Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1995-03-09
2002-11-05
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C435S320100, C435S471000, C510S392000, C536S023200
Reexamination Certificate
active
06475765
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences 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 DY 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 DY variants having a modified amino acid sequence of wild-type Subtilisin DY amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type Subtilisin DY (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Subtilisin DY. The present invention also relates to DNA sequences encoding such Subtilisin DY variants. The present invention also relates to compositions comprising such Subtilisin DY variants for cleaning a variety of surfaces.
DESCRIPTION
I. Subtilisin DY Variants
This invention pertains to subtilisin enzymes, in particular Subtilisin DY, 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, “Subtilisin DY 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 DNA sequences encoding for such Subtilisin DY 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,
Langmur
, “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
Polymer Solutions, Blends And Interfaces
, 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 Subtilisin DY, certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 5865 form the first loop region; positions 94-106 form the second loop region; positions 125-132 form the third loop region; positions 153-166 form the fourth loop region; positions 186-190 form the fifth loop region; and positions 199-219 form the sixth loop region (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin Subtilisin DY (SEQ ID NO:1)).
It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Subtilisin DY molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the Subtilisin DY molecule gives them 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 Subtilisin DY DNA” means a DNA sequence coding for a Subtilisin DY variant.
As used herein, “wild-type Subtilisin DY” refers to an enzyme represented by SEQ ID NO:1. The amino acid sequence for Subtilisin DY is further described by Nedkov, P., Oberthur, W. and Braunitzer, G.,
Biol. Chem
., Vol. 366, pp. 421430 (1985), incorporated herein by reference.
As used herein, the term “Subtilisin DY wild-type amino acid sequence”
Barnett Bobby Lee
Brode, III Philip Frederick
Rubingh Donn Nelton
Cook C. Brant
Moore William W.
Patterson Jr. Charles L.
Procter & Gamble Company
Taffy Frank
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