MXPA00008969A - HALOPEROXIDASES WITH ALTERED pH PROFILES - Google Patents

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

HALOPEROXIDASAS WITH ALTERED pH PROFILE FIELD OF THE INVENTION The present invention relates to variants of haloperoxidases with altered optimal pH compared to wild type.

BACKGROUND OF THE INVENTION Haloperoxidases form a class of enzymes that are capable of oxidizing halides (X = Cl ", Br", or I ~) in the presence of hydrogen peroxide to the corresponding hypohalide acid (HOX) according to: H £ 02 + X '+ H ~ .- H20 + HOX If a suitable nucleophilic acceptor is present, a reaction with HOX will occur, by means of which a variety of halogenated products of the reaction could 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 H20. A bromide peroxidase is an enzyme capable of oxidizing bromide and iodide ions with the consumption of H202. An iodide peroxidase (EC 1.11.1.8) is an enzyme capable of oxidizing iodide ions with the consumption of H202.

REF .: 122033 Vanadio haloperoxidases are different from other haloperoxidases in that the prosthetic group in these enzymes has structural characteristics similar to vanadate (vanadium V), while the other haloperoxidases are hemeperoxidases. Haloperoxidases have been isolated from several organisms: mammals, marine animals, plants, algae, lichen, fungi and bacteria (for reference see Biochimica et Biophvsica Acta 1161. 1993, pp. 249-256). It is generally accepted that haloperoxidases are enzymes responsible for the formation of halogenated compounds in nature, although other enzymes could be involved. The amino acid sequence has been published (SEC No. 1) for the chloroperoxidase containing vanadium from the fungus Curvul ari a ina equal i s (see S ISS-PROT: P49053). The amino acid sequence has been published (SEC No. 2) for the vanadium-containing chloroperoxidase of the fungus Curvularia verruculosa (see WO 97/04102). The X-ray structure of the vanadium-containing chloroperoxidase of the fungus Curvularia ina equal i s has been published (Proc. Nati, Acad.Sci.U S, 93 (1), 1996, 392-396, and pdblvnc.ent). Haloperoxidases are of common interest because of their wide range of potential industrial uses. For example, haloperoxidases have been proposed for use as an anti-microbial agent.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to variants of haloperoxidases containing vanadium with altered optimal pH compared to parental haloperoxidase, so in particular the present invention relates to: A variant of a parent haloperoxidase containing vanadium, this variant has haloperoxidase activity and an altered optimal pH 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 parental haloperoxidase has an amino acid sequence that is at least 80% homologous to SEQ ID NO. 1.

DETAILED DESCRIPTION OF THE INVENTION Haloperoxidases homologs sue contain vanadium A number of vanadium-containing haloperoxidases produced by different fungi are homologous at the amino acid level. Haloperoxidases of Curvularia ir.aequali s and Curvularia verruculosa were ordered. The ordering uses the amino acid sequence of the haloperoxidase obtained from the 3D structure record of C. inaequalí s (Brookhaven databank file pdblvnc.ent). When using the homology percent obtained from the UWGCG program that uses the GAP program with the preset parameters (penalties: opening width = 3.0, width of length = 0.1, WISCONSIN PACKAGE Version 8.1-Unix, August 1995, Genetics Computer Gorup, 575 Science Drive, Madison, Wisconsin, USA 53711) the following homology was found: vanadium-containing haloperoxidase from Curvularia ina equali comprising the amino acid sequence shown in SEQ ID No. 1: 100%; the vanadium-containing haloperoxidase from Curvularia verruculosa 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 haloperoxidase containing vanadium produced or produced by a strain of the organism in question, but also a haloperoxidase containing vanadium encoded by a DNA sequence isolated from such strain and produced in a host organism that contains the DNA sequence. Finally, the term is intended to indicate a haloperoxidase containing vanadium which is encoded by a DNA sequence of synthetic origin and / or cDNA, and which has the identifying characteristics of the vanadium-containing haloperoxidase in question.

