Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1997-11-04
2001-10-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S221000, C435S222000, C435S253100, C435S320100, C435S471000, C435S476000, C510S300000, C510S320000, C536S023200
Reexamination Certificate
active
06300116
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority of Danish application Ser. Nos. 1235/96, 1240196 and 0284/97 filed on Nov. 4, 1996, Nov. 5, 1996 and Mar. 14, 1997, respectively, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
This invention relates to novel mutant protease enzymes or enzyme variants useful in formulating detergent compositions and exhibiting increased autoproteolytic stability; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.
BACKGROUND OF THE INVENTION
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
Although proteases have been used in the detergent industry for more than 30 years, much remains unknown as to details of how these enzymes interact with substrates and/or other substances present in e.g. detergent compositions. Some factors related to specific residues of the proteases and influencing certain properties, such as oxidative and thermal stability in general, of the proteases have been elucidated, but much remains to be found out. Also, it is still not exactly known which physical or chemical characteristics are responsible for a good washing performance or stability of a protease in a specific detergent composition.
The currently used proteases have for the most part been found by isolating proteases from nature and testing them in detergent formulations.
At present at least the following proteases are known to be commercially available and many of them are marketed in large quantities in many countries of the world.
Subtilisin BPN′ or Novo (available from e.g. SIGMA, St. Louis, U.S.A.), and Subtilisin Carlsberg, ALCALASE® (NOVO NORDISK A/S)) and MAXATASE® (Genencor).
A
Bacillus lentus
subtilisin, subtilisin 309, marketed by NOVO NORDISK A/S as SAVINASE®. A protein engineered variant of this enzyme is marketed as DURAZYM®.
Enzymes closely resembling SAVINASE®, such as subtilisin PB92, MAXACAL® marketed by Genencor Inc. (a protein engineered variant of this enzyme is marketed as MAXAPEM®), OPTICLEAN® marketed by SOLVAY et Cie. and PURAFECT® marketed by GENENCOR International.
A
Bacillus lentus
subtilisin, subtilisin 147, marketed by NOVO NORDISK A/S as ESPERASE®;
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
PROTEASES
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979
, Enzymatic Reaction Mechanisms
. W.H. Freeman and Company, San Francisco, Chapter 3). Bacteria of the Bacillus species secrete two extracellular types of protease, neutral proteases (or metalloproteases), and aLkaline proteases among which the most important functionally is a serine endopeptidase and usually referred to as subtilisin.
SERINE PROTEASES
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973
“Principles of Biochemistry
,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymottypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977)
Bacteriological Rev
. 41 711-753).
SUBTILASES
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al.,
Protein Engng
. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilisins have been identified, and the amino acid sequence of a number of subtilisins have been determined. These include more than six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus, and mesentericopeptidase (Kurihara et al. (1972)
J. Biol. Chem
. 247 5629-5631; Wells et al. (1983)
Nucleic Acids Res
. 11 7911-7925; Stahl and Ferrari (1984)
J. Bacteriol
. 159 811-819, Jacobs et al. (1985)
Nucl. Acids Res
. 13 8913-8926; Nedkov et al. (1985)
Biol. Chem. Hoppe-Seyler
366 421-430, Svendsen et al. (1986)
FEBS Lett
. 196 228-232), one subtilisin from an actinomycetales, thermitase from
Thermoactinomyces vulgaris
(Meloun et al. (1985)
FEBS Lett
. 198 195-200), and one fungal subtilisin, proteinase K from
Tritirachium album
(Jany and Mayer (1985)
Biol. Chem. Hoppe-Seyler
366 584-492). for further reference Table I from Siezen et al. has been reproduced below.
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisins have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors, and define the structural consequences for natural variation (Kraut (1977)
Ann. Rev. Biochem
. 46 331-358).
One subgroup of the subtilases, I-S1, comprises the “classical” subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A/S), subtilisin 147 (ESPERASE®, NOVO NORDISK A/S), and alkaline elastase YaB.
Random and site-directed mutations of the subtilase gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilase's catalytic activity, substate specificity, tertiary structure, etc. (Wells et al. (1987)
Proc. Natl. Acad. Sci. U.S.A
. 84; 1219-1223; Wells et al. (1986)
Phil. Trans. R. Soc. Lond.A
. 317 415-423; Hwang and Warshel (1987)
Biochem
. 26 2669-2673; Rao et al., (1987)
Nature
328 551-554.
More recent publications covering this area are Carter et al. (1989)
Proteins
6 240-248 relating to design of variants that cleave a specific target sequence in a substrate (positions 24 and 64); Graycar et al. (1992)
Annals of the New York Academy of Sciences
672 71-79 discussing a number of previously published results; and Takagi (1993)
Int. J. Biochem
. 25 307-312 also reviewing previous results.
Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in the following patent applications and patents:
PREVIOUSLY CHARACTERIZED PROTEASE VARIANTS
Numerous references describe construction of Protease variants. In order to make it easier to get an overview of the overall prior art status, the references are ordered in two sections.
Section one deals with references describing the t
Andersen Carsten
Bauditz Peter
Halkier Torben
Hansen Peter Kamp
Osten Claus von der
Achutamurthy Ponnathapu
Lambiris Esq Elias J.
Moore William W.
Novozymes A/S
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