Transglutaminase from oomycetes

Details Transglutaminase from oomycetes Transglutaminase from oomycetes

1997-06-24

2002-08-06

Bugaisky, Gabrielle (Department: 1653)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S251000, C435S911000, C536S023200

Reexamination Certificate

active

06428993

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel transglutaminase preparations derivable from the class Oomycetes, a novel transglutaminase derived from
Phytophthora cactorum
, CBS 618.94 or IFO 30474, a DNA construct encoding the transglutaminase enzyme, a method of producing the novel transglutaminase and the novel transglutaminase preparation, a method for producing a gel or protein gelation composition, and the use thereof.
2. Description of Related Art
Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the &egr;-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transglutaminases form intramolecular or intermolecular &egr;-(&ggr;-Glu)-Lys crosslinks.
This peptide crosslinking activity is useful for a variety of industrial purposes, including gelling of proteins, reduction of antigenicity of proteins, improvement of baking quality of flour, producing paste type food materia from protein, fat and water, preparation of cheese from milk concentrate, binding of chopped meat product, improvement of taste and texture of food proteins, producing jelly, gel cosmetics etc.
A wide array of transglutaminases have been isolated and characterized from animals and plants. The animal derived TGases are Ca
2+
-dependent and often multi-subunit enzymes. The most widely used mammalian transglutaminase, FXIIIa, is product inhibited, difficult to obtain in high amounts and thus expensive, and therefore not useful for all applications.
A few microbial TGases have been described, including the Ca
2+
-independent TGases from Streptoverticillia disclosed in U.S. Pat. No. 5,156,956 and related species disclosed in U.S. Pat. No. 5,252,469.
The yields of the microbial transglutaminases in shake-flasks and fermentors are far below those seen for other industrial enzymes. Thus, better production methods, including new high-yielding producers are needed. Previously, this goal has been pursued by applying conventional recombinant DNA techniques for cloning and expression in
E. coli, S. cerevisiae
and
S. lividans
(Washizu et al.; Tahekana et al.; Takagi et al.) but without success.
Klein et al. found and partially characterized a transglutaminase from the slime mold
Physarum polycephalum
which is a homodimer having a total molecular weight of 77 kDa. JP 6078783 Kokai relates to the use of this transglutaminase for protein gelation. However, it is well-known that slime molds are unsuited for large-scale industrial fermentation. Further, Physarum is not a fungus; it belongs to the Myxomycetes (Entrez NIH data base, current version January 1996). Taxonomically, the only common feature of Oomycetes, Myxomycetes and Eumycota (fungi) is that they all are mitochondrial eukaryotes.
The object of the invention is to provide a novel transglutaminase, a novel transglutaminase preparation, a method for producing the transglutaminase or transglutaminase preparation in a better yield and higher purity than hitherto possible which transglutaminase can be used either alone or in combination with other enzymes for industrial purposes.
SUMMARY OF THE INVENTION
Surprisingly, it has been found that organisms belonging to the class Oomycetes produce transglutaminase and that high-level expression is achievable by growing these coenocytium forming organisms.
In particular, isolates belonging to the class Oomycetes have been shown to express transglutaminases in unprecedented high amounts, including isolates belonging to the order Peronosporales, family Pythiaceae, and the genera Pythium and Phytophthora.
Accordingly, the present invention relates to transglutaminase preparations producible by cultivation of a transglutaminase producing strain of the class Oomycetes and to novel transglutaminases derived from transglutaminase producing strains of the class Oomycetes. Preferably, the novel transglutaminase and the transglutaminase preparation of the invention are derived from or producible by transglutaminase producing strains belonging to the class Oomycetes.
Further, the present invention relates to a parent transglutaminase derived from or producible by a species selected from
Phytophthora cactorum
, CBS 618.94 or IFO 30474
, Phytophthora cryptogea
, CBS 651.94
, Pythium periilum
(or
P. periplocum
), CBS 620.94
, Pythium irregulars
, CBS 701.95, Pythium sp., CBS 702.95
, Pythium intermedium
, CBS 703.95, Pythium sp., CBS 704.95
, Pythium ultimum
, CBS 705.95 or a functional analogue thereof.
The present invention also relates to a method for the production of a transglutaminase preparation according to the invention by cultivating, in a suitable medium, a strain belonging to the class Oomycetes, preferably belonging to an order selected from Peronosporales, Saprolegniales, Leptomitales and Lagenidiales, more preferably belonging to a family selected from Pythiaceae, Peronosporaceae, Saprolegniaceae, Leptomitaceae, Rhiphidiaceae and Lagenidiaceae, especially belonging to a genus selected from Pythium and Phytophthora.
Further, the present inventors have now surprisingly succeeded in isolating and characterizing a DNA sequence from a strain of the oomycetes
Phytophthora cactorum
exhibiting transglutaminase activity, thereby making it possible to prepare a recombinant transglutaminase.
Accordingly, in yet another aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting transglutaminase activity, which DNA sequence comprises
a) the DNA sequence shown in SEQ ID No. 1, and/or the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256 or
b) an analogue of the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256, which
i) is homologous with the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256, or
ii) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
Eschefichia coli
DSM 10256, or
iii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256, or
iv) encodes a polypeptide which is immunologically reactive with an antibody raised against the purified transglutaminase encoded by the DNA sequence shown in SEQ ID No 1 and/or the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256.
It is believed that the DNA sequence shown in SEQ ID No. 1 is identical to the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 10256.
The strain
Escherichia coli
was deposited under the deposition number DSM 10256 on Sep. 18, 1995 at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maascheroder Weg 1b, D-38125 Braunschweig, Germany, according to the Budapest Treaty.
In another aspect, the invention relates to a method of crosslinking proteins comprising contacting a proteinaceous substrate with a transglutaminase or transglutaminase preparation of the present invention.
In yet another aspect, the invention relates to use of the transglutaminase or transglutaminase preparation of the present invention in flour, baked products, meat products, fish products, cosmetics, cheese, milk products, gelled food products and leather finishing.
DETAILED DESCRIPTION OF THE INVENTION
In the present specification and claims, the term “transglutaminase” is intended to be understood as an enzyme capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor.
In the present context the term “derivable”

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