Variants with Altered Optimal PH The desired optimal pH of a vanadium-containing haloperoxidase depends on which is the application of interest, eg, if the vanadium-containing haloperoxidase is to be used to flush denim the preferred optimum pH will be around pH 5-8 , whereas if the vanadium-containing haloperoxidase is used for washing purposes the preferred optimum pH will be around pH 8-10. It is possible to alter the optimum pH of a vanadium-containing parental haloperoxidase, where the variant is the result of a mutation, i.e. one or more amino acid residues have been removed from, replaced or added to the parent haloperoxidase containing vanadium. By introducing charge changes in the vicinity of the residues of the active site, the pKa of the residue of interest can be changed to provide an altered activity profile of the haloperoxidase in question. It is a common opinion that by introducing more negative charged residues near the His (the active site of the haloperoxidase), its pKa rises and thus will be able to act on the catalysis at a higher pH than before. The His of the active site, introducing more positive charged residues near the His, will alter its pKa to a lower pKa than before and in this way will be able to act in the catalysis at a lower pH than the previous one. The increase in pKa can also be obtained by decreasing the accessibility of the solvent to the active site (His). The decrease in pKa can also be obtained by increasing the accessibility of the solvent at the active site (His). But according to the present invention it is found that more important is that the residues are within 10 Á 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 optimal pH of the enzyme. In this way, the residues can be mutated in e.g. the haloperoxidase of Curvul aria ina equali s. The homologous structures that are supposed to have a similar structure can be constructed with models (see Example 1) and the regions of interest can be found in the same way. The preferred positions for the 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; K394A, 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 having an amino acid sequence that is at least 80% homologous to SEQ ID No. 1, or homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 85% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase having an amino acid sequence that is at least 90% homologous to the SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 95% homologous to S? C ID No. 1, or the homologous positions in a parent haloperoxidase having a sequence of amino acid that is at least 96% homologous to SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 97% homologous to SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 98% homologous to SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 99% homologous to SEQ ID NO 1. In particular, the following mutations are 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; A498 S; W347F, Y; V492A, T, V, S; K391A, L, I, Q, M, E, D; wherein the parent halide peroxidase has the amino acid sequence given in SEQ ID No. 2. In a preferred embodiment two or more amino acid residues could 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, wherein the parental haloperoxidase has the amino acid sequence given in SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 80% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase having an amino acid sequence that is at least 85% homologous to SEQ ID No. 1, or homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 90% homologous to SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 95% homologous to SEQ ID No. 1, or the homologous positions in a parent haloperoxidase having an amino acid sequence that is at least 96% homologous to SEC ID No. 1, or the positions s homologues in a parental haloperoxidase having an amino acid sequence that is at least 97% homologous to SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 98% homologous to the SEQ ID No. 1, or the homologous positions in a parental haloperoxidase having an amino acid sequence that is at least 99% homologous to S? C ID No. 1. In a preferred embodiment two or more amino acid residues could be substituted as follows : 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 has the amino acid sequence given in SEQ ID No. 2.

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

Cloning a DNA sequence encoding a haloperoxidase containing vanadium The DNA sequence encoding a vanadium-containing parent haloperoxidase could be isolated from any cell or microorganism that produces the haloperoxidase in question, using several methods well known in the art, first , a genomic DNA and / or cDNA library must be constructed using chromosomal DNA or messenger RNA from the organism that produces the haloperoxidase to be studied. Then, if the amino acid sequence of the haloperoxidase is known, labeled, homologous oligonucleotide probes could be synthesized and used to identify clones encoding haloperoxidase from a genomic library prepared from the organism in question. Alternatively, a labeled oligonucleotide probe containing sequences homologous to a known haloperoxidase gene could be used as a probe to identify clones encoding haloperoxidase, using conditions of hybridization and washing of lower stringency. One method to identify clones encoding haloperoxidase involves inserting cDNA into an expression vector, such as a plasmid, transforming the haloperoxidase-negative fungus with the resulting cDNA library, and then plating the transformed fungus on agar containing a substrate. for the haloperoxidase, thus allowing the clones to express the haloperoxidase to be identified. Alternatively, the DNA sequence encoding the enzyme could be prepared synthetically by established standard methods, e.g. the phosphoramidite method. In the phosphoramidite method, oligonucleotides, e.g., are synthesized. in an automatic DNA synthesizer, they are purified, fixed, ligated and cloned into appropriate vectors. Finally, the DNA sequence could be of mixed genomic and synthetic origin, synthetic mixed origin and cDNA or mixed genomic origin and cDNA, prepared by ligament fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to several parts of the total DNA sequence), according to standard techniques. The DNA sequence could also be prepared by polymerase chain reaction (PCR) using specific primers.Targeted site mutagenesis Once a DNA sequence encoding haloperoxidase has been isolated, and desirable sites for mutation identified, mutations could be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; the mutant nucleotides are inserted during the synthesis of the oligonucleotide. In a specific method, a range of a strand of DNA, which bridges the sequence encoding the haloperoxidase, is created in a vector carrying the haloperoxidase gene. Then the synthetic nucleotide, which carries the mutation the desired mutation, is fixed to a homologous portion of the DNA of a strand. The remaining gap is then filled in with T7 DNA polymerase and the construct ligated using T4 ligase (Morinaga method - see Biotechnology, 2, 1984, pp. 626-639). US 4,760,025 discloses the introduction of oligonucleotides that encode multiple mutations by performing minor alterations of the cassette. However, an even larger variety of mutations can be introduced at any time by the Morinaga method, because a multitude of oligonucleotides of various lengths can be introduced. Another method for introducing mutations into DNA sequences encoding haloperoxidase is the generation in 3 steps of a PCR fragment containing the desired mutation introduced using a DNA strand synthesized chemically as one of the primers in the PCR reactions. From the fragment generated by PCR, a fragment of DNA carrying the mutation could be isolated by cutting with restriction endonucleases and reinserted into an expression plasmid.

