Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-07-18
2003-07-01
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S221000, C435S222000, C435S252300, C435S320100, C435S471000, C536S023200, C510S350000
Reexamination Certificate
active
06586223
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to genetically engineered subtilisin proteases which are useful in compositions such as, for example, personal care compositions, laundry compositions, hard surface cleansing compositions, and light duty cleaning compositions.
BACKGROUND OF THE INVENTION
Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating naturally occurring and genetically engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases.
Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen, Roland J. et al., “Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases”,
Protein Engineering
, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such modified proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause an immunogenic and/or allergenic response (herein collectively described as immunogenic response) in mammals.
Furthermore, while genetic engineering has been prominent in the continuing search for more highly effective proteases for use in laundry applications, genetically engineered proteases have not been commercially utilized in personal care compositions and light duty detergents. A primary reason for the absence of engineered proteases in products such as, for example, soaps, gels, body washes, and shampoos, is due to the problem of human sensitization leading to undesirable immunological responses. It would therefore be highly advantageous to provide a personal care composition or light duty detergent which provides the cleansing properties of engineered proteases with minimized provocation of an immunological response.
One approach toward alleviating the immunological activity of a protease is through the redesign of one or more epitopes of the protease. Epitopes are those amino acid regions of an antigen which evoke an immunological response through the binding of antibodies or the presentation of processed antigens to T cells via a major histocompatibility complex protein (MHC). Changes in the epitopes can affect their efficiency as an antigen. See Walsh, B. J. and M. E. H. Howden, “A Method for the Detection of IgE Binding Sequences of Allergens Based on a Modification of Epitope Mapping”,
Journal of Immunological Methods
, Vol. 121, pp. 275-280 (1989).
The present inventors have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPN′, have prominent epitope regions at amino acid positions 103-126 and 220-246, as well as at amino acid positions 70-84 corresponding to subtilisin BPN′. The present inventors have herein genetically redesigned such subtilisins to alleviate the immunogenic properties attributed to this epitope region. In so doing, the present inventors have discovered subtilisins which evoke a decreased immunological response yet maintain their activity as an efficient cleansing protease. Accordingly, the present proteases are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral, and contact lens compositions.
SUMMARY OF THE INVENTION
The present invention relates to variants of serine proteases having decreased immunogenicity relative to their corresponding wild-type proteases. More particularly, the present invention relates to variants having a modified amino acid sequence of a wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution of one or more epitope regions, wherein the epitope regions are 103-126 and 220-246 corresponding to subtilisin BPN′. Additionally, the present invention relates to modified amino acid sequences comprising one or more substitutions in two or more of epitope regions, wherein the epitope regions are 70-84, 103-126, and 220-246 corresponding to subtilisin BPN′. The invention further relates to mutant genes encoding such variants and cleaning and personal care compositions comprising such variants.
DETAILED DESCRIPTION OF THE INVENTION
The essential components of the present invention are herein described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any of the required or optional components and/or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.
All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, that may be present in commercially available sources.
All documents referred to herein, including all patents, patent applications, and printed publications, are hereby incorporated by reference in their entirety.
As used herein, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein:
TABLE I
Amino Acid
Three-letter Abbreviation
One-letter Abbreviation
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic Acid
Asp
D
Cysteine
Cys
C
Glutamine
Gln
Q
Glutamic Acid
Glu
E
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
Definitions
As used herein, the term “mutation” refers to an alteration in a gene sequence and/or an amino acid sequence produced by those gene sequences. Mutations include deletions, substitutions, and additions of amino acid residues to the wild-type protein sequence.
As used herein, the term “wild-type” refers to a protein, herein specifically a protease, produced by unmutated organisms.
As used herein, the term “variant” means a protein, herein specifically a protease, having an amino acid sequence which differs from that of the corresponding wild-type protein.
As referred to herein, while the variants of the present invention are not limited to those of subtilisin BPN′, all amino acid numbering is with reference to the amino acid sequence for subtilisin BPN′ which is represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPN′ is further described by Wells et al.,
Nucleic Acids Research
, Vol. 11, 7911-7925 (1983), incorporated herein by reference.
Variants of the Present Invention
The present inventors have discovered three epitope regions in serine proteases which correspond to positions 103-126 (referred to herein as the first epitope region), 220-246 (referred to herein as the second epitope region), and 70-84 (referred to herein as the third epitope region) of subtilisin BPN′. The present inventors have further discovered that one or more amino acid substitutions in one or more of the epitope
Rubingh Donn Nelton
Sikorski Elizabeth Ellen
Achutamurthy Ponnathapu
Frieko Laura L.
Kendall Dara M.
Moore William W.
Peebles Brent M.
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