CN1293708A

CN1293708A - Haloperoxidases with altered pH profiles

Info

Publication number: CN1293708A
Application number: CN99804012A
Authority: CN
Inventors: A·斯文德森; L·乔根森
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1998-03-18
Filing date: 1999-03-16
Publication date: 2001-05-02
Also published as: EP1062327A1; KR20010041989A; AU2714599A; WO1999047651A1; BR9908830A; JP2002506638A; CA2322781A1

Abstract

Variants 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; K394A,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.

Description

Haloperoxidases with altered pH profiles

Technical Field

The present invention relates to haloperoxidase (haloperoxidase) variants with altered pH optima compared to the wild type.

Background

Haloperoxidases constitute a class of enzymes which are capable of reacting a halide (X = C) in the presence of hydrogen peroxide according to the following formulal^-、Br^-Or I^-) Oxidation to the corresponding hypohalous acid (HOX):

if a convenient nucleophilic acceptor is present, it will react with HOX and may form a variety of halogenation reaction products.

Chloride peroxidase (EC 1.11.1.10) is a highly potent H consuming enzyme₂O₂Chlorine, bromine and iodide ions.

Bromide peroxidase (Bromide peroxidase) is a substance which consumes H₂O₂Enzymes that oxidize bromine and iodine ions.

Iodide peroxidase (EC1.11.1.8) is a potent H consuming enzyme₂O₂An enzyme that oxidizes iodide ions.

Vanadium haloperoxidases differ from other haloperoxidases in that the former have prosthetic group structural features similar to vanadate (vanadium V) and the latter are heme peroxidases.

Haloperoxidases have been isolated from a variety of organisms: mammals, marine animals, plants, algae, lichen, fungi and bacteria (cf. Biochemical and biophysical reports 1161, 1993: 249-. It is generally accepted that haloperoxidases are enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may also be involved in this process.

The amino acid sequence (SEQ NO. 1) of the enzyme chloroperoxidase containing vanadium (chloroperoxidase) from the fungus Curvularia anisopliae has been disclosed (see SWISS-PROT: P49053).

The amino acid sequence of vanadium-containing chloroperoxidases from the fungus Curvularia verruculosa (SEQ ID NO. 2) has been disclosed (see WO 9704102).

The X-ray structure of the vanadium-containing chloroperoxidase from the fungus Campylobacter anisotropis has been disclosed (proceedings of the American academy of sciences USA 93(1), 1996, 392-.

The current interest in haloperoxidases is due to their wide range of potential industrial uses. For example, haloperoxidases have been suggested as an antimicrobial agent.

Brief description of the invention

The present invention relates to vanadium-containing haloperoxidase variants having an altered pH optimum relative to the parent haloperoxidase, and thus in particular the present invention relates to:

a variant of a parent vanadium-containing haloperoxidase, the variant having haloperoxidase activity, an altered pH optimum and comprising a mutation at 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 comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence contained in the parent haloperoxidase that is identical to the amino acid sequence given in SEQ ID NO:1 at least 80% homologous. Detailed description of the invention homologous vanadium-containing haloperoxidases

Many vanadium-containing haloperoxidases produced by different fungi are homologous at the amino acid level.

The anisomerosa was aligned with haloperoxidase from c. The haloperoxidase amino acid sequence from the 3D structural diagram of Curvularia anisopliae (Brookhavandatabank file pdblvnc. ent) was used for the alignment.

Using the percentage homology obtained by the UWGCG program (which uses the GAP program with system-set parameters: penalty: GAP weight) = 3.0, length (1engthweight) = 0.1; WISCONSIN PACKAGE version 8.1-UNIX, 8.1995, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA53711), the following homology was found:

comprises the amino acid sequence of SEQ ID NO:1, the anisotropium vanadiferous haloperoxidase of the amino acid sequence shown in 1: 100 percent; comprises the amino acid sequence of SEQ ID NO: 2 c.verruculosa vanadium-containing haloperoxidase having the amino acid sequence shown in seq id no: 96 percent.

In this context, "derived from" is used to mean not only the vanadium-containing haloperoxidase produced or producible by the microorganism strain in question, but also vanadium-containing haloperoxidases which are encoded by a DNA sequence isolated from this strain and which are produced in a host organism containing this DNA sequence. Finally, the term is also used to denote a vanadium-containing haloperoxidase encoded by a DNA sequence of synthetic and/or cDNA origin having the identifying characteristics of the vanadium-containing haloperoxidase in question. Variants with altered pH optima

The expected optimum pH of the vanadium-containing haloperoxidase depends on its application purpose, e.g. if the vanadium-containing haloperoxidase is to be used for bleaching denim, the preferred optimum pH should be around 5-8, whereas if the vanadium-containing haloperoxidase is to be used for washing purposes, the preferred optimum pH should be around 8-10.

It is possible to alter the pH optimum of a parent vanadium-containing haloperoxidase wherein the variant is the result of a mutation, i.e.deletion, substitution or addition of 1 or more amino acid residues in the parent vanadium-containing haloperoxidase. By introducing a charge change near the active site residue, the pKa of the residue of interest can be altered, thereby altering the activity profile of the haloperoxidase being targeted.

It is generally accepted that the pKa of His (haloperoxidase) can be increased by introducing more negatively charged residues near His active site, so that the enzyme can be catalyzed at higher pH than before. By introducing more positively charged residues near His, the active site His lowers its pKa, enabling catalysis at lower than previous pH values.

The pKa can also be increased by reducing the solvent accessibility of the active site (His). pKa can also be decreased by increasing solvent accessibility of the active site (His).

However, according to the present invention, it has been found that the most important residues are those located within 10 Å around His496 and His404, which are residues 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, it has been found that changes in these regions alter the pH-dependent activity of the enzyme or change its optima pH. can mutate residues in such a way in, for example, Isocurvularia haloperoxidase.