Random mutagenesis Random mutagenesis of a DNA sequence encoding a parental haloperoxidase could be conveniently performed by the use of any method known in the art. For example, random mutagenesis could be performed by the use of a suitable mutagenic physical or chemical agent, by the use of a suitable oligonucleotide, or by subjecting the DNA sequence to mutagenesis generated by PCR. In addition, random mutagenesis could be performed by using any combination of these mutagenic agents.

The mutagenic agent could be, e.g., one that induces transitions, transversions, inversions, confusing mixtures, deletions and / or insertions. Examples of a mutagenic physical or chemical agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N '-nitro-nitrosoguanidine (MNNG), 0-methyl hydroxylamine, nitrous acid, ethyl methane sulfonate (EMS) ), sodium bisulfite, formic acid and analogous nucleotides. When such agents are used, mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the selected mutagenic agent under conditions suitable for mutagenesis to be carried out, and selecting the mutated DNA that is mutated. It has the desired properties. When performing mutagenesis through the use of an oligonucleotide, the oligonucleotide could be mixed or enclaved with the three non-parental nucleotides during the synthesis of the oligonucleotide at the positions to be changed. Mixing or nesting could be done to avoid unwanted amino acid codons. The mixed or nailed oligonucleotide could be incorporated into the DNA encoding the haloperoxidase enzyme by any published technique, using e.g. PCR, LCR or any polymerase and DNA ligase. When mutagenesis generated by PCR is used, a gene either chemically treated or untreated which encodes a parental haloperoxidase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992, Leung et al., Technique, Vol. 1, 1989, pp. 11-15). A mutant strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, p. 179-191), S. Cerevi e e or any other microbial organism could be used for the random mutagenesis of the DNA encoding the haloperoxidase enzyme, e.g. transforming a plasmid containing the parent enzyme into the mutant strain, the mutant strain growing with the plasmid and isolating the mutated plasmid from the mutant strain. The mutated plasmid could subsequently be transformed in the expression organism. The DNA sequence to be mutagenized could conveniently be presented in a genomic or cDNA library prepared from an organism expressing the parent haloperoxidase enzyme. Alternatively, the DNA sequence could be presented in a suitable vector such as a plasmid or a bacteriophage, which as such could be incubated with or otherwise exposed to the mutagenic agent. The DNA to be mutagenized could also be presented in a host cell, whether it is integrated into the genome of the cell or is present in a vector housed in the cell. Finally, the DNA that is going to be mutagenized could be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a sequence of cDNA or genomic DNA. In some cases it may be convenient to amplify the mutated DNA sequence before the expression step or the screening step is performed. Such amplification could be carried out according to methods known in the art, the currently preferred method which is amplification generated by PCR using oligonucleotide primers is prepared on the basis of the DNA or amino acid sequence of the parent enzyme. Subsequent to incubation with or exposure to the mutagenic agent, the mutated DNA is expressed by culturing a suitable host cell that carries the DNA sequence under conditions that allow expression to be carried out. The host cell used for this purpose could be one that has been transformed with the mutated DNA sequence, optionally present in a vector, or one that carried 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 oryza. The mutated DNA sequence could further comprise a DNA sequence encoding functions that allow the expression of the mutated DNA sequence.

