JP2002506638A - Haloperoxidase with modified pH characteristics - Google Patents

Abstract

  (57) [Summary] A mutant derived from the parent vanadium-containing haloperoxidase, having haloperoxidase activity, having an optimal pH changed, and having 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; K394A, L, I, Q, M, E, D. However, the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 1 or has at least 80% homology to SEQ ID NO: 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to a haloperoxidase mutant having an optimal pH changed as compared to a wild type.

BACKGROUND OF THE INVENTION Haloperoxidase oxidizes halides (X = Cl-, Br-, or I-) in the presence of hydrogen peroxide to give the corresponding hypohalous acid ( HOX) is a group of enzymes that: H ₂ O ₂ + X- + H +-> H ₂₀ + HOX If a convenient nucleophilic receptor is present, it reacts with HOX to produce various halogens. A reaction product is formed.

[0003] Chloride peroxidase (EC 1.11.1.10) is an enzyme that consumes H ₂ O ₂ and oxidizes chloride, bromide and iodide ions. Bromide peroxidase is an enzyme that consumes H ₂ O ₂ and oxidizes bromide and iodide ions. Iodide peroxidase (EC 1.11.1.8) is an enzyme that consumes H ₂ O ₂ and oxidizes iodide ions. Vanadium haloperoxidase is composed of vanadate (Vanadium V)
Is different from other haloperoxidases in that it has structural characteristics similar to that of haloperoxidase, whereas other haloperoxidases are heme peroxidases.

[0004] Haloperoxidases have been isolated from a variety of organisms: mammals, marine animals, plants, algae, lichens, fungi and bacteria (Biochimica et Biophysica Acta 1161,
1993, 249-256). Haloperoxidase is considered in nature to be an enzyme responsible for forming halogenated compounds together with other enzymes.

[0005] The amino acid sequence of vanadium-containing chloroperoxidase derived from the fungus Curvularia inaequalis (SEQ ID NO: 1) has been published (SWISS-PROT: P49053).
. The amino acid sequence of vanadium-containing chloroperoxidase from the fungus Curvularia verruculosa (SEQ ID NO: 2) has been published (WO 97/04102).

The X-ray structure of vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis has been published (Proc. Natl. Acad. Sci. USA, 93 (1), 1996,
392-396; pdb1vnc.ent). Haloperoxidases are currently receiving attention because of their potential for industrial applicability. For example, haloperoxidase has been proposed for use as an antimicrobial agent.

SUMMARY OF THE INVENTION [0007] The present invention relates to vanadium-containing haloperoxidase variants having an optimized pH as compared to the parent haloperoxidase, and thus, in particular, the present invention relates to:

A mutant derived from the parent vanadium-containing haloperoxidase, which has haloperoxidase activity, has an optimal pH changed, and has 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; K394A, L, I, Q, M, E, D; provided that the haloperoxidase of the present invention has the amino acid sequence of SEQ ID NO: 1, or Has an amino acid sequence at least 80% homologous to SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE INVENTION Homologous Vanadium-Containing Haloperoxidase A large number of vanadium-containing haloperoxidases produced by various fungi are:
Homologous at the amino acid level. Curvularia inaequalis and Curvularia verruculosa haloperoxidase were aligned. For this alignment, the three-dimensional structure file of C. inaequalis (Brookha
The amino acid sequence of haloperoxidase obtained from the ven databank file pdb1vnc.ent) is used.

[0010] Default parameters (penalties: gap weight = 3.0, length weight = 0.1; W
ISCONSIN PACKAGE Version 8.1-UNIX, August 1995, Genetics Computer Group,
575 Science Drive, Madison, Wisconsin, USA 53711), using the percent homology obtained from the UWGCG program, the following homology was found: Curvularia inaequalis consisting of the amino acid sequence of SEQ ID NO: 1 Vanadium-containing haloperoxidase: 100%; Curvularia v consisting of the amino acid sequence of SEQ ID NO: 2
erruculosa vanadium-containing haloperoxidase: 96%.

