Haloperoxidases with altered pH profiles

Details Haloperoxidases with altered pH profiles Haloperoxidases with altered pH profiles

2001-02-20

2002-04-16

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S252300, C435S320100, C536S023200, C530S350000

Reexamination Certificate

active

06372465

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to haloperoxidase variants with an altered pH optimum compared to the wild type.
BACKGROUND OF THE INVENTION
Haloperoxidases form a class of enzymes which are able to oxidize halides (X═Cl—, Br—, or I—) in the presence of hydrogen peroxide to the corresponding hypohalous acid (HOX) according to:
H
2
O
2
+X−+H+→H
2
O+HOX
If a convenient nucleophilic acceptor is present, a reaction will occur with HOX whereby a diversity of halogenated reaction products may be formed.
A chloride peroxidase (EC 1.11.1.10) is an enzyme capable of oxidizing chloride, bromide and iodide ions with the consumption of H
2
O
2
.
A bromide peroxidase is an enzyme capable of oxidizing bromide and iodide ions with the consumption of H
2
O
2
.
A iodide peroxidase (EC 1.11.1.8) is an enzyme capable of oxidizing iodide ions with the consumption of H
2
O
2
.
Vanadium haloperoxidases are different from other haloperoxidases in that the prosthetic group in these enzymes have structural features similar to vanadate (vanadium V), whereas the other haloperoxidases are hemeperoxidases.
Haloperoxidases have been isolated from various organisms: mammals, marine animals, plants, algae, a lichen, fungi and bacteria (for reference see
Biochimica et Biophysica Acta
1161, 1993, pp. 249-256). It is generally accepted that haloperoxidases are the enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may be involved.
The amino acid sequence (SEQ No. 1) for the vanadium-containing chloroperoxidase from the fungus
Curvularia inaequalis
has been published (see SWISS-PROT:P49053).
The amino acid sequence (SEQ No. 2) for the vanadium-containing chloroperoxidase from the fungus
Curvularia verruculosa
has been published (see WO 97/04102).
The X-ray structure of the vanadium-containing chloroperoxidase from the fungus
Curvularia inaequalis
has been published (Proc. Natl. Acad. Sci. U S A, 93(1), 1996, 392-396; and pdb1vnc.ent).
Haloperoxidases are of current interest because of their broad range of potential industrial uses. For example, haloperoxidases have been proposed for use as an anti-microbial agent.
BRIEF DISCLOSURE OF THE INVENTION
The present invention relates to vanadium-containing haloperoxidase variants with an altered pH optimum compared to the parent haloperoxidase, so in particular the present invention deals with:
A variant of a parent vanadium-containing haloperoxidase, which variant has haloperoxidase activity and an altered pH optimum and comprises a mutation in a position corresponding to at least one of the following positions:
R490A,L,I,Q,M,E,D;
A399G;
F397N,Y,E,Q;
P395A,S;
R360A,L,I,Q,M,E,D;
K353Q,M;
S402A,T,V,S;
D292L;
A501S;
W350F,Y;
V495A,T,V,S;
K394 A,L,I,Q,M,E,D;
wherein the parent haloperoxidase has the amino acid sequence given in SEQ ID No. 1 or the parent haloperoxidase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1.
DETAILED DISCLOSURE OF THE INVENTION
Homologous Vanadium-Containing Haloperoxidases
A number of vanadium-containing haloperoxidases produced by different fungi are homologous on the amino acid level.
An alignment of the
Curvularia inaequalis
and the
Curvularia verruculosa
haloperoxidases was performed. The alignment uses the haloperoxidase amino acid sequence obtained from the 3D structure file of
C. inaequalis
(Brookhaven databank file pdb1vnc.ent).
When using the homology percent obtained from UWGCG program using the GAP program with the default parameters (penalties: gap weight=3.0, length weight=0.1; WISCONSIN PACKAGE Version 8.1-UNIX, August 1995, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) the following homology was found:
Curvularia inaequalis
vanadium-containing haloperoxidase comprising the amino acid sequence shown in SEQ ID No. 1: 100%;
Curvularia verruculosa
vanadium-containing haloperoxidase comprising the amino acid sequence shown in SEQ ID No. 2: 96%.
In the present context, “derived from” is intended not only to indicate a vanadium-containing haloperoxidase produced or producible by a strain of the organism in question, but also a vanadium-containing haloperoxidase encoded by a DNA sequence isolated from such strain and produced in a host organism containing said DNA sequence. Finally, the term is intended to indicate a vanadium-containing haloperoxidase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the vanadium-containing haloperoxidase in question.
Variants with Altered pH Optimum
The desired pH optimum of a vanadium-containing haloperoxidase depends on which application is of interest, e.g., if the vanadium-containing haloperoxidase is to be used for denim bleaching the preferred pH optimum will be around pH 5-8, whereas if the vanadium-containing haloperoxidase is to be used for washing purposes the preferred pH optimum will be around pH 8-10.
It is possible to alter the pH optimum of a parent vanadium-containing haloperoxidase wherein said variant is the result of a mutation, i.e. one or more amino acid residues have been deleted from, replaced or added to the parent vanadium-containing haloperoxidase. By introducing charge changes in the neighbourhood of the active site residues, the pKa of the residue of interest can be changed in order to accomodate an altered activity profile of the haloperoxidase in question.
It is a common belief that by introducing more negative charged residues close to the His (the active site of the haloperoxidase), its pKa is elevated and it will thus be able to act in catalysis at a higher pH than previously. The active site His will, by introducing more positive charged residues close to His, alter its pKa to a lower pKa than previously and thus be able to act in catalysis at a lower pH than previously.
The increase in pKa can also be obtained by decreasing the solvent accessibility of the active site (His). The decrease in pKa can also be obtained by increasing the solvent accessibility of the active site (His).
But according to the present invention it is found that of most importance is that the residues are within 10 Å around His 496 and His 404. These residues are: 46-48, 193, 257, 259-265, 267-269, 285-294, 297-304, 307, 242, 245-346, 349-350, 353, 358-363, 365, 378, 380-384, 393-412, 441, 443, 482-502, 507, 551-557. Changes within this region are found to alter the pH dependent activity or change the pH-optimum of the enzyme. Residues can in this way be mutated in e.g. the
Curvularia inaequalis
haloperoxidase. Homologous structures which are assumed to have similar structure can be modelbuild (see Example 1) and regions of interest found in the same way.
Preferred positions for mutations are the following:
R490A,L,I,Q,M,E,D;
A399G;
F397N,Y,E,Q;
P395A,S;
R360A,L,I,Q,M,E,D;
K353Q,M;
S402A,T,V,S;
D292L,E;
A501S;
W350F,Y;
V495A,T,V,S;
K394 A,L,I,Q,M,E,D;
wherein the parent haloperoxidase has the amino acid sequence given in SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 85% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 90% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 95% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 96% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 97% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase which has an amino acid sequence which is at least 98% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase whic

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