Localized random mutagenesis Random mutagenesis could be located advantageously in a part of the parent haloperoxidase in question. This could be advantageous, e.g., when certain regions of the enzyme have been identified as being of particular importance for a given property of the enzyme, and when modified it is expected to result in a variant having improved properties. Such regions could normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme. Localized random mutagenesis is conveniently performed by the use of mutagenesis techniques generated by PCR as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified, e.g., could be isolated. which is inserted into an appropriate vector, and the part could subsequently be subjected to mutagenesis by the use of any mutagenesis method discussed above. With respect to the screening step in the method of the invention mentioned above, this could be conveniently performed by using a filter test based on the following principle: A microorganism capable of expressing the mutated haloperoxidase enzyme of interest is incubated in a suitable medium and under conditions suitable for the enzyme to be secreted, the medium is provided with a double filter comprising a first filter that laces the protein and on top of that a second filter having a low protein binding capacity. . The microorganism is located in the second filter. Subsequent to incubation, the first filter as secreted from the microorganisms is separated from the second filter containing the microorganisms. The first filter is screened for the desired enzymatic activity and the corresponding microbial colonies present in the second filter are identified. The filter used to link the enzymatic activity could be any filter that binds e.g. protein. nylon or nitrocellulose. The top filter that carries the colonies of the expression organism could be any filter that does not have or has little affinity for binding proteins, e.g. cellulose acetate or Durapore ™. The filter could be pretreated with any of the conditions that will be used for screening or could be treated during the detection of enzymatic activity. Enzyme activity could be detected by a dye, fluorescence, precipitation, pH indicator, IR absorbance or any other known technique for detection of enzymatic activity. The compound that it detects could be immobilized by means of any immobilization agent, e.g., agarose, agar, gelatin, polyacrylamide, starch, filter paper, cloth; or any combination of immobilization agents.

Expression of Haloperoxidase Variants According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative method known in the art, can be expressed in the form of an enzyme using an expression vector that typically includes controls that encode a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or several activator genes. The recombinant expression vector carrying the DNA sequence encoding a haloperoxidase variant of the invention could be any vector that could be conveniently subjected to recombinant DNA methods, and the selection of the vector will often depend on the host cell in which it is will enter. In this way, the vector could be a self-replicating vector, i.e. a vector that exists as an extrachromosomal entity, replication thereof is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or artificial chromosome. Alternatively, the vector could be one which, when introduced into the host cell, is integrated into the genome of the host cell and replicated together with the chromosome (s) into which it has been integrated. In the vector, the DNA sequence must be operably connected to an appropriate promoter sequence. The promoter could be any DNA sequence that shows transcriptional activity in the selected host cell and could be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding a haloperoxidase variant of the invention, especially in a fungal host, are those derived from the gene encoding TAKA amylase from A. oryza e, aspartic proteinase of Rhi zomucor mi ehei, neutral-amylase of A. ni ger, stable acid-amylase from A. nor ger, glucoamylase from A. ni ger, lipase from Rhi zomu cor mi ehei, alkaline protease from A. oryza e, triose phosphate isomerase from A. oryza e or acetamidase from A. nor sweet ans. The expression vector of the invention could also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding the haloperoxidase variant of the invention. The termination and polyadenylation sequences could be derived appropriately from the same sources as the promoter. The vector could further comprise a sequence of DNA that allows the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBUO, pE194, pAMB1 and pIJ702. The vector could also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as one that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. In addition, the vector could comprise Aspergi l lus selection markers such as amdS, argB, niaD and sC, a marker that gives rise to the hygromycin resistance, or the selection could be made by co-transformation, e.g. as described in WO 91/17243. The methods used to ligate the DNA construct of the invention encoding a variant of haloperoxidase, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to those skilled in the art. art (see, for example, Sambrook et al. (1989)).

