Enzyme with protease activity

Details Enzyme with protease activity Enzyme with protease activity

1999-09-29

2001-02-20

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Cleaning using a micro-organism or enzyme

C435S219000

Reexamination Certificate

active

06190905

ABSTRACT:

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 Rizopus sp. (EP 72978), Schytalidium sp. (JP 48091273), Sulpholobus sp. and Thermoplasma sp. (WO90 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 data base homology search it has been found that the DNA sequence shown in SE 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 the invention.
In a further important aspect the invention relates to an enzyme with protease activity, which is active at a pH below 7.0 and in the presence of up to 3% hydrogen peroxide. In the present context, the term “is active” as used about the enzyme is intended to indicate that the enzyme is capable of hydrolysing a substrate under the above-mentioned conditions, e.g. as described in example 5 herein.
This enzyme of the invention which is active at a pH below 7.0 and in the presence of up to 3% hydrogen peroxidase and which removes more than 80% of the lysozyme from the lenses under the conditions specified in example 5 is termed the “H
2
O
2
-stable protease” in the following disclosure. This enzyme is believed to be generally novel.
Also, the present invention provides an enzyme with protease activity, which enzyme is active at a pH below 7.0 and which is specific towards Phe-Val or Lys-Tyr linkages. The term “specific” is intended to indicate that the enzyme, when the substrate is bovine glucagon, primarily cleaves these linkages.
Protease I and protease II described herein are preferred examples of an enzyme of the invention. The enzymes have been found to be acid proteases, i.e. proteases which has an acid pH optimum.
By the present invention it is possible to provide the protease in a highly purified form, i.e. greater than 75% pure, and more preferably greater than 90% pure as determined by SDS gel electrophoresis as described in the Materials and Methods section herein.
In final aspects the invention relates to an enzyme preparation comprising an enzyme of the invention and the use of the enzyme or enzyme preparation for various purposes in which modification or degradation of protein-containing substances is desirable.
DETAILED DESCRIPTION OF THE INVENTION
The DNA construct, vector and method of the invention
In the present context the term “analogue” used to define the DNA construct of the invention is understood to include any DNA sequence which encodes an enzyme with protease activity and which is at least 80% homologous to the DNA sequence shown in SEQ ID No. 1 or 2, respectively. The analogous DNA sequence may be a DNA sequence which hybridizes to the same probe as the DNA coding for the protease enzyme under the following conditions: presoaking in 5×SSC and prehybridizing for 1 h at −55° C. in a solution of 5×SSC, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 &mgr;g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 &mgr;Ci 32-P-dCTP labelled probe for 18 h at ~55° C. followed by washing three times in 2×SSC, 0.2% SDS at 55° C. for 30 minutes. The analogous DNA sequence is preferably at least 90% homologous to the sequence shown in SEQ ID No. 1 or 2, preferably at least 95% homologous to said sequence.
The analogous DNA sequence may, e.g., be isolated from another organism or may be one prepared on the basis of the DNA sequence shown in SEQ ID No. 1 or 2, such as by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the protease but which correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a protease mutant with different properties than the native enzyme. Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence, or deletion of one or more nucleotides at either end or within the sequence.
Furthermore, it is preferred that the protease encoded by the analogous DNA sequence is immunologically cross-reactive with an antibody raised against a purified protease encoded by the DNA sequence shown in SEQ ID No. 1 or 2.
The nucleotide probe with which the analogue of the DNA sequence shown in SEQ ID No. 1 can hybridize may, e.g. be prepared on the basis of any of the following DNA sequences or any combination thereof:
(a)
AATTAAGCAT CCTCCATCTT
(

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