Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1995-03-02
2002-08-27
Achutamurthy, Ponnathapu (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252350, C435S320100, C435S471000, C536S023200, C510S320000, C510S374000, C510S392000
Reexamination Certificate
active
06440717
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and the genes encoding such enzyme variants.
BACKGROUND
Enzymes make up the largest class of naturally occurring proteins. Each class of enzyme. generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction. One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially, improve removal of such stains.
Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces.
OBJECTS OF THE PRESENT INVENTION
It is an object of the present invention to provide subtilisin enzyme variants having improved hydrolysis versus the wild-type of the enzyme.
It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants.
SUMMARY
The present invention relates to subtilisin BPN′ variants comprising at least one, two or three amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN′ (i.e., substitution) at specifically identified positions, whereby the BPN′ variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN′. The present invention also relates to the genes encoding such subtilisin BPN′ variants. The present invention also relates to compositions comprising such subtilisin BPN′ variants for cleaning a variety of surfaces.
DESCRIPTION
I. Subtilisin Variants
This invention pertains to subtilisin enzymes, in particular BPN′, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, “BPN′ variants”) of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to the mutant genes encoding for such BPN′ variants.
The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.
The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would, increase with increasing surface concentration. This has been shown to be the case. (Brode, P. F. III and D. S. Rauch, L
ANGMUIR
, “Subtilisin BPN′: Activity on an Immobilized Substrate”, Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, “Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface,” in P
OLYMER
S
OLUTLIONS
, B
LENDS AND
I
NTERFACES
, Ed. by I. Noda and D. N. Rubingh,. Elsevier, p. 464 (1992)). Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme, to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).
While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface.
The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils. In BPN′, the amino acids from position 199 to position 220 form a large exterior loop on the enzyme molecule. It has been discovered that this loop plays a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in this loop-have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that this loop is important to the adsorption of the BPN′ molecule for at least two reasons. First, the amino acids which comprise this exterior loop can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of this loop to the active-site and binding pocket of the BPN′ molecule gives it a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).
As used herein, “variant” means an enzyme having an amino acid sequence which differs from that of wild-type.
As used herein, “mutant BPN′ gene” means a gene coding for a BPN′ variant.
As used herein, “wild-type subtilisin BPN′” refers to a subtilisin enzyme represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPN′ is further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell and E. Y. Chen, N
UCLEIC
A
CIDS
R
ESEARCH
, Vol. II, 7911-7925 (1983), incorporated herein by reference.
As used herein, the term “wild-type amino acid sequence” encompasses SEQCID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 199-220.
As used herein, “more hydrophilic amino acid” refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T. P., and Woods, K. R., “Prediction of Protein Antigenic Determinants from Amino Acid Sequences”, P
ROCEEDINGS OF THE
N
ATIONAL
A
CADEMY OF
S
CIENCE
USA, Vol. 78, pp. 3824-3828, 1981, incorporated herein by reference).
TABLE 1
Amino Acid
Hydrophilicity Value
Trp
−3.4
Phe
−2.5
Tyr
&m
Barnett Bobby Lee
Brode, III Philip Frederick
Ghosh Chanchal Kumar
Rubingh Donn Nelton
Achutamurthy Ponnathapu
Clark Karen F.
Corstanje Brahm J.
Hersko Bart S.
Moore William W.
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