The cell of the invention, whether comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of a haloperoxidase variant of the invention. The cell could be transformed with the DNA construct of the invention encoding the variant, by conveniently integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is likely to remain stable in the cell. The integration of the DNA constructs into the host chromosome could be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell could be transformed with an expression vector as described above in connection with the different types of host cells. The cell of the invention could be a cell of a higher organism such as a mammal or an insect, but is preferably a cell, e.g. a fungal cell. Filamentous fungi could advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger. The fungal cells could be transformed by means of a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable method for the transformation of Aspergillus host cells is described in EP 238 023. Yet in a further aspect, the present invention relates to a method for producing a haloperoxidase variant of the invention, this method comprises culturing a host cell as described above, under conditions that lead to the production of the variant and recover the variant of the cells and / or culture medium. The medium used to grow the cells could be any conventional means suitable for growing the host cell in question and obtaining the expression of the haloperoxidase variant of the invention. Suitable means are available from commercial suppliers or could be prepared according to published formulas (e.g. as described in catalogs of the American Type Culture Collection). The haloperoxidase variant secreted from the host cells could be conveniently recovered from the culture medium by well-known methods, including separation of cells from the medium by centrifugation or filtration, and precipitation of proteinaceous components from the medium, by means of a salt such as sodium sulfate. ammonium, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like.

Industrial applications The haloperoxidase of the invention could be incorporated in a detergent or cleaning composition comprising other types of enzymes useful in detergents or cleaning compositions, preferably at least one additional enzyme selected from the group consisting of proteases, amylases, cutinases, peroxidases, oxidases, laccases, cellulases, xylanases and lipases. In particular, the haloperoxidase of the invention could be used for bleaching or for sanitary purposes. When used for the preservation of foods, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouthwashes, contact lens products, enzyme formulations, or food ingredients, haloperoxidase of the invention could be incorporated into eg the non-preserved food, beverages, cosmetics, contact lens products, food ingredients or anti-inflammatory products in an amount effective to kill or inhibit the growth of microbial cells. Thus, the haloperoxidase used in the method of the invention could be useful as a disinfectant, e.g., in the treatment of acne, infections in the eye or mouth, skin infections.; in antiperspirants or deodorants; in salts for foot baths; for disinfection at the end of the cleaning of contact lenses, hard surfaces, teeth (oral care), wounds, bruises and the like. In general it is contemplated that the haloperoxidase of the present invention is useful for cleaning, disinfecting or inhibiting microbial growth on any hard surface. Examples of surfaces that could be contacted with the composition of the invention are processing equipment surfaces used e.g. in dairies, chemical or pharmaceutical process plants, water sanitation systems, paper pulp processing plants, water treatment plants and cooling towers. The haloperoxidase of the invention should be used in an amount that is effective to clean, disinfect or inhibit microbial growth on the surface in question. In addition, it is contemplated that the haloperoxidase of the invention may be advantageously used in an on-site cleaning system (C.I.P.) for the cleaning of process equipment of any kind. The haloperoxidase of the invention may additionally be used for cleaning surfaces and cooking utensils in plants, food processing and in any area in which they are prepared or serving food such as hospitals, private hospitals, restaurants, especially fast food restaurants, shops cold cuts and the like. It could also be used as an antimicrobial in food products and would be especially useful as an antimicrobial surface in cheeses, fruits and vegetables and foods in salad bars. It could also be used as a preservation agent or a disinfection agent in water-based paints: Preservation / preservation of paintings The preservation of paint products in cans has been carried out in the art by adding non-enzymatic organic biocides to the paints. In the context of the invention the paint is constructed as a substance comprising a solid colorant material dissolved or dispersed in a liquid carrier such as water, organic solvent and / or oils, which when spread on a surface, dries to leave a coating thin colored, decorative and / or protective.

Typically isothiazolones, such as 5-chlor-2-methyl-4-thia-zoli-3-on, have been added to the paint as biocides in dosages in the range of about 0.05-0.5% to inhibit / prevent microbial growth in the paint. However, the method of the invention can be suitably applied in this field, thus solving the problem of the ever present environmental bio-hazards of using toxic organic biocides by replacing these toxic biocides with environmentally compatible enzymes. In this way the invention provides a method of preserving a paint, which comprises contacting the paint with a variant of haloperoxidase according to the invention. In addition, the invention provides a paint composition comprising a haloperoxidase variant according to the invention. The paint is preferably a water-based paint, i.e. the solids in the paint are dispersed in an aqueous solution. The paint could contain 0-20% organic solvent, preferably 0-10%, e.g. 0-5%. The enzyme could be added to the paint in an amount of 0.0001-100 mg of active enzyme protein per liter of paint, preferably 0.001-10 mg / liter, e.g. 0.01-1 mg / liter.