As used herein, “derived from” is encoded by a DNA sequence isolated from a strain of the organism of interest, as well as a vanadium-containing haloperoxidase produced by, or capable of being produced by, the strain of the organism in question, and It also refers to vanadium-containing haloperoxidase produced by a host organism containing the DNA sequence. Finally, the term refers to a vanadium-containing haloperoxidase encoded by a DNA sequence of synthetic and / or cDNA origin and having the identity properties of the vanadium-containing haloperoxidase in question.

The desired optimal pH of the mutant vanadium-containing haloperoxidase with altered optimal pH depends on the intended use. For example, when vanadium-containing haloperoxidase is used for denim bleaching, the preferred optimal pH is about 5-8. On the other hand, when vanadium-containing haloperoxidase is used for washing, a preferred optimum pH is about 8 to 10
It is.

[0013] It is possible to alter the optimal pH of the parent vanadium-containing haloperoxidase, and such variants are the result of mutations, ie, one or more amino acid residues are replaced by the parent vanadium-containing haloperoxidase. , Is substituted, or added. Introducing a charge change near an active site residue can change the pKa of the residue of interest, thereby changing the activity profile of the haloperoxidase.

[0014] By introducing a more negatively charged residue near the His residue (the active site of the haloperoxidase), its pKa can be increased and thus exhibit catalytic activity at a higher pH than before. By introducing a more positively charged residue in the vicinity of the active site His, its pKa is reduced and therefore it may exhibit catalytic activity at a lower pH than before.

[0015] An increase in pKa can also be achieved by reducing the solvent accessibility of the active site (His). A reduction in pKa can also be achieved by increasing the solvent accessibility of the active site (His).

However, according to the present invention, the most important sites are residues within 10 ° of His496 and His404. 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, 39
3-412, 441, 443, 482-502, 507, 551-557. Changes in this region induce a change in pH-dependent activity, that is, a change in the optimal pH of the enzyme. For example, Curvularia i
In this haloperoxidase of naequalis, residues can be mutated in this way. A model of a homologous structure presumed to have a similar structure can be constructed (Example 1), whereby a region of interest can be found.

Preferred positions for the mutations are: 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;

Provided that the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 1, or has a homologous position within the parent haloperoxidase having an amino acid sequence at least 80% homologous to SEQ ID NO: 1, or Has a homologous position in the parent haloperoxidase having an amino acid sequence at least 85% homologous to, or has a homologous position in the parent haloperoxidase having an amino acid sequence at least 90% homologous to SEQ ID NO: 1, or Has a homologous position in a parent haloperoxidase having an amino acid sequence at least 95% homologous to SEQ ID NO: 1 or has a homologous position in a parent haloperoxidase having an amino acid sequence at least 96% homologous to SEQ ID NO: 1, Or a parent haloperio having an amino acid sequence at least 97% homologous to SEQ ID NO: 1 Has a homologous position in the oxidase, or at least
Has a homologous position in the parent haloperoxidase having an amino acid sequence 98% homologous, or has a homologous position in the parent haloperoxidase having an amino acid sequence at least 99% homologous to SEQ ID NO: 1.

Particularly preferred are the following mutations: 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; Peroxidase has the amino acid sequence of SEQ ID NO: 2.

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;

Wherein the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 1, or has a homologous position within the parent haloperoxidase having an amino acid sequence at least 80% homologous to SEQ ID NO: 1, or Has a homologous position in the parent haloperoxidase having an amino acid sequence at least 85% homologous to, or has a homologous position in the parent haloperoxidase having an amino acid sequence at least 90% homologous to SEQ ID NO: 1, or Has a homologous position in a parent haloperoxidase having an amino acid sequence at least 95% homologous to SEQ ID NO: 1 or has a homologous position in a parent haloperoxidase having an amino acid sequence at least 96% homologous to SEQ ID NO: 1, Or a parent haloperio having an amino acid sequence at least 97% homologous to SEQ ID NO: 1 Has a homologous position in the oxidase, or at least
Has a homologous position in the parent haloperoxidase having an amino acid sequence 98% homologous, or has a homologous position in the parent haloperoxidase having an amino acid sequence at least 99% homologous to SEQ ID NO: 1.

