Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-04-20
2001-05-08
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C536S023200, C435S320100, C435S252300, C435S254110, C435S254300, C435S254600, C435S219000
Reexamination Certificate
active
06228632
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a recombinant deoxyribonucleic acid (DNA) from
Aspergillus soyae
which codes for a leucine aminopeptidase (LAP), to vectors which contain said DNA as well as further DNA sequences for the expression of the LAP gene, and to filamentous fungi which are transformed with said vectors and which can express the recombinant DNA. The invention also relates to enzyme products which contain a recombinant LAP produced by means of the recombinant filamentous fungi and to methods of producing protein hydrolysates with a low content of bitter substances by means of the recombinant LAP.
PRIOR ART
Protein hydrolysates which are produced enzymatically from difficultly digestible or difficultly soluble animal or vegetable protein, such as gluten, or whey or soya proteins, could have a broad range of application in the food industry, e.g. as additives for whipped foodstuffs, baby foods or animal feedstuffs, and could be of general use for meat and pasta products.
One general problem is that peptides with an unpleasantly bitter accompanying taste are formed as the degree of hydrolysis of the proteins increases. Numerous attempts have already been made to eliminate this bitter accompanying taste. None of the methods developed hitherto has been completely satisfactory, so that a broad range of use has hitherto not existed.
According to more recent findings, the bitter taste is caused by a content of oligopeptides which are formed by endopeptidases as progressive cleavage of the native proteins takes place.
This bitter taste first occurs after a certain degree of hydrolysis of the peptide bonds which are contained. This critical degree of hydrolysis is therefore termed the bitter point. The bitter point is stongly dependent on the hydrolysed protein. Termination of hydrolysis before the bitter point is reached is difficult to achieve technically, and moreover has the effect that the better utilisation capacity which is the aim of hydrolysis is not achieved to its full extent.
European Patent Specification EP 384 303 describes a method of producing protein hydrolysates with a low content of bitter substances. A proteinase (endopeptinase) and an aminopeptidase (exopeptidase) from an Aspergillus culture are used for hydrolysis, in a one- or two-step process. Toxicologically harmless strains of Aspergillus species, for example,
A. oryzae, A. niger
or
A. soyae
, are cited as the source for the peptidase mixture. An excessive formation of bitter-tasting oligopeptides is prevented by restricting the proportion of endopeptidase in relation to aminopeptidase. This is effected by the selective thermal deactivation of the endopeptidases. Using this method, it is possible to delay the occurrence of the bitter point and thus to achieve higher degrees of hydrolysis whilst maintaining a low content of bitter substances.
An improvement has in fact been achieved by said method, but much higher degrees of proteolysis without the simultaneous formation of bitter peptides would be desirable commercially. Another disadvantage is that the thermal deactivation of endopeptidases, and thus the subsequent protein hydrolysis, is not completely reproducible. This applies in particular when the aim is only to effect a partial endopeptidase deactivation by a single-stage process. The poor reproducibility can result in the bitter point being exceeded, or in insufficient hydrolysis. It should also be stated that the deactivation step which has to be performed contributes to a not inconsiderable increase in the cost of the enzyme used and thus of the method as a whole.
In recent years, a series of genes for fungal proteases has been isolated and expressed. WO 90/00192 describes the isolation of the gene for Aspergillopepsin A from
A. awamori
. Aspergillopepsin is a protease which can result in the proteolytic breakdown of an external protein, paticularlyon heterologous gene expression in
A. awamori
. During the expression of calf chymosin in
A. awamori
, the problem also arises that the aspergillopepsin can give rise to unwanted off-tastes during the production of cheese. The object of the aforementioned patent application was therefore the deactivation of the unwanted gene.
Japanese Patent Application JP 90-269370 describes the isolation of the gene for alkaline protease (I) from a chromosomal gene bank of
A. soyae
. This alkaline protease is widely used in the south-east Asia region for the production of soy sauce. However, the present invention does not relate to the production of soy sauce.
OBJECT AND SOLUTION
The object of the present invention was to provide a cost-effective source of an endopeptidase for protein hydrolysis, the use of which further delays the occurrence of the bitter point by a large extent compared with the current state of the art, so that process reliability is significantly improved and costs are reduced at the same time. This object is achieved by a recombinant deoxyribonucleic acid (DNA) which can be isolated from
Aspergillus soyae
, characterised in that it codes for a leucine aminopeptidase (LAP) and comprises a nucleotide sequence corresponding to the nucleotide sequence given in SEQ ID NO: 1 for the mature LAP or to a nucleotide sequence derived therefrom which hybridises under stringent conditions with the nucleotide sequence given in SEQ ID NO: 1 for the mature LAP.
Vectors, particularly plasmids, with which Aspergillus strains or
Trichoderma reesei
strains can be transformed, can be produced by means of this DNA. Those strains which express and secrete LAP in large amounts can then be selected from the transformants obtained. These transformed host organisms in turn enable processes to be carried out for the production of LAP in large amounts. Enzyme products which contain LAP and which are particularly suitable for the hydrolysis of proteins can be produced from the fermentation liquors obtained. Surprisingly, it has been found that protein hydrolysis with the enzyme products according to the invention makes it possible to achieve degrees of hydrolysis, without the occurrence of a bitter taste, which are considerably higher than those which are possible with preparations produced in a conventional manner.
FIGURES, SEQUENCE DESCRIPTIONS AND DEPOSITION OF STRAINS
FIG.
1
: Representation of the vector pKD12
Sequence descriptions:
SEQ ID NO: 1: Chromosomal LAP gene from
A. soyae
RH3782
SEQ ID NO: 2: Protein sequence of the LAP from
A. soyae
RH3782 with signal peptide sequence
Deposition of strains:
The following microorganisms were deposited at the German Collection of Microorganisms and Cell Cultures (DSM), Mascheroder Weg 1b, D-38124 Brunswick, Germany, in accordance with the conditions of the Budapest Agreement (the date of deposition is given in parentheses in each case):
A. soyae
RH3782: deposition number DSM 10090 (Jul. 5, 1995)
E. coli
DH5&agr; pKD12: deposition number DSM 10089 (Jul. 7, 1995)
A. awamori
RH3827: deposition number DSM 10091 (Jul. 5, 1995)
DESCRIPTION OF THE INVENTION
Recombinant DNA
This invention relates to a recombinant deoxyribonucleic acid (DNA) which can be isolated from
Aspergillus soyae
, characterised in that it codes for a leucine aminopeptidase (LAP) and comprises a nucleotide sequence corresponding to the nucleotide sequence given in SEQ ID NO: 1 for the mature LAP or to a nucleotide sequence derived therefrom which hybridises under stringent conditions with the nucleotide sequence given in SEQ ID NO: 1 for the mature LAP.
Sequence description SEQ ID NO: 1 corresponds to the chromosomal LAP gene from
A. soyae
RH3782 with 5′- and 3′-flanking sequences. The nucleotide sequence for the mature LAP is to be understood as the DNA sequence which is contained in SEQ ID NO: 1 and which codes for the structural gene of LAP without the signal peptide. The nucleotide sequence for the mature LAP are thus those exon sequences which code for amino acids 1 (Tyr) to 298 (Leu).
The present invention also relates to a nucleotide sequence derived from the nucleotide sequence for the mature LAP. This i
Gottschalk Michael
Khanh Nguyen Quoc
Plainer Hermann
Schuster Erwin
Sproessler Bruno
Burns Doane Swecker & Mathis L.L.P.
Carlson Karen Cochrane
Roehm GmbH
Srivastava Devesh
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