Carboxypeptidases and nucleic acids encoding the same

Details Carboxypeptidases and nucleic acids encoding the same Carboxypeptidases and nucleic acids encoding the same

1997-10-03

2001-02-13

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

Reexamination Certificate

active

06187578

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
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 erym atic hydrolysis processes.
Enzymatic hydrolysis processes 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.
Acid carboxypeptidases (EC 3.4.16) are serine exopeptidases which catalyze the removal of amino acids from the C-terminus of peptides, oligopeptides or proteins. These carboxypeptidases generally have a narrow substrate specificity, i.e., they can cleave only few amino acids.
Acid carboxypeptidases of
Aspergillus oryzae
have been reported previously. For instance, Nakadai, Nasuno, and Iguchi, 1972
, Agricultural and Biological Chemistry
36: 1343-1352, disclose a carboxypeptidase I with a molecular weight of 120 kDa (gel filtration) and optimal activity in the pH range 3.0 to 4.0. Nakadai, Nasuno, and Iguchi, 1972
, Agricultural and Biological Chemistry
36: 1473-1480, disclose a carboxypeptidase II with a molecular weight of 105 kDa (gel filtration) and optimal activity at pH 3.0. Nakadai, Nasuno, and Iguchi, 1972
, Agricultural and Biological Chemistry
36: 1481-1488, disclose a carboxypeptidase III with a molecular weight of 61 kDa (gel filtration) and a pH optimum of 3.0. Nakadai, Nasuno, and Iguchi, 1972
, Agricultural and Biological Chemistry
37: 1237-1251, disclose a carboxypeptidase IV with a molecular weight of 43 kDa (gel filtration) and optimal activity at pH 3.0. Tekeuchi and Ichishima, 1986, Agricultural and Biological Chemistry 50: 633-638, disclose a carboxypeptidase O with a molecular weight of 73 kDa (SDS-PAGE). Tekeuchi, Ushijima, and Ichishima, 1982
, Current Microbiology
7: 19-23, disclose a carboxypeptidase O-1 and a carboxypeptidase O-2 both with a molecular weight of 63 kDa (gel filtration) and optimal activity at a pH in the range of 3.7 to 4.0. Ichishima et al., 1972
, Journal of Biochemistry
72: 1045-1048, disclose a comparison of the enzymatic properties of several Aspergillus acid carboxypeptidases. Azarenkova et al., 1976, Biokhimiya 41: 20-27, disclose the isolation of an acid carboxypeptidase from
Aspergillus oryzae
with a molecular weight of 37 kDa (SDS-PAGE) and a pH optimum of 4 to 5.
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 carboxypeptidase activity as well as methods of obtaining protein hydrolysates with desirable organoleptic qualities and high degrees of hydrolysis.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having carboxypeptidase 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) a polypeptide having (i) optimal activity in the range of about pH 3.0 to about pH 7.5 at 25° C.; (ii) optimal activity 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) a capability to hydrolyze X from N-CBZ-Ala-X wherein N-CBZ is N-carbobenzoxy and X is any amino acid;
(d) an allelic variant of (a) or (b); and
(e) a fragment of (a), (b) or (d), wherein the fragment retains carboxypeptidase activity.
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 of obtaining hydrolysates from proteinaceous substrates which comprise subjecting the proteinaceous material to a polypeptide with carboxypeptidase activity alone or in combination with an endopeptidase, and to hydrolysates obtained from the method.
The present invention also relates to methods of 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 carboxypeptidase activity.
The present invention further relates to flavor-improving compositions comprising a polypeptide with carboxypeptidase activity. The compositions may further comprise additional enzymatic activities.
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 96/09397 (1996-03-01), None
Choi et al. “Partial characterization ofAspergillus oryzaecell wall fraction-bound enzyme related to immobilized biocatalyst” Journal of Fermentation and Bioengineering, 1991,vol. 72, No. 3, pp. 216-216), Aug. 15, 1991.
Choi et al., Journal of Fermentation and Bioengineering, vol. 72 No. 3, pp. 214-216. (1991).
Azarenkova et al., Biochemistry, vol. 41, No. 1/1, pp. 15-21 (1976).
Svensen et al., FEBS Letters, vol. 333, No. 1/2, pp. 39-43 (1993).
Database WPI, XP 002054568, JP 54 037 894 (Tanabe Seiyaku) No Date Provided.
Database WPI, XP 002054569 JP 48 035 459 (Kikkoman Shoyu KK) No Date Provided).
Ichishima et al., J. Biochem., 1972, vol. 72, pp. 1045-1048.

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