Sources of Hydrogen Peroxide According to the invention the hydrogen peroxide necessary for the reaction with the haloperoxidase could be obtained in many different ways: It could be hydrogen peroxide or a hydrogen peroxide precursor, such as, eg, percarbonate or perborate, or a percarboxylic acid or a salt thereof, or it could be an enzymatic system that generates hydrogen peroxide, such as, eg, an oxidase and its substrate. Useful oxidases could be, e.g., a glucose oxidase, a glycerol oxidase or an amino acid oxidase. An example of an amino acid oxidase is given in WO 94/25574. It could be advantageous to use hydrogen peroxide generated enzymatically, since this source results in a relatively low concentration of hydrogen peroxide under the biologically relevant conditions. Low concentrations of hydrogen peroxide result in an increase in the reaction rate catalyzed by haloperoxidase. According to the invention, the source of hydrogen peroxide necessary for the reaction with the haloperoxidase could be added in a concentration corresponding to a concentration of hydrogen peroxide in the range of O.Ol-lOOOmM, preferably in the range of 0.1-500. mM.

Halide Sources According to the invention the halide source needed for the reaction with the haloperoxidase could be obtained in many different ways, eg, by adding a halide salt: it could be sodium chloride, potassium chloride, sodium bromide, bromide potassium, sodium iodide or potassium iodide. The concentration of the halide source will typically correspond to 0.01-1000 mM, preferably in the range of 0.1-500 mM.

The Composition The composition comprising the haloperoxidase, the source of hydrogen peroxide, and the halide source could be formulated as a solid or a liquid, in particular in the form of a powder-free granulate, or a stabilized liquid. When formulated as a solid all the components could be mixed together, e.g., as a powder, a granulate or a gelled product. When different compositions of the dry form are used and still in that case, it is preferred to use a two part formulation system having the hydrogen peroxide separated from the other components. The composition of the invention could further comprise auxiliary agents such as wetting agents, thickening agents, buffers, stabilizers, perfume, colorants, fillers and the like. Useful wetting agents are surfactants, i.e., non-ionic, anionic, amphoteric or zwitterionic surfactants. The composition of the invention could be a concentrated product or a ready-to-use product.

Haloperoxidase activity According to the present invention the activity of the haloperoxidase could be measured as described in WO 97/04102, p. 13 1. 7-19. The present invention is further illustrated in the following examples which are not intended in any way to limit the scope of the invention as claimed.

EXAMPLE 1 Construction of homology of the 3D structure of the haloperoxidase of Curvularia verruculosa Using the homology of the sequence of Curvulari to ina equali s (Cl) for other sequences, e.g., Curvularia verru culosa, 3D structures similar to Cl can be found. In comparison with the Curvularia ina equal i s, used to elucidate the structure, Curvularia verruculosa differs in the number of residues. The model could be constructed using the INSIGHT and HOMOLOGY programs of Molecular Simulations Inc. The program replaces the amino acids in Curvularia ina equali s with Curvul amino acids to verruculose in the homologous positions defined in the program as structurally conserved regions (SCR). Internal wastes are constructed using the LOOP option with GENÉRATE. Using these steps, an unrefined model could be obtained, which gives information about spatial interactions. The structure can be refined using the method described in the HOMOLOGY package.

EXAMPLE 2 Construction of Haloperoxidase Variants For the construction of variants of the haloperoxidase enzyme of Curvularia verruculosa the commercial kit, Chameleon double-stranded, site-directed mutagenesis kit, can be used according to the manufacturer's instructions. The gene encoding the haloperoxidase enzyme in question is located in the plasmid pElo29. According to the manufacturer's instructions, the Seal site of the Ampicillin gene of pElo29 is changed to an Mlul site by using primer 117996 (SEQ ID NO: 3). At the same time the desired mutation is introduced into the haloperoxidase gene by the addition of an appropriate primer comprising the desired mutation.

Construction of the pElo29 plasmid The pElo29 plasmid was constructed from the following fragments: a) The 4.1 kb fragment of the pCiP vector (described in WO 93/34618) cut with BamHI and Bgl II. b) The haloperoxidase gene of Curvularia verruculosa amplified by PCR with Pwo polymerase, using the plasmid pAJ014-l (WO 97/04102) as a template, and the primers 146063 and 146062 (SEQ ID NO: 4 and 5), introducing the sites BamHI and Bgl II, 5 'and 3' respectively, to the haloperoxidase gene. After ligation of fragments a and b, the complete haloperoxidase gene was sequenced to ensure that undesirable mutations had not been introduced during PCR amplification.

