Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-09-29
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
06541235
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.
Yet a further object of the invention is to provide a mutant subtilisin protein which has the calcium binding loop deleted (i.e. amino acids 75-83) and deletion of amino acids in the N-terminal region (amino acids 1-22). It was discovered that when the calcium binding loop was deleted, the N-terminal part of the molecule is no longer absolutely required for proper folding.
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. Most particularly, the present invention relates to subtilisin proteins which have been modified to eliminate calcium binding and to delete the N-terminal region of the protein.
(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 (such as for biodegradable cleaning agents in laundry detergent) 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
366:485492 (1985)) and thermomycolase from the thermophilic fungus,
Malbranchea pulchella
(Gaucher et al., “Endopeptidases: Thermomycolin,”
Methods Enzymol
. 45:415433 (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 aa away (FIG.
1
). In subtilisin BPN′, calcium binds to site A with an affinity of −10
7
M
−1
(Bryan et al,
Biochemistry
31:4937-4945 (1992)). By binding at a specific site in the tertiary structure, calcium contributes its binding energy to the stability of the native state and makes a large contribution to the overall free energy of folding (Schellman,
Biopolymers
14:999-1018 (1975)). 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-
Achutamurthy Ponnathapu
Fuierer Marianne
Hultquist Steven J.
Moore William W.
University of Maryland Biotechnology Institute
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