Preferred sites for mutagenesis are as follows:

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；

K394A，L，I，Q，M，E，D；

wherein the parent haloperoxidase comprises the amino acid sequence of SEQ ID NO:1, or has an amino acid sequence identical to that given in SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 80% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 96% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 97% homologous to the amino acid sequence of SEQ ID NO:1, or a site of homology in a parent haloperoxidase having an amino acid sequence at least 98% homologous to the amino acid sequence of SEQ ID NO:1 a site of homology in a parent haloperoxidase of an amino acid sequence that is at least 99% homologous.

The following mutations are particularly preferred:

R487A，L，I，Q，M，E，D；

A396G；

F394N，Y，E，Q；

P392A，S；

R357A，L，I，Q，M，E，D；

K350Q，M；

S399A，T，V，S；

D289L，E；

A498S；

W347F，Y；

V492A，T，V，S；

K391A，L，I，Q，M，E，D；

wherein the parent haloperoxidase comprises the amino acid sequence of SEQ ID NO: 2, and 2, amino acid sequence shown in the specification.

In a preferred embodiment, two or more amino acid residues may be substituted as follows:

R490A+D292L；

R490A+D292E；

R490L+D292L；

R490L+D292E；

R490I+D292L；

R490I+D292E；

R490Q+D292L；

R490Q+D292E；

R490M+D292L；

R490M+D292E；

R490E+D292L；

R490E+D292E；

R490D+D292L；

R490D+D292E；

R487A+D289L；

R487A+D289E；

R487L+D289L；

R487L+D289E；

R487I+D289L；

R487I+D289E；

R487Q+D289L；

R487Q+D289E；

R487M+D289L；

R487M+D289E；

R487E+D289L；

R487E+D289E；

R487D+D289L；

R487D+D289E；

wherein the parent haloperoxidase comprises the amino acid sequence of SEQ ID NO: 2, and 2, amino acid sequence shown in the specification. Method for preparing vanadium-containing haloperoxidase variants

Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of the haloperoxidase-encoding DNA sequence, the method of generating mutations at specific sites within the haloperoxidase-encoding sequence will be discussed. Cloning of DNA sequences encoding haloperoxidases

The DNA sequence encoding the parent vanadium-containing haloperoxidase may be isolated from any cell or microorganism capable of producing the haloperoxidase of interest using various methods known in the art. First, a genomic DNA and/or cDNA library is constructed using chromosomal DNA and/or messenger RNA from those organisms that produce the haloperoxidase to be studied. Then, if the amino acid sequence of the haloperoxidase is known, homologous, labeled oligonucleotide probes may be synthesized and used to identify haloperoxidase-encoding clones from a genomic library prepared from the organism under study. Alternatively, haloperoxidase-encoding clones may be identified using low stringency hybridization and wash conditions using labeled oligonucleotides containing sequences homologous to known haloperoxidase genes as probes.

The method for identifying haloperoxidase-encoding clones involves inserting the cDNA into an expression vector, such as a plasmid, transforming a haloperoxidase-negative fungus with the resulting cDNA library, and then plating the transformed fungus on agar containing a haloperoxidase substrate, thereby enabling identification of clones expressing haloperoxidase.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by well-established standard methods, such as the phosphoramidite method. In the phosphoramidite method, oligonucleotides are synthesized, for example on an automated DNA synthesizer, purified, annealed, ligated and cloned into suitable vectors.

Finally, the DNA sequences may be of mixed genomic and synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (each fragment corresponding to a different part of the complete DNA sequence, as the case may be) together in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction using specific primers. Site-directed mutagenesis

Once the haloperoxidase-encoding DNA sequence has been isolated and the desired site of mutation identified, the mutation can be introduced using synthetic oligonucleotides. These oligonucleotides contain a nucleotide sequence flanking the desired mutation site; the mutant nucleotide is inserted during the synthesis of the oligonucleotide. In one embodiment, a single-stranded gap of DNA bridging the haloperoxidase coding sequence is created in a vector carrying the haloperoxidase gene. The synthetic nucleotide with the desired mutation is then annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the constructs are ligated using T4 ligase (Morinaga method-see Biotechnology, 2, 1984, 626-. U.S. patent 4760025 discloses the introduction of oligonucleotides encoding multiple mutations by minor changes in the oligonucleotides. However, the Morinaga method can introduce even more mutations at one time because it can introduce a large number of oligonucleotides of different lengths.

Another method for introducing mutations into the haloperoxidase-encoding DNA sequence is 3 steps to generate a PCR fragment containing the desired mutation by using a chemically synthesized DNA strand as a primer in the PCR reaction. The DNA fragment carrying the mutation can be isolated by cleavage with restriction enzymes and reinserted into the expression plasmid from the PCR-generated fragment. Random mutagenesis

The DNA sequence encoding the parent haloperoxidase may be conveniently subjected to random mutagenesis by any method known in the art.

For example, random mutagenesis may be achieved by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by PCR mutagenesis of a DNA sequence. In addition, random mutagenesis can be performed by using any combination of these mutagens.

The mutagen may be, for example, one that induces transitions, transversions, inversions, rearrangements (scrambling), deletions and/or insertions.

Examples of physical or chemical mutagens suitable for the present purpose include Ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, Ethyl Methane Sulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs.

When these agents are used, mutagenesis is generally carried out by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under conditions suitable for mutagenesis to occur, and selecting out the mutated DNA having the desired properties.

When mutagenesis is performed with an oligonucleotide, 3 non-parent nucleotides can be added or inserted at the positions to be altered during synthesis of the oligonucleotide. Additions or insertions may be made to avoid codons for those undesired amino acids. The added or inserted oligonucleotide may be incorporated into the haloperoxidase-encoding DNA by any of the disclosed techniques, using, for example, PCR, LCR, or any DNA polymerase and ligase.