In a preferred embodiment, two or more amino acid residues may 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; however, the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 2.

Methods for Preparing Vanadium-Containing Haloperoxidase Several methods for introducing mutations into genes are known in the art. After a brief description of the cloning of the DNA sequence encoding haloperoxidase, a method for mutating a specific site in the haloperoxidase coding sequence will be described.

Cloning of the DNA sequence encoding the vanadium-containing haloperoxidase The DNA sequence encoding the parent vanadium-containing haloperoxidase can be isolated by any of a variety of methods well known in the art to any cell or microorganism producing the haloperoxidase in question. Can be isolated from First, a genomic DNA and / or cDNA library is constructed using the chromosomal DNA or mRNA of the organism producing the haloperoxidase to be studied. Next, if the amino acid sequence of the haloperoxidase is known, a homologous labeled oligonucleotide probe is synthesized and used to identify a haloperoxidase-encoding clone from a genomic library prepared from the organism in question. I can do it. Alternatively, labeled oligonucleotide probes having sequences homologous to known haloperoxidase genes can be used under low stringency hybridization and washing conditions to identify clones encoding haloperoxidase.

In a method for identifying clones encoding haloperoxidase, the cDNA is inserted into an expression vector, such as a plasmid, the resulting cDNA library is used to transform haloperoxidase-negative fungi, and contains a haloperoxidase substrate. The transformed fungus is sown on an agar, whereby clones expressing haloperoxidase can be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared by synthesis according to established standard methods, for example the phosphoramidite method. In the phosphoramidite method, oligonucleotides are synthesized, for example, on an automatic DNA synthesizer, purified, annealed, ligated, and cloned into a suitable vector.

Finally, the DNA sequence may be a mixture of genomic and synthetic sources, a mixture of synthetic and cDNA sources, or a mixture of genomic and cDNA sources; And fragments of synthetic, genomic or cDNA origin (if appropriate, fragments corresponding to various portions of the entire DNA sequence). The DNA sequence can also be prepared by PCR using specific primers.

Site-directed mutagenesis Once the DNA sequence encoding haloperoxidase has been isolated and the site desired for mutation has been identified, the mutation can be introduced using synthetic oligonucleotides. These oligonucleotides have a nucleotide sequence flanking the desired mutation site and insert the mutant nucleotide during oligonucleotide synthesis.
In a specific method, in a vector carrying a haloperoxidase gene, a single stranded gap of DNA is created that spans the sequence encoding the haloperoxidase. Next, a synthetic nucleotide having a desired mutation is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the construct is ligated with T4 ligase (Morinaga method; Biotechnology 2, 1984, 6).
26-639). U.S. Pat. No. 4,760,025 discloses a method for introducing oligonucleotides encoding multiple mutations through minor changes in the cassette. However, in the Morinaga method, since a large number of oligonucleotides of various lengths can be introduced, even more diverse mutations can be introduced.

Another method of introducing mutations into the DNA sequence encoding haloperoxidase is to use PC
In the R reaction, a chemically synthesized DNA strand is used as one of the primers to form a PCR fragment containing the desired insertion mutation in three steps. From the PCR product fragment, a DNA fragment carrying the mutation is isolated by restriction endonuclease cleavage and reinserted into the expression plasmid.

Random Mutagenesis The DNA sequence encoding the parent haloperoxidase can be conveniently subjected to random mutation according to methods well known in the art. For example, random mutagenesis can be performed by using a suitable physical or chemical mutagen, using a suitable oligonucleotide, or performing PCR on the DNA sequence. In addition, random mutagenesis can be performed by using any combination of mutagenic agents.

[0031] Mutagenesis agents include, for example, bases transitions, conversions.
, Inversion, scrambling, deletion, and / or insertion. Examples of physical or chemical mutagens suitable for the present invention include ultraviolet radiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrite, ethyl nitrate. There are methanesulfonic acid (EMS), sodium bisulfite, formic acid, and nucleotide analogs.