Site-directed mutagenesis Site-directed mutagenesis as described above was used to construct plasmids harboring genes encoding variants of the haloperoxidase enzyme. 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 complete gene. The resulting plasmids were named pElo30, pElo31, pElo34 and pElo37 respectively.

Transformation of pElo30. pElo31, pElo34 v oElo37 in JaL 228 Aspergillus oryza e JAL 228 (WO 97/27221) is Aspergillus oryzae IFO 4177 eliminated in alkaline protease and neutral metalloprotease I. This strain was transformed with pElo30, pElo31, pElo34 and pElo37 using the plasmid pToC90 (WO 91/17243) which carries the amdS gene as cotransformant. The selection of the transformants was carried out using acetamide as described in patent EP 0531 372 Bl. The transformants that sporulated were reisolated twice. The spores of the second isolation of each transformant were tested for the production of haloperoxidase in small-scale fermentations (shake flasks and microplates of titration).

Fermentation of Curvu aria verruculose haloperoxidase variants in Aspersill usorzae The above isolates were fermented in a tank with 12 g of sucrose, 20 g of 50% yeast extract, 2 g of MgSO4 * 7H20, 2 g of KH2P04, 3 g of K2S04, 4 g of citric acid, 1 ml of pluronic, 182 mg of V205, and 0.5 ml of trace metal solution »per liter, and fed during the course of fermentation with compound medium 250 g of 80% maltose solution, 20 g of 50% yeast extract, 5 g of citric acid, 5 ml of pluronic, 182 mg of V205, and 0.5 ral of trace metal solution per liter. The fermentation broth was harvested after 5 days of fermentation at 34oC, pH above 6.0. n Trace metal solution: 14.3 g of ZnS04 * 7H20, 2.5 g of CuS04 * 5H20, 0.5 of NiC12 * 6H20, 13.8 g of FeS04 * 7H20, 8.5 g of MnS04 * H20, and 3.0 g of citric acid per liter.

Sequence Listing SEQ ID NO 3: Primer 117996; the changed nucleotides are underlined and lead to the elimination of the Sea l site, and the introduction of the Ml ul site into pElo29: 5'-GA ATG ACT TGG TTG ACG CGT CAC CAG TCA C-3 ' SEQ ID NO 4: Primer 146063; PCR primer for the amplification of the haloperoxidase gene from Curvul ari to verruculosa. Underlined nucleotides introduce the BamHI site: 5 '-CGC GGA TCC TCT ATA TAC ACA ACT GG-3' SEQ ID NO 5: Primer 146062; PCR primer for amplification of the haloperoxidase gene of Curvularia verruculosa. Underlined nucleotides introduce the Bgl II site: 5 '-GAA GAT CTC GAG TTA ATT AAT CAC TGG-3' SEQ ID NO 6: Primer 147293; Underlined nucleotides introduce the D289L mutation into the haloperoxidase enzyme in question: 5'-GG TCT GTA TTG GGC CTA CCT TGG GTC AAA CC-3 ' SEQ ID NO 7: Primer 147295; Underlined nucleotides introduce the D289E mutation into the haloperoxidase enzyme in question: 5'-GG TCT GTA TTG GGC CTA CGA GGG GTC AAA CC-3 ' SEQ ID NO 8: Primer 139078; The underlined nucleotides introduce the R487E mutation in the haloperoxidase enzyme in question: 5'-GG CCA TTT CTG AGA TCT TCC TGG GC-3 ' SEQ ID NO 9: Primer 139085; Underlined nucleotides introduce the V492S mutation into the haloperoxidase enzyme in question: 5'-GC ATC TTC CTC GGC AGC CAC TGG CGA TTC GAT GCC G-3 'EXAMPLE 3 PH curves of various haloperoxidase variants Haloperoxidase variants (derived from Verrucous curvularia) were made as described in Example 2.

Experimental: Phenol red test for the determination of the pH profile: In 96-well microtiter plates: 100 μl of 0.008% phenol red in 60 mM Britton-Robinson buffer, pH 4-8.5. To these solutions were added 40 μl of 0.5 M KBr and 50 μl of diluted enzyme solution containing 1 mM of ortho-vanadate. The reaction was started by adding 10 μl of 0.3% hydrogen peroxide and the kinetics were measured for 5 minutes at 595 nm.