In the case of mutagenesis by PCR, the gene encoding the parent haloperoxidase, chemically treated or untreated, is subjected to PCR under conditions which enhance misincorporation of nucleotides (Deshler 1992; Leung et al, technique 1, 1989: 11-15).

An mutator strain of E.coli, Saccharomyces cerevisiae or any other microorganism (Fowler et al, molecular and genetic genetics, 133, 1974: 179-191) can be used for random mutagenesis of the DNA encoding the haloperoxidase by, for example, transforming a plasmid containing the gene for the parent enzyme into the mutator strain, culturing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmids can also subsequently be transformed into expression organisms.

The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent haloperoxidase. Alternatively, the DNA may be present on a suitable vector, such as a plasmid or phage, which may be incubated with or treated with a mutagenizing agent. The DNA to be mutagenized may also be present in the host cell as integrated into the genome of the cell or located on a vector carried by the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be appreciated that the preferred DNA sequence to be subjected to random mutagenesis is a cDNA or genomic DNA sequence.

In some cases, it may be convenient to amplify the mutated DNA sequence prior to performing the expression or screening step. Such amplification may be performed according to methods known in the art, with PCR amplification using oligonucleotide primers prepared based on the DNA or amino acid sequence of the parent enzyme being presently preferred.

After incubation with or treatment with a mutagen, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under suitable conditions for expression to occur. The host cell used for this purpose may be any cell which has been transformed with the mutated DNA sequence, optionally in a vector, or which carries the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae.

The mutated DNA sequence may also comprise a DNA sequence encoding a function enabling expression of the mutated DNA sequence. Localized random mutagenesis

It may be advantageous to localize the random mutagenesis to a certain part of the parent haloperoxidase under investigation. This may be advantageous, for example, when it has been determined that certain regions of an enzyme are particularly important for a given property of the enzyme, and that modifications are desired to produce variants with improved properties. Such regions can be identified generally when the tertiary structure of the parent enzyme is elucidated and linked to the function of the enzyme.

Localized random mutagenesis may be conveniently performed as described above using PCR mutagenesis techniques or other suitable techniques known in the art.

Alternatively, the portion of the DNA sequence to be modified may be isolated, for example by inserting it into a suitable vector, and then mutagenized by any of the mutagenesis methods discussed above.

In view of the above-mentioned screening step of the method of the present invention, this can be carried out using an amino acid membrane filtration method based on the following principle:

culturing a microorganism capable of expressing the mutated haloperoxidase on a suitable culture medium under conditions suitable for secretion of the enzyme, the culture medium being provided with two filter membranes, a first membrane which is protein-binding and a second membrane which exhibits lower protein-binding capacity. The microorganism is located on the second layer of film. After incubation, the first membrane comprising the enzyme secreted by the microorganism is separated from the second membrane comprising the microorganism. The first membrane is screened for the desired enzyme activity and the corresponding microbial colony is identified on the second membrane.

The membrane used to bind the enzyme activity may be any protein binding membrane, such as nylon or nitrocellulose. The upper membrane carrying the expressed biological colonies may be any membrane which has no or only low protein binding capacity, e.g. cellulose acetate or Durapore^TM. The membrane may be pretreated in any condition used for screening, or may be treated in the process of detecting the enzyme activity.

The enzyme activity may be detected using dyes, fluorescence, precipitation, pH indicators, IR-absorption or any known technique for detecting enzyme activity.

The test compound may be immobilized using any immobilizing agent, such as agarose, agar, gelatin, polyacrylamide, starch, filter paper, cloth, or a combination of these immobilizing agents. Expression of haloperoxidase variants

According to the present invention, the DNA sequence encoding the variant produced by the methods described above, or any alternative method known in the art, can be expressed enzymatically using an expression vector which typically includes regulatory sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding the haloperoxidase variant of the present invention may be any vector which can be conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a phage or an extrachromosomal element, a minichromosome, or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell chromosome and replicated together with the chromosome(s) into which it has been integrated.

Suitable promoters for directing transcription of a DNA sequence encoding a haloperoxidase variant of the present invention, particularly in fungal cells, are those derived from genes encoding Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic protease, Aspergillus niger neutral α -amylase, Aspergillus niger acid stable α -amylase, Aspergillus niger glucoamylase, R.miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, or Aspergillus nidulans acetamidase.

The expression vectors of the invention also contain a suitable transcription terminator and, in eukaryotic cells, polyadenylation sequences operably linked to the DNA sequence encoding the haloperoxidase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may additionally comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ 702.

The vector may also contain a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as a gene that confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance) to the host. Alternatively, the vector may comprise Aspergillus selection markers (such as amdS, argB, niaD and sC), a marker for hygromycin resistance, or selection may be achieved by co-transformation, for example as described in WO 9117243.

The procedures for ligating the DNA construct encoding the haloperoxidase variant of the present invention, the promoter, terminator and other elements separately and inserting them into a suitable vector containing the information required for replication are well known to those skilled in the art (see, e.g., Sambrook et al (1989)).

The cells of the invention, whether comprising the DNA construct or the expression vector of the invention as defined above, may advantageously be used as host cells for the recombinant production of the haloperoxidase variants of the invention. The cell may be conveniently transformed with a DNA construct of the invention encoding the variant by integrating the DNA construct (in 1 or more copies) into the host chromosome. Integration is generally considered to be advantageous because the DNA sequence is more likely to be stably retained in the cell. The DNA construct may be integrated into the host chromosome according to conventional methods, for example by homologous or heterologous recombination. Alternatively, the cells may be transformed with an expression vector as described above for the different types of host cells.

The cell of the invention may be a cell of a higher organism, such as a mammal or an insect, but is preferably a microbial cell, e.g. a fungal cell.

