Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1999-04-20
2001-02-20
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S072000, C435S193000, C426S573000
Reexamination Certificate
active
06190879
ABSTRACT:
The present invention relates to microbial transglutaminases, a DNA construct encoding a transglutaminase, a method of producing the transglutaminases, a composition comprising the transglutaminase and a method for producing a gel or protein gelation composition; and the use thereof.
BACKGROUND OF THE INVENTION
Transglutaminases (EC 2.3.2.13) are enzymes capable of catalyzing an acyl transfer reaction in which a &ggr;-carboxy-amide 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 &ggr;-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 &ggr;-glutamyl-&egr;-lysyl crosslinks.
This peptide crosslinking activity has shown useful for a variety of industrial purposes, including gelling 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, casein finishing in leather processing etc.
A wide array of transglutaminases have been identified and characterized from a number of animals and a few plant species. The most widely used animal derived transglutaminase, FXIIIa, is a Ca
2+
-dependent multi-subunit enzyme which is product inhibited, properties which are a disadvantage for many industrial applications and for production. A Ca
2+
-dependent transglutaminase from the slime mould
Physarum polycephalum
has been described in Klein et al., (1992).
Only few microbial transglutaminases have been disclosed, namely tranglutaminases from the species
Streptoverticillium mobaraense, Streptoverticillium cinnamoneum,
and
Streptoverticillium griseocarneum
(in U.S. Pat. No. 5,156,956) and from the species contemplated to be
Streptomyces lavendulae
(in U.S. Pat. No. 5,252,469).
U.S. Pat. No. 5,156,956 discloses that, after an extensive search for transglutaminases including screening a wide range of organisms and more than 5000 isolates of microbial origin, only the above-mentioned three Streptoverticillium species were found to produce transglutaminase. Members of this former genus
Streptoverticillium are now generally included within the genus Streptomyces (Kaempfer et al. (
1991), and Ochi et al. (1994)).
U.S. Pat. No. 5,252,469 discloses transglutaminase from what was believed to be two related species: Streptomyces sp., and
Streptomyces lavendulae.
However, from the disclosed data for the contemplated
S. lavendulae
strain it is evident to the skilled person that the disclosed strain is not
S. lavendulae.
Streptoverticillia are classified together in Cluster group F (clusters 55 to 67) of Streptomyces and related genera (Williams et al.). Therefore the known microbial transglutaminases all originate from members of this Cluster group as defined in Williams et al.
Streptomyces lavendulae
is also classified in Cluster group F.
All known microbial transglutaminases have been identified by using a conventional enzyme assay in which hydroxylamine is converted to hydroxamic acid (Folk, J. E. & Cole, P. W. (1966)).
In order to construct strains overproducing different enzymes, recombinant DNA techniques are widely used. For the same purpose, the
Streptoverticillium mobaraense
transglutaminase gene has been cloned for expression in
Escherichia coli, Streptomyces lividans,
and
Saccharomyces cerevisiae
(Washizu et al., Tahekana et al., and EP-A-0 481 504). Even the most succesful of these approaches (Washizu et al.) resulted in a production yield much lower than the yield in the wildtype
S. mobaraense
strain, in spite of extensive experimentation and optimization. Thus, none of the efforts to overproduce the
S. mobaraense
enzyme have been successful, although they included a number of different approaches such as chemical synthesis of a codon-optimized gene and its subsequent expression (as a cleavable heterologous signal peptide fusion to the mature transglutaminase) to the periplasm of
E. coli;
or expression as a similar fusion to the mature transglutaminase in
S. cerevisiae;
or expression as a similar fusion to pro-transglutaminase in
S. cerevisiae;
or traditional isolation and expression of the natural DNA sequence encoding the preproenzyme in
S. lividans.
U.S. Pat. No. 5,252,469 discloses strains closely related to
S. mobaraense
which produce higher amounts of transglutaminase by conventional techniques.
The object of the invention is to provide novel microbially derived transglutaminases, preferably in single-component or mono-component form, a novel gene encoding a transglutaminase, and a method for producing the transglutaminase in a better yield and higher purity than hitherto possible by recombinant DNA technology, as well as the use of the transglutaminase either alone or in combination with other enzymes for the use in a variety of industrial purposes, including gelling of proteins; improvement of baking quality of flour; producing paste type food or food ingredients from protein, fat and water; preparation of cheese from milk concentrate; binding of chopped meat or fish products; improvement of taste and texture of food proteins; casein finishing in leather processing; shoe shine, etc.
SUMMARY OF THE INVENTION
It has been found that, by screening a wide array of bacterial and fungal strains, often screening of the same extract which in the traditional hydroxamate assay gave rise to a negative result, in a modified putrescine assay resulted in a positive reaction. Accordingly, the modified version of the putrescine incorporation assay was applied in a screening procedure which surprisingly resulted in detection of transglutaminase activity in a wide array of organisms.
Therefore, and opposite to what has hitherto been known, it has now been found that transglutaminases (TGases) are produced by an overwhelming array of phylogenetically dispersed microorganisms. Also, it has been found that even within Cluster groups other than Cluster group F, e.g. Cluster groups A and G, members have been found which produce transglutaminases.
Several of the provided enzymes may be useful for industrial applications. The industrial potential is underlined by three circumstances:
1. The novel transglutaminases of the invention may be obtained in the higher production yields than obtained for any other microbial transglutaminase;
2. A number of the TGase-producing strains provided by the mentioned assay are closely related to industrial production strains in current use, and can hence be subjected to recombinant DNA expression in closely related species; e.g. members of the genera Bacillus, Streptomyes, Aspergillus, and Trichoderma;
3. The novel transglutaminases of the invention may be found extracellularly.
By applying a number of different growth conditions for the organisms to be screened, the inventors also surprisingly found that these conditions, in several instances, were decisive for detection of TGase activity in the extract.
The inventors also succeeded in isolating and characterizing a DNA sequence from a strain of
Streptomyces lydicus,
exhibiting transglutaminase activity, thereby making it possible to prepare a single-component transglutaminase.
Accordingly, in 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
E. coli
DSM 10175, or
b) an analogue of the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
E. coli
DSM 10175, which
i) is at least 80% homologous with the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in
E. coli
DSM 10175, or
ii) encodes a polypeptide which is at least 79% homolog
Andersen Jens Tønne
Bech Lisbeth
Halkier Torben
Nørrevang Iben Angelica
Rasmussen Grethe
Green Reza
Lambiris Elias J.
Novo Nordisk A S
Slobodyansky Elizabeth
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