Carboxypeptidases and nucleic acids encoding same
View Patent ↗The present invention relates to polypeptides having carboxypeptidase 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 the polypeptides. The present invention further relates to methods of obtaining protein hydrolysates useful as flavor improving agents.
1. A method of producing a hydrolysate from a proteinaceous substrate which comprises subjecting the substrate to an endopeptidase and a polypeptide having carboxypeptidase activity, wherein the polypeptide having carboxypeptidase activity is selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 95% identity with amino acids 19 to 555 of SEQ ID NO: 2;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions nucleotides 55 to 1662 of SEQ ID NO: 1, or its complementary strand, wherein medium stringency conditions are defined as prehybridization and hybridization a 42° C. in 5×SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmon sperm DNA, and 35% formamide; and
(c) a fragment of (a) or (b), wherein the fragment has carboxypeptidase activity.
2. The method of claim 1 , wherein the polypeptide has an amino acid sequence which has at least 95% identity with amino acids 19 to 555 of SEQ ID NO: 2.
3. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
4. The method of claim 1 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof having carboxypeptidase activity.
5. The method of claim 4 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2.
6. The method of claim 4 , wherein the polypeptide consists of amino acids 19 to 555 of SEQ ID NO: 2.
7. The method of claim 1 , wherein the polypeptide is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with nucleotides 55 to 1662 of SEQ ID NO: 1, or its complementary strand, wherein medium stringency conditions are defined as prehybridization and hybridization a 42° C. in 5×SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmon sperm DNA, and 35% formamide.
8. The method of claim 1 , wherein the polypeptide is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with nucleotides 55 to 1662 of SEQ ID NO: 1, or its complementary strand, , wherein high stringency conditions are defined as prehybridization and hybridization a 42° C. in 5×SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmon sperm DNA, and 50% formamide.
9. The method of claim 1 , wherein the polypeptide having carboxypeptidase activity has (i) an optimal activity with respect to pH in the range of about pH 4.0 to about pH 5.0 at 25° C.; (ii) an optimal activity with respect to temperature in the range of about 55° C. to about 60° C. at pH 4; (iii) a residual activity of at least about 65.5% after 30 minutes at pH 4.0 and 60° C.; and (iv) an ability to hydrolyze X from N-CBZ-Ala-X wherein N-CBZ is N-carbobenzoxy and X is any amino acid.
10. The method of claim 9 , wherein X is selected from the group consisting of IIe, Glu, Lys, Arg, Asp, Asn, Phe, and Tyr.
11. The method of claim 1 , wherein the polypeptide having carboxypeptidase activity is encoded by the nucleic acid sequence contained in plasmid pEJG12 which is contained in E. coli NRRL B-21616.
12. A protein hydrolysate produced by the method of claim 1 .
13. A food product comprising the protein hydrolysate of claim 12 .