Advantageously, the filamentous fungus may belong to a species of Aspergillus, such as Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed in a known manner by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall. Suitable procedures for transforming Aspergillus host cells are described in EP 238023.

In a further aspect, the present invention relates to a method of preparing a haloperoxidase variant of the present invention comprising culturing a host cell as described above under conditions conducive to the production of the variant and purifying the variant from the cell and/or the culture medium.

The medium used to culture the cells may be any conventional medium suitable for the growth of the host cell in question. Suitable media can be purchased from commercial suppliers or prepared according to published recipes (e.g., as described in catalogues of the American type culture Collection).

The haloperoxidase variants secreted by the host cells may be conveniently purified from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt, such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, and the like.

Industrial use

The haloperoxidases of the present invention may be incorporated into a stain removal or cleaning composition comprising other enzymes useful in stain removal or cleaning compositions, preferably comprising at least one enzyme selected from the group consisting of proteases, amylases, cutinases, peroxidases, oxidases, laccases, cellulases, xylanases and lipases. In particular, the haloperoxidases of the present invention may be used for bleaching and hygiene purposes.

For preserving foods, beverages, cosmetics (such as skin lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouthwashes), contact lens products, enzyme preparations or food ingredients, the haloperoxidases of the present invention may be added to, for example, unpreserved foods, beverages, cosmetics, contact lens products, food ingredients or anti-infective products in an amount effective to kill or inhibit microbial cell growth.

Thus, the haloperoxidases useful in the methods of the present invention may be used as disinfectants, e.g., for the treatment of acne, eye or oral infections, skin infections; in antiperspirants or deodorants; used in foot washing saline; it can be used for cleaning and disinfecting contact lens, hard surface, teeth (oral cavity health promotion), wound, bruise, etc.

In general, it is contemplated that the haloperoxidases of the present invention may be used to clean, disinfect, or inhibit microbial growth of any hard surface. Examples of such surfaces which can advantageously be treated with the composition according to the invention are surfaces of processing equipment used in, for example, dairy products, chemical or pharmaceutical processing plants, water purification systems, pulp processing plants, water treatment plants and cooling towers. The haloperoxidases of the present invention should be used in an amount effective to clean, disinfect, or inhibit microbial growth on the surface.

In addition, it is contemplated that the haloperoxidases of the present invention may be advantageously used in clean-in-place (CIP) systems for cleaning any kind of process equipment.

In addition, the haloperoxidases of the present invention may be used to clean surfaces and cookware in food processing plants or in any location where food is produced or provided (e.g., hospitals, nursing homes, restaurants, particularly fast food restaurants, deli restaurants, etc.). It can also be used as an antimicrobial agent in food products, particularly as a surface antimicrobial agent for cheese, fruits and vegetables, and food on salad bars.

It can also be used as a preservative or disinfectant in water-based coatings. Preservation/corrosion protection of coatings

The prior art has preserved canned paint products by adding non-enzymatic organic biocides to the paint. In the context of the present invention, a coating is to be understood as a substance comprising a solid colouring substance dissolved or dispersed in a liquid carrier, such as water, an organic solvent and/or an oil, which when applied to a surface leaves a thin coloured, decorative and/or protective layer after drying. Isothiazolinones (isothiazolinones), such as 5-chloro-2-methyl-4-thiazolin-3-one, are commonly added to coatings as fungicides at a dosage of about 0.05-0.5% to inhibit/prevent microbial growth in the coating. The method of the present invention can be suitably used in this field to solve the problem of environmental biohazard which has long existed in the use of toxic germicides by replacing the toxic organic germicides with environmentally compatible enzymes. The present invention thus provides a method for preserving a coating comprising contacting the coating with a haloperoxidase variant of the present invention. The present invention further provides a coating composition comprising a haloperoxidase variant of the present invention.

Preferably the coating is water based, i.e. the solids of the coating are dispersed in an aqueous solution. The coating may contain 0-20% organic solvent, preferably 0-10%, for example 0-5%.

The enzyme may be added to the coating in an amount of 0.0001-100 mg of active enzyme protein per liter of coating, preferably 0.001-10 mg/l, e.g.0.01-1 mg/l. Hydrogen peroxide source

According to the present invention, the hydrogen peroxide required for the reaction with the haloperoxidase may be obtained in a number of different ways: may be hydrogen peroxide or a hydrogen peroxide precursor such as, for example, percarbonate or perborate, or a percarboxylic acid or a salt thereof, or may be a hydrogen peroxide generating enzyme system such as, for example, an oxidase and its substrate. Useful oxidizing enzymes may be, for example, glucose oxidase, glycerol oxidase or amino acid oxidase. An example of an amino acid oxidase is given in WO 9425574.

The use of enzymatically produced hydrogen peroxide may be advantageous because this source produces lower concentrations of hydrogen peroxide under biologically relevant conditions. The low concentration of hydrogen peroxide results in an increased rate of haloperoxidase-catalyzed reaction.

According to the invention, the source of hydrogen peroxide required for the reaction with the haloperoxidase may be added at a concentration corresponding to a hydrogen peroxide concentration in the range of 0.01-1000 mM, preferably 0.1-500 mM. Halide source

According to the present invention, the halide source required for the reaction with the haloperoxidase may be obtained in a number of different ways: may be sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide or potassium iodide.

The concentration of the halide source is usually 0.01 to 1000mM, preferably 0.1 to 500 mM. Composition comprising a metal oxide and a metal oxide

The composition comprising the haloperoxidase, the hydrogen peroxide source and the halide source may be formulated as a solid or liquid, especially as a non-dusting granulate or a stabilized liquid.

When formulated as a solid, all the components may be mixed together, for example, as a powder, granules or gel-like product.

When compositions other than dry form are used, and even when dry compositions are used, it is preferred to use a two-part formulation system to separate the hydrogen peroxide from the other components.