When the drug is used, it typically encodes the parent enzyme to be mutated
Mutagenesis is performed by incubating the DNA sequence in the presence of a mutagenic agent under conditions suitable for mutagenesis, and selecting a mutated DNA having the desired properties.

When mutagenesis is performed using an oligonucleotide, three nucleotides different from the parent nucleotide are introduced into the site to be mutated during oligonucleotide synthesis.
(doped or spiked). This introduction can be made to remove unwanted amino acid codons. After the introduction of the oligonucleotide, any known method such as PCR, LCR
Alternatively, it can be incorporated into the DNA encoding the haloperoxidase enzyme by a DNA polymerase and ligase.

When performing mutagenesis by PCR, the chemically treated or untreated gene encoding the parent haloperoxidase enzyme is subjected to PCR under conditions that increase nucleotide misincorporation (Deshler 1992). ; Leung et al., Technique, Vo
l.1, 1989, 11-15).

To randomly mutagenize the DNA encoding the haloperoxidase enzyme,
E. Coli (Fowler et al., Moelc. Gen. Genet., 133, 1974, 179-191), S. cere.
viseae or any other microbial mutagenized strain can be used, including the parent enzyme DN
By transferring the plasmid containing A to the mutagenized strain, growing the mutagenized strain carrying the plasmid, and isolating the mutated plasmid from the mutagenized strain. An expression organism is transformed with the mutated plasmid.

The mutagenic DNA sequence may conveniently be present in a genomic or cDNA library prepared from an organism that expresses the parent haloperoxidase enzyme. Alternatively,
The DNA sequence may be present on a suitable vector, such as a plasmid or bacteriophage, which may be incubated with the mutagen or otherwise contacted. The mutagenic DNA may be present in the host cell,
It is either integrated into the genome of the cell or present on a vector present in the cell. Finally, the mutagenic DNA sequence may be in an isolated state. The DNA sequence to be subjected to random mutagenesis is preferably a cDNA or genomic DNA sequence.

It may be advantageous to amplify the mutated DNA sequence before the expression or screening process. This amplification can be performed according to methods well known in the art, and the presently preferred method is PCR amplification using oligonucleotide primers made based on the DNA or amino acid sequence of the parent enzyme.

Following incubation or contact with a mutagen, a suitable host cell harboring the DNA sequence is cultured under conditions permitting expression, thereby allowing the mutant DN to be expressed.
A is expressed. The host cell used for this purpose is a cell transformed with the mutant DNA and, if necessary, the mutant DNA present on a vector, or a cell carrying the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Can be Examples of suitable host cells are fungal hosts, such as Aspergillus niger or Aspergillus oryzae. The mutated DNA sequence may further comprise a DNA sequence encoding a function that enables expression of the sequence.

Localization of random mutagenesis Advantageously, random mutagenesis can be localized to a portion of the parent haloperoxidase in question. This is the case, for example, when certain regions of the enzyme have been identified as being particularly important for certain properties of the enzyme, and where the modifications are expected to form variants with improved properties. Will be useful. Such regions can usually be identified when the tertiary structure of the parent enzyme has been elucidated and linked to enzyme function.

Conveniently, this localized random mutagenesis is performed using the PCR mutagenesis technique described above, or any suitable technique known in the art. Alternatively, the DNA sequence encoding the portion of the DNA sequence to be modified is isolated, for example by insertion into a suitable vector, and the portion is then mutagenized by any of the mutagenesis methods described above. It can be performed.

The screening process of the above method can be conveniently performed using an aa filter test based on the following principles. A microorganism capable of expressing a haloperoxidase enzyme mutant of interest,
Incubate on a suitable medium and under conditions suitable for secreting the enzyme. Two filters are placed on the medium. A second filter having a low protein binding ability is placed on the first protein binding filter. Microorganisms are present on the second filter. Following the incubation,
The first filter containing the enzyme secreted from the microorganism is separated from the second filter containing the microorganism. The first filter is screened for the desired enzyme activity and the corresponding microbial colonies present on the second filter are identified. The filter used to bind the enzyme activity may be any protein binding filter, such as a nylon or nitrocellulose filter. The upper filter that retains the colonies of the expressing microorganisms can be any filter that has no or low protein binding capacity, such as cellulose acetate or Durapore ^™ . The filter can be pre-treated under any conditions used for screening or can be processed during detection of enzyme activity.

