, C., Wilke, M., and Fletterick, R. J. (1992) J. Mol. Biol. 228, 580-595; Heinz, D. W., Priestle, J. P., Rahuel, J., Wilson, K. S., and Grütter, M. G. (1991) J. Mol. Biol. 217, 353-371; Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Neidhart, D. J. and Petsko, G. A. (1988) Protein Eng. 2, 271-276; Teplyakov, A. V., Kuranova, I. P., Harutyunyan, E. H., Vainshtein, B. K., Frbömmel, C., Höhne, W.-E., and Wilson, K. S. (1990) J. Mol. Biol. 214, 261-279). In spite of this wealth of information the structure/function differences between these closely related subtilisins have not been explained.
Subtilisins are widely used in commercial products (for example, in laundry and dish washing detergents, contact lens cleaners) and for research purposes (catalysts in synthetic organic chemistry). One member of the subtilisin family, a highly alkaline protease for use in detergent formulations has been described in patent application WO 91/02792. This
Bacillus lentus
alkaline protease (BLAP) can be obtained in commercial quantities from
Bacillus licheniformis
ATCC 53926 strain transformed by an expression plasmid harboring the wild type BLAP gene under the control of the
B. licheniformis
ATCC 53926 alkaline protease gene promoter. The crystal structure of BLAP has been deduced (Goddette, D. W., et al. (1992) J. Mol. Biol. 228, 580-595; WO 92/21760), and the coordinates have been deposited with the Brookhaven Protein Data Bank.
Unless other wise noted the numbering of the amino acid positions is according to the sequence in BLAP (269 amino acids), which differs from that of subtilisin BPN′ (275 amino acids). When aligned for optimal sequence homology the following pattern emerges. In 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′.
In order to describe protease variants according to the invention, the following nomenclature is used: [Original Amino Acid; Position from N-terminus of the mature enzyme; Substituted Amino Acid]. For example, the substitution of valine with isoleucine at position 4 in BLAP is designated as V4I. The list of abbreviations for amino acids is shown in Table 1.
TABLE 1
Amino Acid Nomenclature
A = Ala = Alanine
C = Cys = Cysteine
D = Asp = Aspartic acid
E = Glu = Glutamic acid
F = Phe = Phenylalanine
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
Multiple mutations within the same protein molecule are indicated as the sum of individual mutations, i.e. S3T+V4I+A188P+V193M+V199I.
Protection against thermal and chemical inactivation and improvement of washing and cleaning performance are primary objectives in research and development of proteases for technical as well as for commercial applications. A large number of enzymes including subtilisins have been generated by random and site-specific mutagenesis and they provide some guidelines for a rational approach to the improvement of thermal and chemical stability (Estell, D. A., Graycar, T. P., and Wells, J. A. (1985) J. Biol. Chem. 260, 6518-6521; Matsumura, M., Becktel, W. J., Levitt, M., and Matthews, B. W. (1989) Proc. Natl. Acad. Sci. USA 86, 6562-6566; Pantoliano, M. W., Whitlow, M., Wood, J. F., Rollence, M. L., Finzel, B. C., Gilliland, G. L., Poulos, T. L., and Bryan, P. N. (1988) Biochemistry 27, 8311-8317; Russell, A. J. and Fersht, A. R. (1987) Nature 328, 496-500; Siezen, R. J., De Vos, W. M., Leunissen, J. A. M., and Dijkstra, B. W. (1991) Protein Eng. 4, 719-737; van Ee, J. H. (1991) Chimicaoggi (7/8), 31-35; Wells, J. A. and Estell, D. A. (1988) Trends Biochem. Sci. 13, 291-297). The modulation of enzymic activity, in particular rate enhancement or optimization for a subset of substrates, is a far more complex problem. EP 0260105 teaches the construction of subtilisin BPN′ mutants with altered transesterification rate/hydrolysis rate ratios and nucleophile specificities by changing specific amino acid residues within 15 Å of the catalytic triad. Russell, A. J., and Fersht, A. R. (1987) J. Mol. Biol. 193: 803-813, teach the isolation of a subtilisin BPN′ mutant (D099S) that had a change in the surface charge 14 to 15 Å from the active site. This substitution causes an effect on the pH dependence of the subtilisin's catalytic reaction. Neither of these publications teach how to predict amino acid alterations that will improve the wash performance of the protease. EP 0130756, EP 0247647, and U.S. Pat. No. 4,760,025 teach a saturation mutation method where one or multiple mutations are introduced into the subtilisin BPN′ at amino acid residues (BPN′ numbering) Asp32, Asn155, Tyr104, Met222, Gly166, His64, Ser221, Gly169, Glu156, Ser33, Phe189, Tyr217, and/or Ala152. Using this approach mutant proteases exhibiting improved oxidative stability, altered substrate specificity, and/or altered pH activity are obtained. These publications also teach that mutations within the active site region of the protease are the most likely to influence activity. However, neither EP0130756, EP 0247647, nor U.S. Pat. No. 4,760,025 teach a method for predicting amino acid alterations that will improve the wash performance of the protease.
Most of the information on the catalytic activity of subtilisins has been collected by examining the hydrolysis of small, well defined peptide substrates. Yet, little is known about interactions with large protein substrates. This is especially true for the wash performance of proteases where the substrate is attached to a textile surface and catalysis takes place in presence of interfering compounds such as bleach, tensides, and builders.
EP 0328229 teaches the isolation and characterization of PB92 subtilisin mutants with improved properties for laundry detergent applications based upon wash test results. It teaches that biochemical properties are not reliable parameters for predicting enzyme performance in the wash. Methods for selection of mutations involve the substitution of amino acids by other amino acids in the same category (pola
Christianson Teresa M.
Goddette Dean W.
Maurer Karl-Heinz
Paech Christian G.
Tang Maria R.
Achutamurthy Ponnathapu
Henkel Kommanditgesellschaft auf Aktien
Jaeschke Wayne C.
Moore William W.
Murphy Glenn E.J.
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