Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-02-01
1998-12-29
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435212, 435223, 435224, 435225, 4352523, 43525233, 43525411, 4352543, 4353201, 536 231, 536 232, 536 2374, C12N 962, C07H 2104
Patent
active
058540504
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/DK94/00274 filed Jul. 5, 1994, which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a DNA construct encoding an enzyme with protease activity, a method of producing the enzyme, an enzyme with protease activity, and an enzyme preparation containing the enzyme.
BACKGROUND OF THE INVENTION
Proteases are enzymes capable of cleaving peptide bonds. Acid proteases (i.e. proteases having an acidic pH optimum) have been found to be produced by a number of different organism including mammals and microbes. For instance, microbial acid proteases have been found to be produced by bacterial strains such as strains of Bacillus sp. (JP 01240184), fungal strains, e.g. of Rhizopus sp. (EP 72978), Schytalidium sp. (JP 48091273), Sulpholobus sp. and Thermoplasma sp. (WO/90 10072) and Aspergillus sp. (JP 50121486, EP 82 395).
JP 3058794 discloses the cloning of a gene encoding an acid protease from R. niveus and the recombinant expression thereof. The cloning and expression of a gene from Cryphonectira parasitica encoding an aspartic protease is described by Choi et al. (1993). Takahashi et al. (1991), Inoue et al. (1991), and JP 407 5586 discloses the cloning of a gene from Aspergillus niger encoding an acid proteinase (Protease A).
Berka et al. (1990) disclose a gene encoding the aspartic proteinase aspergillopepsin A from Aspergillus awamori. The cloning of a gene encoding the aspartic proteinase aspergillopepsin O from Aspergillus oryzae is described by Berka et al. (1993). The cloning of a gene encoding the acid protease (PEPA) from Aspergillus oryzae is disclosed by Gomi et al. (1993).
Acid proteases are widely used industrially, e.g. in the preparation of food and feed, in the leather industry (e.g. to dehair hides), in the production of protein hydrolysates and in the wine making and brewing industry.
There is a need for single-component acid proteases for many different applications, especially in the food and feed industry.
SUMMARY OF THE INVENTION
It is an object of the present invention to prepare a single-component protease.
Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting protease activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1 or an analogous sequence thereof being at least 80% homologous to the DNA sequence shown in SEQ ID No. 1.
In a second aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting protease activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 2 or an analogous sequence thereof being at least 80% homologous to the DNA sequence shown in SEQ ID No. 2.
The DNA sequence shown in SEQ ID No. 1 encodes an enzyme which in the following disclosure is referred to as Protease I. The enzyme encoded by the DNA sequence shown in SEQ ID No. 2 is referred to as Protease II.
By a database homology search it has been found that the DNA sequence shown in SEQ ID Nos. 1 and 2 are generally novel. The highest homology of the DNA sequence shown in SEQ ID No. 1 to known protease genes was found to be 74.7% to the Aspergillus niger acidic proteinase A as determined for an overlap of 538 nucleotides. The highest homology to protease II was found to be 75.5% to the Aspergillus oryzae aspergillopepsin O as determined for an overlap of 343 nucleotides.
In further aspects the invention relates to an expression vector harbouring a DNA construct of the invention, a cell comprising the DNA construct or expression vector and a method of producing an enzyme exhibiting protease activity which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
In a still further aspect the invention relates to an enzyme exhibiting protease activity, which enzyme is encoded by a DNA construct of the invention as defined above or produced by the method of t
REFERENCES:
patent: 3492204 (1970-01-01), Koaze et al.
patent: 3509024 (1970-04-01), Jurgens et al.
Takahashi et al., The J. of Biological Chem., vol. 266, No. 29, pp. 19480-19483, 1991.
Berka et al., Gene, vol. 125, pp. 195-198, 1993.
Inoue et al. "The gene and deduced protein sequence of the zymogen of Aspergillus niger acid proteinase A" J. Biol. Chem. 266, 19484-18489, Oct. 1991.
Berka et al. "Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori" Gene 86, 153-162, 1990.
Andersen Lene Nonboe
Christgau Stephan
Dalb.o slashed.ge Henrik
Dambmann Claus
Kauppinen Markus Sakari
Agris, Esq. Cheryl H.
Nashed Nashaat T.
Novo Nordisk A S
Wax Robert A.
Zelson Esq. Steve T.
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