The enzyme activity can be detected using a dye, fluorescence, precipitation, pH indicator, IR absorption, or any known technique for detecting enzyme activity. The detection compound can be immobilized by any immobilizing agent, for example, agarose, agar, gelatin, polyacrylamide, starch, filter paper, cloth, or any combination of immobilizing agents.

Expression of Haloperoxidase Variants In accordance with the present invention, DNA sequences encoding variants produced by the methods described above, or any of the alternatives known in the art, can be expressed in enzymatic form. To that end, typically an expression vector is used that contains a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a repressor gene or various activator genes.

The recombinant expression vector carrying a DNA sequence encoding a haloperoxidase variant of the present invention is one that can conveniently employ recombinant DNA techniques. The vector is often chosen depending on the host cell into which it is to be introduced. Thus, the vector is an autonomously replicating vector, i.e., a vector that is extrachromosomal and whose replication is independent of chromosomal replication, such as a plasmid, bacteriophage, or extrachromosomal element, minichromosome or artificial chromosome. May be. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated along with the integrated chromosome.

In the vector, the DNA sequence is operably linked to a suitable promoter sequence. The promoter can be any DN that has transcriptional activity in the selected host cell.
A sequence, which is derived from a gene encoding a protein of the same or different type with respect to the host cell. Suitable promoters that direct transcription of the DNA sequence encoding the haloperoxidase variants of the present invention, particularly in fungal hosts, include, for example, A. oryzae Taka amylase, Rhizomucor miehei aspartic proteinase, and A. niger neutral. α-amylase, A. niger acid-stable α-amylase, A. niger
niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase, or A. nid
Some are derived from the gene encoding acetamidase of ulans.

The expression vector of the present invention may further contain a suitable transcription terminator and, in eukaryotic cells, a polyadenylation sequence. They are operably linked to DNA encoding a haloperoxidase variant of the invention. The terminator and polyadenylation sequence will suitably be obtained from the same source as the promoter. The vector may further carry a DNA sequence enabling its replication in the host cell in question. Examples of such sequences are the plasmids pUC19, pACYC177, pUB110, pE194
, pAMB1 and pIJ702 are the origins of replication.

The vector further carries a selectable marker, for example, a gene for a product that complements the deficiency of the host cell, for example, a gene that confers resistance to an antibiotic such as ampicillin, kanamycin, chloramphenicol or tetracycline. obtain. In addition, the vector contains Aspergillus selectable markers such as amdS, argB, niaD and sC.
May carry a marker conferring hygromycin resistance. Alternatively, the selection can be performed by co-transformation, for example as described in WO 91/17243. The method of ligating the DNA construct of the present invention encoding the haloperoxidase mutant, the promoter, the terminator, and other elements, and the method of inserting them into an appropriate vector having information necessary for replication are described in the present invention. It is well known to those skilled in the art (Sambrook et al. (1989)).

The cells of the present invention carrying either the DNA construct or the expression vector of the present invention
In recombinant production of the haloperoxidase mutant of the present invention, it is advantageous to use it as a host cell. Cells may be transformed with a DNA construct of the present invention encoding the variant, conveniently by integration of the DNA construct (in one or more copies) into the host chromosome. Generally, this integration is considered beneficial since the DNA constructs are likely to be stably maintained in cells. Integration of the DNA construct into the host chromosome can be performed by conventional methods, for example, by homologous or heterologous recombination. Alternatively, the cells can be transformed in different types of host cells by the expression vectors described above.

