Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-07-13
2003-06-17
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S252310, C435S252330, C435S320100, C536S023100, C530S350000, C510S108000
Reexamination Certificate
active
06579698
ABSTRACT:
TECHNICAL FIELD
This invention relates to protein or proteinaceous protease inhibitors and to novel variants thereof, useful in conjunction with enzymes in cleaning compositions. The invention provides inhibitor variants greater proteolytic stability in detergent. The present invention also relates a variety of cleaning compositions comprising these inhibitors, and the genes encoding them.
BACKGROUND
Enzymes make up the largest class of naturally occurring proteins. Cleaning compositions may include many different enzymes to accomplish stain removal. For example, a liquid laundry detergent may contain proteases, lipases, amylases, peroxidases, and cellulases. The protease's ability to hydrolyze proteins has been exploited in cleaning compositions by incorporating proteases as an additive to aid in removing peptide or protein stains.
However, a commonly encountered problem in such protease-containing liquid aqueous detergents is the degradation by protease of the protease itself or of other enzymes (such as, lipase, amylase and cellulase) in the composition during storage. As a result of this degradation, the detergent composition consequently performs less well. Therefore it is commercially useful to incorporate into the cleaning composition a protease inhibitor.
There is a need to provide inhibitors that are stable enough to be useful. This “usefulness” is measured in terms of the need to provide a long shelf life for the cleaning composition, and an improved yield of the protease from the biological host.
Additionally, these inhibitors could be useful for cleaning compositions, regardless of the composition type, e.g., liquid, gel, granular, or solid compositions.
It would be advantageous to provide reversible inhibitors of the protease, so that upon dilution of the composition during cleaning, or in the cleaning environment, the protease is no longer inhibited, but rather is able to hydrolyze the peptide stains.
Synthetic Protease Inhibitors
Various synthetic protease inhibitors or stabilizers have been proposed for such uses. Panandiker et al. (U.S. Pat. No. 5,422,030) discloses an aromatic borate ester to stabilize enzymes in laundry detergents. For instance, U.S. Pat. No. 4,566,985 proposes to use benzamidine hydrochloride, EP 376 705 proposes to use lower aliphatic alcohols or carboxylic acids, EP 381 262 proposes to use a mixture of a polyol and a boron compound. Such synthetic approaches to enzyme inhibition may provide longer shelf life, but may be expensive and may not improve isolation yield due to proteolysis in the fermentor.
Recognizing these shortcomings, those in the art have experimented with proteinaceous protease inhibitors in hopes of stabilizing enzymes in cleaning compositions, without the drawbacks of the synthetic inhibitors.
Proteinaceous Protease Inhibitors
Nature provides proteinaceous protease inhibitors to regulate the protease in its natural environment (i.e., in vivo). However, these proteinaceous protease inhibitors tend to be unstable, therefore, their commercial use in the presence of proteases and detergents may be somewhat limited.
Proteinaceous protease inhibitors are typically long peptides (often over 28 amino acids), which bind to the active site of a protease and inhibit its activity. These inhibitors have been classified into several families (Families I to IX) based on primary amino acid sequence homologies (Laskowski, M., Jr., and I. Kato, “Protein Inhibitors of Proteinases”,
Ann. Rev. Biochemistry,
(1980) 49: 593-626). Included in these inhibitors are those commonly referred to as family VI inhibitors, such inhibitors include eglin and barley chymotrypsin inhibitor, and family III inhibitors, such as Streptomyces subtilisin inhibitor (SSI), and plasminostreptin.
Such inhibitors tend to bind to specific proteases better than others. Thus it is convenient to consider the inhibitor with a specific protease in mind. For this reason, the art often discusses them as “protease/peptide inhibitor pairs.” An example of a known protease/peptide inhibitor pair is subtilisin BPN′/SSI. See for example, Y. Mitsui, Y. et al, “Crystal Structure of a Bacterial Protein Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) at 2.6 A Resolution”,
J. Mol. Biol.
131: 697-724, (1979) and S. Hirono, H. Akagawa, Y. Mitsui, and Y. Iitaka, “Crystal Structure at 1.6 A Resolution of the Complex of Subtilisin BPN′ with Streptomyces Subtilisin Inhibitor”,
J. Mol. Biol.
178: 389-413, (1984). Mikkelsen (published application WO 92/03529) discloses peptide inhibitors of family VI. It is said that these inhibitors stabilize lipase and cellulase to proteolysis. Mikkelsen recognizes that many natural inhibitors have a high affinity for the protease and that the inhibitor-enzyme complex does not dissociate upon dilution into the wash environment. Mikkelsen discloses the use of proline in the Family VI P1 position. It is recognized that if the protease is completely inhibited in the product, then only a small fraction of the protease would be active even after dilution in the cleaning environment.
SSI is stable in the presence of subtilisin BPN′, as long as the inhibitor is present in sufficient amounts to inhibit all protease activity. However, SSI is unstable in the presence of excess protease.
Tamura et al, (
Biochemistry
30: 5275-5286, 1991) suggests that SSI's instability in the presence of excess protease is due to dissociation and conformational change of hydrophobically formed SSI dimers. Tamura discloses a D83C variant of SSI displaying a higher T
m
than native SSI using DSC (Tamura et al.,
Biochemistry
33:14512-14520, 1994), but Tamura apparently did not test protease resistance in the presence or absence of detergent.
However, if the binding constant (Ki) of an inhibitor provides for some protease activity in the cleaning composition containing the enzyme/inhibitor pair, the protein inhibitor, as well as enzymes in the composition, may be hydrolyzed. Therefore, it would be advantageous to find variants of SSI or other inhibitors which are suitably stable in the presence of protease as well as detergents. In addition, it is preferred that these inhibitors have a binding constant for the particular protease to be inhibited. This binding constant (Ki) should allow for inhibition of the protease in the cleaning composition and during its storage. However, upon diluting the cleaning composition or during the cleaning process, the protease and inhibitor dissociate, and the uninhibited protease becomes active.
The binding of some protease inhibitors has been investigated. Halkier et al. (WO 93/20175) discloses protease inhibitors (e.g., eglin and barley chymotrypsin inhibitors) with lowered affinity compared to naturally occurring family VI inhibitors, due to changes at the P1 and P4 to P2 positions of the inhibitor.
Since the amino acid sequence of any protein or peptide determines its characteristics, a change in the amino acid sequence may alter the protein's or peptide's properties depending upon the location and nature of the amino acid change. Thus mutagenesis has been employed on some protein protease inhibitors in an attempt to determine the structure or function of certain amino acids therein.
For example, Kojima et al. (S. Kojima, Y. Nishiyama, I. Kumagai, and K. Miura,
J. Biochem.
109: 377-382, 1991) made and measured the Ki of 19 SSI P1 position variants against wild-type SSI using subtilisin BPN′. As another example, Mikkelsen discloses mutations in family VI inhibitors that are said to lower binding affinity. Nielsen et al. (WO 93/17086) discloses changes to plasminostreptin that are said to lower binding affinity.
The art describes the need for proteinaceous protease inhibitors that are useful. For example, Feder and Kochavi (FR208475 1) disclose a reversible alkaline protease inhibitor which is said to stabilize an enzyme in the presence of detergents. Estell (U.S. Pat. No. 5,178,789) discloses the use of turkey ovomucoid as a reversible inhibitor said to be useful for stabilizing subtilisin.
Correa Paul E.
Laskowski, Jr. Michael
Saunders Charles Winston
Lewis Leonard W.
McDow-Dunham Kelly
Miller Steven W.
Nashed Nashaat T.
The Procter & Gamble & Company
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