Results: The activity was taken with respect to the highest value of each enzyme: It can be seen from the Table that the wild type has an optimum pH at pH 7.0; the variant D289E and R487E and V492S have an optimum pH at 6.0; and the variant D289L has an optimum pH at pH 7.5.

EXAMPLE 4 PH curve of the purified haloperoxidase variant (V492S) The haloperoxidase variant (V492S) described above and the wild type verrucous curvularia were purified and tested with "Chicago Skye Blue": Haloperoxidase purification: The fermentation broth containing the haloperoxidase activity was filtered GF / F (Whatmann) and 0.22 μm (GS, Millipore) before concentration in the Filtron (10 kDa cut). The pH was adjusted to pH 7.5 and the sample was loaded onto a Q-Sepharose column (Pharmacia) equilibrated in 50 mM Tris-HCl, pH 7.5. The haloperoxidase was eluted in a linear gradient of 0-1 M NaCl in 50 mM Tris-HCl, pH 7.5. The haloperoxidase containing fractions was concentrated in an Amicon cell (membrane YM10) and loaded onto MonoQ column (Pharmacia) equilibrated in 50 mM Tris-HCl, pH 8.5 and eluted in a linear gradient of 0-1 M NaCl in Tris- 50 mM HCl. The haloperoxidase containing fraction was collected and then purified on a Superdex75 16/60 Pharmacia) column equilibrated in 50 mM sodium acetate, 0.1 M NaCl, pH 5.5.

Experimental: In 96-well microtiter plates: 100 μl of 60 mM Britton-Robinson pH 4-8 + 50 μl of enzyme solution + 25 μl of 0.4 M NaCl + "Chicago Skye Blue" diluted in water to OD610 = 5. The reaction was started by adding 10 μl of 2 mM H202. The activity was taken as the linear decrease in absorption at 595 nm.

Relative activities pH wt V492S 4 0 0.11 4. 5 0 0.62 0.25 0.93 . 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 8 0 0.01 Conclusion: V492S clearly shows increased activity in the low pH range compared to the wt enzyme (wild type). It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (22)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A variant of a vanadium-containing parental haloperoxidase, characterized in that this variant has haloperoxidase activity and an altered optimum pH 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, E; 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 parental haloperoxidase has an amino acid sequence that is at least 80% homologous to SEQ ID NO.

1. 2. A variant of a parental haloperoxidase containing vanadium in accordance with the claim 1, characterized in that the variant comprises a mutation in 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; wherein the parent haloperoxidase has the amino acid sequence given in SEQ ID No. 2.

3. A variant according to any of claims 1-2, characterized in that the parental haloperoxidase is derived from Curvularia.

4. A variant according to claim 1, characterized in that the parental haloperoxidase is derived from Curvulari to inaequalis.

5. A variant according to claim 2, characterized in that the parental haloperoxidase is derived from Curvul aria verruculosa.

6. A DNA construct, characterized in that it comprises a DNA sequence encoding a haloperoxidase variant according to any of claims 1-5.

7. A recombinant expression vector, characterized in that it carries a DNA construct according to claim 6.

8. A cell, characterized in that it is transformed with a DNA construct according to claim 6 or a vector according to the invention. claim 7.

9. A cell in accordance with the claim 8, characterized because it is a microorganism.

10. A cell according to claim. 9, characterized because it is a bacterium or a fungus.

11. A cell in accordance with the claim 10, characterized in that it is an Aspergillus niger cell or an Aspergillus oryza e cell.

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

13. The use according to claim 12 for bleaching spots.

14. The use according to claim 12 for cotton bleaching.

15. The use according to claim 12 for bleaching dyes.

16. Use of a haloperoxidase according to any of claims 1-5 for microbial control.

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

18. A detergent additive, characterized in that it comprises a haloperoxidase variant according to any of claims 1-5 in the form of a powder-free granulate, a stabilized liquid or a protected enzyme.

19. A detergent additive according to claim 18, characterized in that it additionally comprises one or more enzymes such as a protease, a lipase, an amylase and / or a cellulase.

20. A detergent composition, characterized in that it comprises a haloperoxidase variant according to any of claims 1-5 and a surfactant.

21. A detergent composition according to claim 20, characterized in that it additionally comprises one or more enzymes such as a protease, a lipase, an amylase and / or a cellulase.

22. A paint, characterized in that it comprises a haloperoxidase variant according to any of claims 1-5.