Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-09-11
2003-04-01
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S222000, C435S252300, C435S320100, C435S471000, C536S023200
Reexamination Certificate
active
06541234
ABSTRACT:
GENERAL OBJECTS OF THE INVENTION
A general object of the invention is to provide
subtilisin
mutants which have been mutated such that they do not bind calcium.
Another object of the invention is to provide DNA sequences which upon expression provide for
subtilisin
mutants which do not bind calcium.
Another object of the invention is to provide
subtilisin
mutants which comprise specific combinations of mutations which provide for enhanced thermal stability.
Another object of the invention is to provide a method for the synthesis of a
subtilisin
mutant which does not bind calcium-by the expression of a
subtilisin
DNA which comprises one or more substitution’, deletion or addition mutations in a suitable recombinant host cell.
A more specific object of the invention is to provide class I subtilase mutants, in particular BPN′ mutants which have been mutated such that they do not bind calcium.
Another specific object of the invention is to provide DNA sequences which upon expression result in class I subtilase mutants, and in particular BPN′ mutants which do not bind calcium.
Another specific object of the invention is to provide a method for making
subtilisin
I-S1 or I-S2 mutants, and in particular BPN′ mutants which do not bind calcium by expression of a class I subtilase mutant DNA sequence, and more specifically a BPN′ DNA coding sequence which comprises one or more substitution, addition or deletion mutations in a suitable recombinant host cell.
Yet another specific object of the invention is to provide mutant
subtilisin
I-S1 or I-S2, and more specifically BPN′ mutants which do not bind calcium and which further comprise particular combinations of mutations which provide for enhanced thermal stability, or which restore cooperativity to the folding reaction.
The
subtilisin
mutants of the present invention are to be utilized in applications where
subtilisins
find current usage. Given that these mutants do not bind calcium they should be particularly well suited for use in industrial environments which comprise chelating agents, e.g. detergent compositions, which substantially reduces the activity of wild-type calcium binding
subtilisins.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to
subtilisin
proteins which have been modified to eliminate calcium binding. More particularly, the present invention relates to novel
subtilisin
I-S1 and I-S2
subtilisin
mutants, specifically BPN′ mutants wherein the calcium A-binding loop has been deleted, specifically wherein amino acids 75-83 have been deleted, and which may additionally comprise one or more other mutations, e.g.,
subtilisin
modifications, which provide for enhanced thermal stability and/or mutations which restore cooperativity to the folding reaction.
(2) Description of the Related Art
Subtilisin
is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. A well known example thereof,
subtilisin
BPN′ is a 275 amino acid serine protease secreted by
Bacillus amyloliquefaciens
. This enzyme is of considerable industrial importance and has been the subject of numerous protein engineering studies (Siezen et al.,
Protein Engineering
4:719-737 (1991); Bryan,
Pharmaceutical Biotechnology
3(B): 147181 (1992); Wells et al.,
Trends Biochem. Sci.
13:291-297 (1988)). The amino acid sequence for
subtilisin
BPN′ is known in the art and may be found in Vasantha et al.,
J. Bacteriol.
159:811-819 (1984). The amino acid sequence as found therein is hereby incorporated by reference [SEQUENCE ID NO:1]. Throughout the application, when Applicants refer to the amino acid sequence of
subtilisin
BPN′ or its mutants, they are referring to the amino acid sequence as listed therein.
Subtilisin
is a serine protease produced by Gram positive bacteria or by fungi. The amino acid sequences of numerous
subtilisins
are known. (Siezen et al.,
Protein Engineering
4:719-737 (1991)). These include five
subtilisins
from Bacillus strains, for example,
subtilisin
BPN′,
subtilisin
Carlsberg,
subtilisin
DY,
subtilisin
amylosacchariticus, and mesenticopeptidase. (Vasantha et al., “Gene for alkaline protease and neutral protease from
Bacillus amyloliquefaciens
contain a large open-reading frame between the regions coding for signal sequence and mature protein,”
J. Bacteriol.
159:811-819 (1984); Jacobs et al., “Cloning sequencing and expression of
subtilisin
Carlsberg from
Bacillus licheniformis, Nucleic Acids Res.