The cells of the invention may be cells of higher organisms, such as mammals or insects,
Preferably, it is a cell of a microorganism, for example, a fungal cell. Preferably, the filamentous fungus is an Aspergillus species, such as Aspergillus oryzae.
Or Aspergillus niger. Fungal cells can be transformed by known methods consisting of protoplast formation, protoplast transformation, and cell wall regeneration. Suitable methods for transforming Aspergillus host cells are described in EP 238 023.

In yet another aspect, the invention relates to a method of producing a haloperoxidase variant of the invention, comprising culturing said host cell under conditions that induce the production of said variant. And recovering the mutant from the cells and / or medium. The medium in which the cells are cultured can be any conventional medium suitable for growing the host cell in question and for expressing the haloperoxidase variants of the present invention. Suitable media are obtained from commercial sources or have published compositions (eg, Americ
an Type Culture Collection).

The haloperoxidase variant secreted from the host cell can be conveniently recovered from the culture medium by well known techniques, for example by separating the cells from the culture medium by centrifugation or filtration, and adding a salt such as ammonium sulfate. To precipitate a protein component in the medium, and chromatography such as ion exchange chromatography and affinity chromatography is performed.

Industrial Use The haloperoxidase according to the invention can be incorporated in detergents or cleaning agents.
The detergent or cleaning agent may contain other useful enzymes, such as proteases, amylases,
It contains at least one enzyme selected from the group consisting of cutinase, peroxidase, oxidase, laccase, cellulase, xylanase, and lipase. The haloperoxidase of the invention can be used in particular for bleaching and hygiene.

Foods, beverages, cosmetics, such as lotions, creams, gels, ointments, soaps,
When used for the preservation of shampoos, conditioners, antiperspirants, deodorants, mouthwashes, contact lens products, enzyme preparations, or food ingredients, the haloperoxidase of the present invention can kill or multiply microorganisms. Can be incorporated in an amount effective to control, for example, unpreserved foods, beverages, cosmetics, contact lens products, food ingredients, or anti-inflammatory agents.

The haloperoxidase used in the method of the present invention may be used as a bactericide, for example,
In the treatment of acne, eye or mouth infections, skin infections; in antiperspirants or deodorants; in footbath salts; in cleaning and disinfection of contact lenses, hard surfaces, teeth (mouth care), wounds, and bruises; Will be useful.

In general, the haloperoxidases of the invention can be used for cleaning, disinfecting,
Or it may be useful for microbial growth control. Examples of such surfaces where it is beneficial to contact the compositions of the present invention include, for example, dairy, chemical or pharmaceutical manufacturing plants,
Surface of process equipment used in water purification systems, paper mills, water treatment plants, and cooling towers. The haloperoxidases of the invention need to be used in an effective amount for washing, disinfecting or inhibiting microbial growth on the surface in question. Furthermore, it would be beneficial to use the haloperoxidases of the present invention in a cleaning-in-place (CIP) for cleaning all types of process equipment.

In addition, the haloperoxidase of the present invention can be used to apply the haloperoxidase of the present invention to food processing plants and any place where food is prepared or supplied, such as hospitals, nursing homes, restaurants, especially fast food restaurants, and delicatessens. Can be used for washing. It can also be used as an antimicrobial in foods, and will be particularly useful as a surface antimicrobial in cheese, fruits and vegetables, and salad bar foods. It can also be used as a preservative or bactericide in aqueous paints.

Preservation of Paint Preservation of paint in cans is accomplished in the art by the addition of non-enzymatic organic biocides. In the context of the present invention, paint means a substance containing a solid colored substance dissolved or dispersed in a liquid solvent, for example water, organic solvents and / or oils. Spreading it on a surface and drying it results in a thin colored decorative and / or protective coating. Typically, isothiazolones, such as 5-chloro-2-methyl-4-thia-zoli-3, are used to inhibit / suppress microbial growth in paints.
-Add ON as a biocide at a dose of about 0.05-0.5%. However, the method of the present invention can be appropriately used in this field. Thus, the existing environmental crises due to toxic organic biocides can be resolved by replacing the toxic biocides with environmentally compatible enzymes. Accordingly, the present invention provides a method of preserving a paint, the method comprising contacting the paint with a haloperoxidase variant of the present invention. The present invention further provides a coating composition containing the haloperoxidase variant of the present invention.