The compositions of the present invention may also contain adjuvants such as wetting agents, thickeners, buffers, stabilizers, fragrances, colorants, fillers, and the like.

Useful wetting agents are surfactants, i.e. nonionic, anionic, amphoteric or zwitterionic surfactants.

The composition of the invention may be a concentrated product or a ready-to-use product. Haloperoxidase activity

According to the invention, the haloperoxidase activity may be measured as described in WO9704102, pages 13, lines 7-19.

The invention is further illustrated by the following examples, which are in no way intended to limit the scope of the invention as claimed.

Example 1 homology construction of Curvularia verruculosa haloperoxidase 3D-Structure

Using sequence homology of Curvularia Inequalis (CI) with other sequences (e.g., C. verruculosa), a 3D-structure resembling CI can be found.

There are differences in many residues of c.verruculosa when compared to campylobacter anisopliae for illustrating its structure. Models can be constructed using the INSIGHT and HOMOLOGY programs available from Molecular diagnostics Inc. At homologous positions where the program identified Structural Conserved Regions (SCRs), the program replaced amino acids in anisomerous with amino acids from c. The middle residues were constructed using the LOOP option of GENERATE. Using these steps, a rough model can be obtained that provides information on the stereo interaction.

The structure can be refined using the methods described in the HOMOLOGY package.

EXAMPLE 2 construction of haloperoxidase variants

To construct variants of c.verruculosa haloperoxidase, commercial kits may be used according to the manufacturer's instructions: chameleon double-stranded site-directed mutagenesis kit.

The gene encoding the haloperoxidase of interest was located on plasmid pElo 29. The ScaI site of the ampicillin gene in pElo29 was changed to the MluI site using primer 117996(SEQ ID NO: 3) according to the manufacturer's instructions. While introducing the desired mutation into the haloperoxidase gene by adding suitable primers containing the desired mutation. Construction of plasmid pElo29

Plasmid pElo29 was constructed from the following fragments:

a) a4.1 kb fragment was excised from the vector pCiP (described in WO 9334618) with BamHI and BglII.

b) C.verruculosa haloperoxidase gene PCR amplified with Pwo polymerase, using plasmid pAJ014-1(WO9704102) as template, primers 146063 and 146062(SEQ ID NO: 4 and 5) BamHI and BglII sites were introduced into 5 'and 3' of the haloperoxidase gene, respectively.

After ligation of fragments a and b, the entire haloperoxidase gene was sequenced to ensure that no unwanted mutations were introduced during the PCR amplification. Site-directed mutagenesis

Site-directed mutagenesis as described above was used to construct plasmids carrying the gene encoding the haloperoxidase variant. The following primers were used:

primer 147293(SEQ ID NO: 6) was used to introduce D289L.

Primer 147295(SEQ ID NO: 7) was used to introduce D289E.

Primer 139078(SEQ ID NO: 8) was used to introduce R487E.

Primer 139085(SEQ ID NO: 9) was used to introduce V492S.

The mutations in each case were verified by sequencing the entire gene. The resulting plasmids were designated pElo30, pElo31, pElo34 and pElo37, respectively. Transformation of pElo30, pElo31, pElo34 and pElo37 into JaL228

Aspergillus oryzae JaL228(WO9727221) is Aspergillus oryzae IFO4177 deficient in alkaline protease and neutral metalloprotease I. The strains were transformed with pElo30, pElo31, pElo34 and pElo37 using plasmid pToC90(WO9117243) carrying the amdS gene as a co-transformant. Transformants were selected using acetamide as described in patent EP 0531372B 1. Transformants were twice sporulated. Small scale fermentations (shake flasks and microplates) were performed to examine the haloperoxidase production from spores from the 2 nd isolate of each transformant. Fermentation of C.verruculosa haloperoxidase variants in Aspergillus oryzae

Fermenting the above isolate in a tank, wherein each liter of culture medium comprises 12g sucrose, 20g 50% yeast extract, 2g MgSO 2g₄*7H₂O、2g KH₂PO₄、3g K₂SO₄4g citric acid, 1ml pluronic, 182mg V₂O₅And 0.5 ml of a trace metal solution, and a medium comprising 250g per liter of an 80% maltose solution, 20g per liter of a 50% yeast extract,5g citric acid, 5ml pluronic, 182mg V₂O₅And 0.5 ml of trace metal solution. Fermenting at 34 deg.C and pH of 6.0 for 5 days, and collecting the fermentation liquid.

Trace metal solution: 14.3 g ZnSO per liter₄*7H₂O、2．5g CuSO₄*5H₂O、0．5gNiCl₂*6H₂O、13．8g FeSO₄*7H₂O、8．5g MnSO₄*H₂O and 3.0 g citric acid. Sequence listing

SEQ ID NO 3: a primer 117996; the changed nucleotides are underlined and result in the removal of the ScaI site in pElo29, and the introduction of the MluI site.

5＇-GA ATG ACT TGG TTG ACG CGT CAC CAG TCA C-3＇

SEQ ID NO 4: a primer 146063; PCR primers for amplifying C.verruculosa haloperoxidase gene. Underlined nucleotides introduce a BamHI site:

5＇-CGC GGA TCC TCT ATA TAC ACA ACT GG-3＇

SEQ ID NO 5: a primer 146062; PCR primers for amplifying C.verruculosa haloperoxidase gene. Underlined nucleotides were introduced into the BglII site:

5＇-GAA GAT CTC GAG TTA ATT AAT CAC TGG-3＇

SEQ ID NO 6: a primer 147293; the underlined nucleotides introduce a D289L mutation in the haloperoxidase studied:

5＇-GG TCT GTA TTG GGC CTA CCT TGG GTC AAA CC-3＇

SEQ ID NO 7: a primer 147295; the underlined nucleotides introduce a D289E mutation in the haloperoxidase studied:

5＇-GG TCT GTA TTG GGC CTA CGA GGG GTC AAA CC-3＇

SEQ ID NO 8: a primer 139078; the underlined nucleotides introduce the R487E mutation in the haloperoxidase studied:

5＇-CG CCA TTT CTG AGA TCT TCC TGG GC-3＇

SEQ ID NO 9: a primer 139085; the underlined nucleotides introduce the V492S mutation in the haloperoxidase under investigation:

5＇-GC ATC TTC CTC GGC AGC CAC TGG CGA TTC GAT GCC G-3＇

EXAMPLE 3 pH profiles of various haloperoxidase variants

Haloperoxidase variants (from c. verruculosa) experiments were prepared as described in example 2:

phenol red test to determine pH characteristics:

in 96-well microplates:

100 μ l of 0.008% phenol red dissolved in 60mM Britton-Robinson buffer, pH 4-8.5. To these solutions were added 40. mu.l of 0.5M KBr and 50. mu.l of a diluted enzyme solution containing 1mM orthovanadate. The reaction was initiated by the addition of 10. mu.l of 0.3% hydrogen peroxide and the kinetics were measured at 595nm over 5 minutes. As a result:

the activity of each enzyme was recorded relative to the maximum enzyme activity value:

as can be seen from the table, the pH optimum of the wild type is at pH 7.0; the optimum pH of variants D289E, R487E and V492S is at 6.0; variant D289L has a pH optimum at pH 7.5.

Example 4 pH Profile of purified haloperoxidase variant (V492S)

The haloperoxidase variant described above (V492S) and c.verruculosa wild type were purified and detected with Chicago Skye Blue:

purification of haloperoxidase:

the fermentation broth containing haloperoxidase activity was filtered through GF/F (Whatmann) and 0.22 μm (GS, Millipore) and then concentrated on Filtron (molecular weight cut-off 10 kDa). The pH was adjusted to pH 7.5 and loaded onto a Q-Sepharose column (Pharmacia) equilibrated with 50mM Tris-HCl (pH 7.5). The haloperoxidase was eluted in a linear gradient of 0-1M NaCl in 50mM Tris-HCl (pH 7.5). The haloperoxidase-containing fractions were concentrated on an Amicon cell (YM10membrane) and loaded onto a MonoQ column (Pharmacia) equilibrated with 50mM Tris-HCl (pH 8.5) and eluted in a linear gradient of 0-1M NaCl in 50mM Tris-HCl. The fractions containing haloperoxidase were collected and further purified on Superdex75 column 16/60 (Pharmacia) equilibrated with 50mM sodium acetate, 0.1M NaCl (pH 7.5). Experiment:

in 96-well microplates:

mu.l of 60mM Britton-Robinson buffer (pH 4-8) + 50. mu.l enzyme solution + 25. mu.l 0.4M NaCl + 25. mu.l Chicago Skye Blue (diluted to OD610=5 in water). The reaction was initiated by the addition of 10. mu.l of 2mM hydrogen peroxide and activity was recorded as a linear decrease in absorbance at 595 nm. Relative activity:

pH wt V492S

4 0 0．11

4．5 0 0．62

5 0．25 0．93

5．5 1 1

6 0．89 0．70

6．5 0．41 0．34

7 0．11 0．08

7．5 0．02 0．02

conclusion 800.01: V492S clearly shows increased activity in the low pH range compared to the wild-type enzyme.

Haloperoxidase with modified pH characteristics of sequence Listing & #60110& #62NOVO NORDISK A/S & #60120& #62& # 8630 & #625516-WO & #60140& #62& #60141& #62& #60160& #629& #60170& #62PatentIn Ver.2.0 & # 10& #621& #60211& #62609& #60212& #62PRT & # 13& #602 # 13& #62 Isotropic ovaries & #60400& #60 621Met Gly Ser Val Thr Pro Ile Pro Leu Pro Lys Ile Asp Glu Pro Glu 151015 # 015 151015 Glu Tyr Asn Thr Asn Tyr Ile Leu Phe Trp Asn His Val Gly Leu Glu

20 25 30Leu Asn Arg Val Thr His Thr Val Gly Gly Pro Leu Thr Gly Pro Pro

35 40 45Leu Ser Ala Arg Ala Leu Gly Met Leu His Leu Ala Ile His Asp Ala

50 55 60Tyr Phe Ser Ile Cys Pro Pro Thr Asp Phe Thr Thr Phe Leu Ser Pro 65 70 75 80Asp Thr Glu Asn Ala Ala Tyr Arg Leu Pro Ser Pro Asn Gly Ala Asn

85 90 95Asp Ala Arg Gln Ala Val Ala Gly Ala Ala Leu Lys Met Leu Ser Ser

100 105 110Leu Tyr Met Lys Pro Val Glu Gln Pro Asn Pro Asn Pro Gly Ala Asn

115 120 125Ile Ser Asp Asn Ala Tyr Ala Gln Leu Gly Leu Val Leu Asp Arg Ser

130 135 140Val Leu Glu Ala Pro Gly Gly Val Asp Arg Glu Ser Ala Ser Phe Met145 150 155 160Phe Gly Glu Asp Val Ala Asp Val Phe Phe Ala Leu Leu Asn Asp Pro

165 170 175Arg Gly Ala Ser Gln Glu Gly Tyr His Pro Thr Pro Gly Arg Tyr Lys

180 185 190Phe Asp Asp Glu Pro Thr His Pro Val Val Leu Ile Pro Val Asp Pro

195 200 205Asn Asn Pro Asn Gly Pro Lys Met Pro Phe Arg Gln Tyr His Ala Pro

210 215 220Phe Tyr Gly Lys Thr Thr Lys Arg Phe Ala Thr Gln Ser Glu His Phe225 230 235 240Leu Ala Asp Pro Pro Gly Leu Arg Ser Asn Ala Asp Glu Thr Ala Glu

