Polypeptides having aminopeptidase activity and nucleic acids encoding same
AssigneeNovozymes Biotech, Inc.
InventorsAlexander Blinkovsky, Tony S. Byun, Alan V. Klotz, Alan Sloma, Kimberly Brown, Maria Tang, Mikio Fujii, Chigusa Marumoto, Lene Venke Kofod
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Abstract
The present invention relates to isolated polypeptides having aminopeptidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
Claims56
An isolated polypeptide having aminopeptidase activity, selected from the group consisting of:
- (a) a polypeptide having an amino acid sequence which has at least 50% identity with amino acids 33 to 674 of SEQ ID NO:2;
- (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or (ii) its complementary strand;
- or a subsequence thereof of at least 100 nucleotides;
- (c) an allelic variant of (a) or (b);
- (d) a fragment of (a), (b), or (c), wherein the fragment has aminopeptidase activity;
- and
- (e) a polypeptide which (i) has aminopeptidase activity in the pH range between pH 5.0-8.5 measured at 37° C., (i) has an isoelectric point in the range of 7.4-8.5;
- (iii) has aminopeptidase activity in the temperature range of 20-55° C., measured at pH 7.5 using Gly-pNA in Tris-HCl buffer;
- (iv) hydrolyzes Ala-pNA, Gly-pNA, Leu-pNA, Glu-pNA, Asp-pNA, Lys-pNA, Ile-pNA and Val-pNA;
- (v) does not hydrolyze Phe-pNA nor Pro-pNA;
- (vi) is not inhibited by phenylmethanesulfonyl fluoride, slightly inhibited by EDTA, di-isopropyl fluoro phosphate, p-chloromercuribenzoic acid and iodoacetic acid, completely inhibited by o-phenanthroline, and/or (vii) is obtained from a strain belonging to Sphingomonas and has a molecular mass of 67±5 kDa.
An isolated nucleic acid sequence comprising a nucleic acid sequence having at least one mutation in the mature polypeptide coding nucleic acid sequence of SEQ ID NO:1, in which the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 33 to 674 of SEQ ID NO:2.
An isolated nucleic acid sequence produced by (a) hybridizing a DNA with the sequence of SEQ ID NO:1, or its complementary strand, or a subsequence thereof of at least 100 nucleotides under low stringency conditions;
- and (b) isolating the nucleic acid sequence.
A method for producing a mutant nucleic acid sequence, comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO:1, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 33 to 674 of SEQ ID NO:2;
- and (b) recovering the mutant nucleic acid sequence.
A method for producing a polypeptide comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence of SEQ ID NO:1 having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO:1, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 33 to 674 of SEQ ID NO:2, and (b) recovering the polypeptide.
A nucleic acid construct comprising a gene encoding a protein operably linked to a nucleic acid sequence encoding a signal peptide consisting of nucleotides 574 to 669 of SEQ ID NO:1, wherein the gene is foreign to the nucleic acid sequence.
Dependencies are inferred from the claim text.