13:8913-8926 (1985); Nedkov et al.,” Determination of the complete amino acid sequence of
subtilisin
DY and its comparison with the primary structures of the
subtilisin
BPN′, Carlsberg and
amylosacchariticus,
Biol. Chem. Hoope-Seyler 366:421-430 (1985); Kurihara et al., “
Subtilisin amylosacchariticus,” J. Biol. Chem.
247:5619-5631 (1972); and Svendsen et al., “Complete amino acid sequence of alkaline mesentericopeptidase,”
FEBS Lett.
196:228-232 (1986)).
The amino acid sequences of
subtilisins
from two fungal proteases are known: proteinase K from
Tritirachium albam
(Jany et al., “Proteinase K from
Tritirachium albam
Limber,”
Biol. Chem. Hoppe-Seyler
366:485-492 (1985)) and thermomycolase from the thermophilic fungus,
Malbranchea pulchella
(Gaucher et al., “Endopeptidases: Thermomycolin,”
Methods Enzymol.
45:415-433 (1976)).
These enzymes have been shown to be related to
subtilisin
BPN′, not only through their primary sequences and enzymological properties, but also by comparison of x-ray crystallographic data. (McPhalen et al., “Crystal and molecular structure of the inhibitor eglin from leeches in complex with
subtilisin
Carlsberg,”
FEBS Lett.,
188:55-58 (1985) and Pahler et al., “Three-dimensional structure of fungal proteinase K reveals similarity to bacterial
subtilisin,” EMBO J.
3:1311-1314 (1984)).
Subtilisin
BPN′ is an example of a particular
subtilisin
gene secreted by
Bacillus amyloliquefaciens.
This gene has been cloned, sequenced and expressed at high levels from its natural promoter sequences in
Bacillus subtilis.
The
subtilisin
BPN′ structure has been highly refined (R=0.14) to 1.3 Å resolution and has revealed structural details for two ion binding sites (Finzel et al.,
J. Cell. Biochem. Suppl.
10A:272 (1986); Pantoliano et al.,
Biochemistry
27:8311-8317 (1988); McPhalen et al.,
Biochemistry
27: 6582-6598 (1988)). One of these (site A) binds Ca
2+
with high affinity and is located near the N-terminus, while the other (site B) binds calcium and other cations much more weakly and is located about 32 A away (FIG.
1
). Structural evidence for two calcium binding sites was also reported by Bode et al.,
Eur. J. Biochem.
166:673-692 (1987) for the homologous enzyme,
subtilisin
Carlsberg.
Further in this regard, the primary calcium binding site in all of the
subtilisins
in groups I-S1 and I-S2 (Siezen et al., 1991, Table 7) are formed from almost identical nine residue loops in the identical position of helix C. X-ray structures of the I-S1
subtilisins
BPN′ and Carlsberg, as well as the I-S2
subtilisin
Savinase, have been determined to high resolution. A comparison of these structures demonstrates that all three have almost identical calcium A-sites.
The x-ray structure of the class I subtilase, thermitase from
Thermoactinomyces vulgaris,
is also known. Though the overall homology of BPN′ to thermitase is much lower than the homology of BPN′ to I-S1 and I-S2
subtilisins
, thermitase has been shown to have an analogous calcium A-site. In the case of thermitase, the loop is a ten residue-interruption at the identical site in helix C.
Calcium binding sites are common features of extracellular microbial proteases probably because of their large contribution to both thermodynamic and kinetic stability (Matthews et al.,
J. Biol. Chem.
249:8030-8044 (1974); Voordouw et al.,
Biochemistry
15:3716-3724 (1976); Betzel et al.,
Achutamurthy Ponnathapu
Fuierer Marianne
Hultquist Steven J.
Moore William W.
University of Maryland Biotechnology Institute
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