245 250 255Tyr Asp Asp Ala Val Arg Val Ala Ile Ala Met Gly Gly Ala Gln Ala

260 265 270Leu Asn Ser Thr Lys Arg Ser Pro Trp Gln Thr Ala Gln Gly Leu Tyr

275 280 285Trp Ala Tyr Asp Gly Ser Asn Leu Ile Gly Thr Pro Pro Arg Phe Tyr

290 295 300Asn Gln Ile Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Glu Asp305 310 315 320Leu Ala Asn Ser Glu Val Asn Asn Ala Asp Phe Ala Arg Leu Phe Ala

325 330 335Leu Val Asp Val Ala Cys Thr Asp Ala Gly Ile Phe Ser Trp Lys Glu

340 345 350Lys Trp Glu Phe Glu Phe Trp Arg Pro Leu Ser Gly Val Arg Asp Asp

355 360 365Gly Arg Pro Asp His Gly Asp Pro Phe Trp Leu Thr Leu Gly Ala Pro

370 375 380Ala Thr Asn Thr Asn Asp Ile Pro Phe Lys Pro Pro Phe Pro Ala Tyr385 390 395 400Pro Ser Gly His Ala Thr Phe Gly Gly Ala Val Phe Gln Met Val Arg

405 410 415Arg Tyr Tyr Asn Gly Arg Val Gly Thr Trp Lys Asp Asp Glu Pro Asp

420 425 430Asn Ile Ala Ile Asp Met Met Ile Ser Glu Glu Leu Asn Gly Val Asn

435 440 445Arg Asp Leu Arg Gln Pro Tyr Asp Pro Thr Ala Pro Ile Glu Asp Gln

450 455 460Pro Gly Ile Val Arg Thr Arg Ile Val Arg His Phe Asp Ser Ala Trp465 470 475 480Glu Leu Met Phe Glu Asn Ala Ile Ser Arg Ile Phe Leu Gly Val His

485 490 495Trp Arg Phe Asp Ala Ala Ala Ala Arg Asp Ile Leu Ile Pro Thr Thr

500 505 510Thr Lys Asp Val Tyr Ala Val Asp Asn Asn Gly Ala Thr Val Phe Gln

515 520 525Asn Val Glu Asp Ile Arg Tyr Thr Thr Arg Gly Thr Arg Glu Asp Pro

530 535 540Glu Gly Leu Phe Pro Ile Gly Gly Val Pro Leu Gly Ile Glu Ile Ala545 550 555 560Asp Glu Ile Phe Asn Asn Gly Leu Lys Pro Thr Pro Pro Glu Ile Gln

565 570 575Pro Met Pro Gln Glu Thr Pro Val Gln Lys Pro Val Gly Gln Gln Pro

580 585 590Val Lys Gly Met Trp Glu Glu Glu Gln Ala Pro Val Val Lys Glu Ala

595600605 Pro & #60210& #622& #60211& #62600& #60212& #62PRT & #60213& #62 curved spore & #60400& #622Met Gly Ser Val Thr Pro Ile Pro Leu Pro Thr Ile Asp Glu Pro Glu 151015 Glu Tyr Asn Asn Asn Tyr Ile Leu Phe Trp Asn Asn Val Gly Leu Glu

20 25 30Leu Asn Arg Leu Thr His Thr Val Gly Gly Pro Leu Thr Gly Pro Pro

35 40 45Leu Ser Ala Arg Ala Leu Gly Met Leu His Leu Ala Ile His Asp Ala

50 55 60Tyr Phe Ser Ile Cys Pro Pro Thr Glu Phe Thr Thr Phe Leu Ser Pro 65 70 75 80Asp Ala Glu Asn Pro Ala Tyr Arg Leu Pro Ser Pro Asn Gly Ala Asp

85 90 95Asp Ala Arg Gln Ala Val Ala Gly Ala Ala Leu Lys Met Leu Ser Ser

100 105 110Leu Tyr Met Lys Pro Ala Asp Pro Asn Thr Gly Thr Asn Ile Ser Asp

115 120 125Asn Ala Tyr Ala Gln Leu Ala Leu Val Leu Glu Arg Ala Val Val Lys

130 135 140Val Pro Gly Gly Val Asp Arg Glu Ser Val Ser Phe Met Phe Gly Glu145 150 155 160Ala Val Ala Asp Val Phe Phe Ala Leu Leu Asn Asp Pro Arg Gly Ala

165 170 175Ser Gln Glu Gly Tyr Gln Pro Thr Pro Gly Arg Tyr Lys Phe Asp Asp

180 185 190Glu Pro Thr His Pro Val Val Leu Val Pro Val Asp Pro Asn Asn Pro

195 200 205Asn Gly Pro Lys Met Pro Phe Arg Gln Tyr His Ala Pro Phe Tyr Gly

210 215 220Met Thr Thr Lys Arg Phe Ala Thr Gln Ser Glu His Ile Leu Ala Asp225 230 235 240Pro Pro Gly Leu Arg Ser Asn Ala Asp Glu Thr Ala Glu Tyr Asp Asp

245 250 255Ser Ile Arg Val Ala Ile Ala Met Gly Gly Ala Gln Asp Leu Asn Ser

260 265 270Thr Lys Arg Ser Pro Trp Gln Thr Ala Gln Gly Leu Tyr Trp Ala Tyr

275 280 285Asp Gly Ser Asn Leu Val Gly Thr Pro Pro Arg Phe Tyr Asn Gln Ile

290 295 300Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Asp Asp Leu Ala Asn305 310 315 320Ser Glu Val Asn Asn Ala Asp Phe Ala Arg Leu Phe Ala Leu Val Asn

