Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-05-15
2003-12-16
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252330, C435S254300, C435S320100, C536S023200, C536S023100, C536S023740, C426S533000, C426S549000
Reexamination Certificate
active
06664092
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to polypeptides having dipeptidyl 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 the polypeptides. The present invention further relates to methods of obtaining protein hydrolysates useful as flavour improving agents.
2. Description of the Related Art
Various food products, e.g., soups, sauces and seasonings, contain flavoring agents obtained by hydrolysis of proteinaceous materials. This hydrolysis is conventionally accomplished using strong hydrochloric acid, followed by neutralization with sodium hydroxide. However, such chemical hydrolysis leads to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of this chemical reaction. Increasing concern over the use of flavoring agents obtained by chemical hydrolysis has led to the development of enzymatic hydrolysis processes.
Enzymatic hydrolysis processes of proteinaceous materials aim at obtaining a high degree of hydrolysis (DH), and this is usually attained using a complex of unspecific acting proteolytic enzymes (i.e., unspecific-acting endo- and exo-peptidases). For example, WO 94/25580 describes a method for hydrolyzing proteins by use of an unspecific acting enzyme preparation obtained from
Aspergillus oryzae
. Specific acting proteolytic enzymes have not been used for this purpose because such enzymes only lead to an inadequate degree of hydrolysis.
Polypeptides having dipeptidyl aminopeptidase activity catalyze the removal of dipeptides from the N-terminus of peptides, polypeptides, and proteins. Such polypeptides are classified under the Enzyme Classification Number E.C. 3.4.14.—of the International Union of Biochemistry and Molecular Biology.
Beauvais et al. (1997
, Journal of Biological Chemistry
272: 6238-6244) disclose a dipeptidyl-peptidase from
Aspergillus fumigatus
which has a molecular weight of 88 kDa by SDS-PAGE and a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum. Tachi et al. (1992
, Phytochemistry
31: 3707-3709) disclose an X-prolyl dipeptidyl aminopeptidase from
Aspergillus oryzae
which has a molecular weight of 145 kDa by SDS-PAGE and a substrate specificity toward the peptide bond at the carboxyl site of a proline residue in the penultimate position of N-terminal free dipeptides and amides at a neutral pH optimum.
The production of protein hydrolysates with desirable organoleptic properties and high degrees of hydrolysis generally requires the use of a mixture of peptidase activities. It would be desirable to provide a single component peptidase enzyme which has activity useful for improving the organoleptic properties and degree of hydrolysis of protein hydrolysates used in food products either alone or in combination with other enzymes.
It is an object of the present invention to provide improved polypeptides having dipeptidyl aminopeptidase activity as well as methods for obtaining protein hydrolysates with desirable organoleptic qualities and high degrees of hydrolysis.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having dipeptidyl aminopeptidase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 50% identity with the amino acid sequence of SEQ ID NO:2;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 1, (ii) its complementary strand, or (iii) a subsequence thereof;
(c) an allelic variant of (a) or (b); and
(d) a fragment of (a), (b), or (c), wherein the fragment has dipeptidyl aminopeptidase activity; and
(e) a polypeptide having dipeptidyl aminopeptidase activity with physicochemical properties of (i) a pH optimum in the range of from about pH 4.4 to about pH 9.8 determined after incubation for 5 minutes at ambient temperature in the presence of Ala-Pro-para-nitroanilide; (ii) a temperature stability of 90% or more, relative to initial activity, at pH 7.5 determined after incubation for 20 minutes at 65° C. in the absence of substrate; and (iii) an activity towards Xaa-Pro-para-nitroanilide or Xaa-Ala-para-nitroanilide wherein Xaa is selected from the group consisting of Ala, Arg, Asp, Gly, and Val.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides.
The present invention also relates to methods for obtaining hydrolysates from proteinaceous substrates which comprise subjecting the proteinaceous material to a polypeptide with dipeptidyl aminopeptidase activity alone or in combination with an endopeptidase, and to hydrolysates obtained from the method.
The present invention also relates to methods for obtaining from a proteinaceous substrate a hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues, which methods comprise subjecting the substrate to a deamidation process and to the action of a polypeptide having dipeptidyl aminopeptidase activity.
The present invention further relates to flavor-improving compositions comprising a polypeptide with dipeptidyl aminopeptidase activity.
In a final aspect, the methods of the invention may be used in food related applications to improve flavor, such as baking. Alternatively, flavor improvement in foods may be achieved by the addition of hydrolysates obtained by the methods of the invention.
REFERENCES:
patent: WO 94/25580 (1994-11-01), None
patent: WO 96/28542 (1996-09-01), None
patent: WO 97/43910 (1997-11-01), None
Tachi et al., “An X-Prolyl Dipeptidyl-Aminopeptidase FromAspergillus oryzae”, Phytochemistry vol. 31, No. 11, pp. 3707-3709, 1992.
Beauvais et al., “Biochemical and Antigenic Characterization of a New Dipeptidyl-Peptidase Isolated FromAspergillys fumigatus” Journal of Biological Chemistry 272 (10), 1997, pp 6238-6244.
Heymann et al., “Complementary Action of Dipeptidyl Peptidase IV and Aminopeptidase M In The Digestion of &bgr;-Casein”, J. Dairy Res 53, 1986, pp. 229-236.
Chemical Abstracts, vol. 124, No. 21, May 20, 1996 (XP-002051874).
Database WPI, Section Ch, Week 9527, May 9, 1995 (XP-002047737).
Database WPI, Section Ch, Week 9710, (XP-002076623) Nov. 17, 1993.
Beauvais et al., “Dipeptidyl-Peptidase IV Secreted byAspergillus fumigatus, A Fungus Pathogenic to Humans” Infection and Immunity, vol. 65, No. 8, Aug. 1997, pp. 3042-3047.
EMBL/Genbank Databases, Accession No. AJ002369, Sequence Reference No. A0DPPIV, Mar. 2, 1998 (XP-002076626).
Blinkovsky Alexander
Brown Kimberly
Byun Tony
Klotz Alan
Rey Michael W.
Nashed Nashaat T.
Novozymes Biotech Inc.
Starnes Robert L.
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