Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1995-11-03
1997-12-02
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435223, 4352523, 43525231, 43525233, 43525235, 43525411, 4352542, 43525421, 4352543, 4352547, 4353201, 4351723, 536 232, C12N 1577, C12N 976, C12N 958, C12N 115
Patent
active
056935200
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an active recombinant trypsin-like protease, a proteolytic composition comprising said trypsin-like protease, a DNA construct comprising a DNA sequence encoding said trypsin-like protease, and a vector and cell harbouring the DNA construct. Furthermore, the present invention relates to a method of preparing the trypsin-like protease by use of recombinant DNA techniques.
BACKGROUND OF THE INVENTION
Proteases are widely used as ingredients in commercial detergents to improve the detergency towards proteinaceous soiling. For a number of years proteases with a broad specificity, such as subtilisins (Bacillus serine proteases) have been the most widely used detergent proteases. However, in recent years proteases having a more narrow specificity have been of increasing interest for detergent purposes.
WO 89/06270 discloses a detergent composition comprising a protease with a narrow substrate specificity, namely a trypsin-like protease capable of cleaving peptide bonds at the C-terminal side of lysine or arginine. The trypsin-like protease disclosed in said reference is produced by conventional fermentation of a strain of the Fusarium sp. F. oxysporum DSM 2672, and is inevitably produced together with another protease (Protease I), the existence of which is undesirable in detergent compositions. Thus, production of the trypsin-like protease from the F. oxysporum strain involves a step in which the undesired protease is separated from the trypsin-like protease, which makes the large scale production of the latter protease more expensive than what is desirable.
It would be desirable to facilitate the production of said Fusarium trypsin-like protease, in particular by avoiding the co-production of the above mentioned undesired Protease I, to be able of producing both larger amounts of the enzyme and to produce it in a more economical manner than what is possible by the prior art methods.
BRIEF DISCLOSURE OF THE INVENTION
The present inventors have now succeeded in cloning a DNA sequence encoding a Fusarium trypsin-like protease and in obtaining expression of an active trypsin-like protease from said DNA sequence.
Accordingly, in a first aspect the present invention relates to an active recombinant trypsin-like protease comprising the amino acid residues 1-224 of the amino acid sequence shown in the appended SEQ ID No. 2.
In the present context the term "trypsin-like protease" is intended to indicate an enzyme having an activity similar to that of trypsin, i.e. an enzyme capable of cleaving peptide bonds at the C-terminal side of lysine or arginine, The trypsin-like protease activity may be determined in an assay based on cleavage of a trypsin substrate such as N-Benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA or L-BAPNA), e.g. as described in the Materials and Methods section below.
The term "recombinant" as used about the trypsin-like protease of the invention is intended to indicate that it is produced by a cell transformed with a DNA sequence encoding the protease. Thus, the recombinant trypsin-like protease is produced by another organism than its parent organism and accordingly essentially free from components derived from said parent organism, i.e. components produced by the F. oxysporum strain DSM 2672. Such components may confer undesirable properties to the protease, for instance by giving rise to undesirable enzymatic activities. In this respect, the recombinant trypsin-like protease of the invention is easily produced without Protease I, and no costly separation similar to that performed according to WO 89/06270 is required.
In the course of the research leading to the present invention, which is described in detail in the following examples, it was quite unexpectedly found to be difficult to obtain any substantial production of an active trypsin-like protease by cultivation of a conventionally used production organism (Aspergillus oryzae) transformed with the DNA sequence shown in SEQ ID No. 1. On the basis of analyses of the DNA sequ
REFERENCES:
S.L. Berger et al. (eds.) "Guide to Molecular Cloning Techniques", Meth. Enzymol. 152: 393-399, 415-423, 432-447, 661-704, 1987.
M.P. Deutscher (ed.) "Guide to Protein Purification", Meth. Enzymol. 182: 602-613, 738-751, 1991.
Rypniewski et al, Protein Engineering, vol. 6, No. 4, pp. 341-348, 1993.
Branner Sven
Hastrup Sven
Gregg, Esq. Valeta A.
Novo Nordisk A S
Prouty Rebecca E.
Zelson Esq. Steve T.
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