325 330 335Val Ala Cys Thr Asp Ala Gly Ile Phe Ser Trp Lys Glu Lys Trp Glu

340 345 350Phe Glu Phe Trp Arg Pro Leu Ser Gly Val Arg Asp Asp Gly Arg Pro

355 360 365Asp His Gly Asp Pro Phe Trp Leu Thr Leu Gly Ala Pro Ala Thr Asn

370 375 380Thr Asn Asp Ile Pro Phe Lys Pro Pro Phe Pro Ala Tyr Pro Ser Gly385 390 395 400His Ala Thr Phe Gly Gly Ala Val Phe Gln Met Val Arg Arg Tyr Tyr

405 410 415Asn Gly Arg Val Gly Thr Trp Lys Asp Asp Glu Pro Asp Asn Ile Ala

420 425 430Ile Asp Met Met Ile Ser Glu Glu Leu Asn Gly Val Asn Arg Asp Leu

435 440 445Arg Gln Pro Tyr Asp Pro Thr Ala Pro Ile Glu Asp Gln Pro Gly Ile

450 455 460Val Arg Thr Arg Ile Val Arg His Phe Asp Ser Ala Trp Glu Met Met465 470 475 480Phe Glu Asn Ala Ile Ser Arg Ile Phe Leu Gly Val His Trp Arg Phe

485 490 495Asp Ala Ala Ala Ala Arg Asp Ile Leu Ile Pro Thr Asn Thr Lys Asp

500 505 510Val Tyr Ala Val Asp Ser Asn Gly Ala Thr Val Phe Gln Asn Val Glu

515 520 525Asp Val Arg Tyr Ser Thr Lys Gly Thr Arg Glu Gly Arg Glu Gly Leu

530 535 540Phe Pro Ile Gly Gly Val Pro Leu Gly Ile Glu Ile Ala Asp Glu Ile545 550 555 560Phe Asn Asn Gly Leu Arg Pro Thr Pro Pro Glu Leu Gln Pro Met Pro

565 570 575Gln Asp Thr Pro Val Gln Lys Pro Val Gln Gly Met Trp Asp Glu Gln

580 585 590Val Pro Leu Val Lys Glu Ala Pro

Description of the artificial sequence of 595600 & #60210& #623& #60211& #6226& #60212& #62DNA & #60213& #62 artificial sequence & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #623cgcggatcct ctatatacac aactgg 26& #60210& #624& #60211& #6227& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #624gaagatctcg agttaattaa tcactgg 27& #60210& #625& #60211& #6230& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #625gaatgacttg gttgacgcgt caccagtcac 30& #60210& #626& #60211& #6231& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #626ggtctgtatt gggcctacct tgggtcaaac c 31& #60210& #627& #60211& #6231& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #627ggtctgtatt gggcctacga ggggtcaaac c 31& #60210& #628& #60211& #6225& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: description of artificial sequences of primers & #60400& #628cgccatttct gagatcttcc tgggc 25& #60210& #629& #60211& #6236& #60212& #62DNA & #60213& #62 and artificial sequences & #60220& #62& #60223& # 62: primer & #60400& #629gcatcttcct cggcagccac tggcgattcg atgccg 36

Claims

1. A variant of a parent vanadium-containing haloperoxidase, the variant having haloperoxidase activity, an altered pH optimum and comprising a mutation at 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，E；

A501S；

W350F，Y；

V495A，T，V，S；

K394A，L，I，Q，M，E，D；

wherein the parent haloperoxidase comprises the amino acid sequence of SEQ ID NO:1, or the amino acid sequence of the parent haloperoxidase is substantially identical to the amino acid sequence given in SEQ ID NO:1 at least 80% homologous.

2. A variant of a parent vanadium-containing haloperoxidase according to claim 1 comprising a mutation at least one of the following positions:

R487A，L，I，Q，M，E，D；

A396G；

F394N，Y，E，Q；

P392A，S；

R357A，L，I，Q，M，E，D；

K350Q，M；

S399A，T，V，S；

D289L，E；

A498S；

W347F，Y；

V492A，T，V，S；

K391A，L，I，Q，M，E，D；

3. The variant of any of claims 1-2, wherein the parent haloperoxidase is derived from Curvularia.

4. The variant of claim 1, wherein the parent haloperoxidase is derived from Curvularia inequalis.

5. The variant of claim 2, wherein the parent haloperoxidase is derived from Curvularia verruculosa.

6. A DNA construct comprising a DNA sequence encoding a haloperoxidase variant of any one of claims 1-5.

7. A recombinant expression vector carrying the DNA construct of claim 6.

8. A cell transformed with the DNA construct of claim 6 or the vector of claim 7.

9. The cell of claim 8, which is a microorganism.

10. The cell of claim 9 which is a bacterium or a fungus.

11. The cell of claim 10, which is an aspergillus niger or aspergillus oryzae cell.

12. Use of a haloperoxidase variant of any of claims 1-5 for bleaching.

13. Use according to claim 12 for bleaching stains.

14. Use according to claim 12 for bleaching cotton.

15. Use according to claim 12 for bleaching dyes.

16. Use of a haloperoxidase variant according to any one of claims 1-5 for controlling microorganisms.

17. Use of a haloperoxidase variant of any of claims 1-5 for hard surface cleaning.

18. A detergent additive comprising the haloperoxidase variant of any of claims 1-5 in the form of a non-dusting granulate, a stable liquid or a protected enzyme.

19. The detergent additive of claim 18, additionally comprising 1 or more other enzymes such as protease, lipase, amylase and/or cellulase.

20. A detergent composition comprising a haloperoxidase variant of any of claims 1-5 and a surfactant.

21. The detergent composition of claim 20, additionally comprising 1 or more other enzymes such as protease, lipase, amylase and/or cellulase.

22. A coating comprising the haloperoxidase variant of any one of claims 1-5.