As the paint, a water-based paint, that is, a paint in which an individual paint is dispersed in an aqueous solution is preferable. The coating may contain 0-20%, preferably 0-10%, for example 0-5%, of organic solvent. The enzyme is used in an amount of 0.0001 to 100 mg active enzyme protein / 1L paint, preferably 0.001 to
A dose of 10 mg / L, for example 0.01-1 mg / L, can be added to the paint.

Hydrogen Peroxide Source In the present invention, the hydrogen peroxide required for the haloperoxidase reaction can be supplied in a number of different ways. This may be hydrogen peroxide, or a hydrogen peroxide precursor, such as percarbonate or perborate, or peroxycarboxylic acid or a salt thereof, or a hydrogen peroxide generating enzyme system, such as oxidase and its substrate. There may be. Useful oxidases include, for example, glucose oxidase, glycerol oxidase or amino acid oxidase. Examples of amino acid oxidases are described in WO94 / 25574.

It may be advantageous to use enzymatically generated hydrogen peroxide. This source produces relatively low concentrations of hydrogen peroxide under biologically relevant conditions. Low concentrations of hydrogen peroxide increase the rate of haloperoxidase catalyzed reactions. In the present invention, the source of hydrogen peroxide required for the reaction of haloperoxidase is 0.01
It can be added to a concentration corresponding to a hydrogen peroxide concentration in the range of -1000 mM, preferably 0.1-500 mM.

Halide Source In the present invention, the halide required for the reaction of haloperoxidase can be supplied in a number of different ways, for example by the addition of a halide salt. It can be sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, or potassium iodide. The concentration of this halide source is typically 0.01-1000 mM, preferably 0.1-1000 mM.
Equivalent to 500 mM.

Compositions Compositions containing haloperoxidase, a source of hydrogen peroxide, and a source of halide can be formulated as solids or liquids, in particular in non-spattering granules, or in stabilized liquids. When formulated as a solid, all components can be mixed, for example, into a powder, granule, or gel. When a composition other than a dried product is used, and even in the case of a dried product, it is preferable to use a two-component preparation system in which hydrogen peroxide and other components are separated.

The compositions of the present invention may further comprise adjuvants such as wetting agents, thickening agents, buffers, stabilizers, fragrances, colorings, fillers and the like. Useful wetting agents are surfactants, ie, nonionic, anionic, zwitterionic surfactants. The composition of the invention may be a concentrated preparation or a ready-to-use preparation.

Haloperoxidase activity In the present invention, haloperoxidase activity can be measured as described in WO 97/04102, p.13, l.7-19. The following examples further illustrate the invention, but do not limit the scope of the invention.

[Example 1] Construction of three-dimensional structure of Curvularia verruculosa haloperoxidase by homology Based on sequence homology with Curvularia ineaqualis (CI), for example, Curvularia verrucu
From the losa sequence, a CI-like three-dimensional structure can be found. Curvularia ineaqualis used to elucidate the structure
verruculosa differs in several residues. This model is called Molecular Simu
It can be constructed using the INSIGHT and HOMOLOGY programs of lations Inc. At the homologous positions identified as structurally conserved regions (SCRs) by the program, the program replaces the amino acids of the Curvularia ineaqualis sequence with Curvulia
Replace with amino acids of aria verruculosa sequence. The residue in between is determined by GENERATE
Build using the LOOP option. Through these processes, a rough model having information on spatial interaction can be obtained. The structure can be refined by the method described in HOMOLOGY.

Example 2 Construction of Haloperoxidase Mutant To construct a haloperoxidase mutant of Curvularia verruculosa,
A commercially available Chameleon double-stranded site-directed mutagenesis kit is used as per the instructions. The gene encoding the haloperoxidase enzyme in question is on plasmid pElo29. Using primer 117996 (SEQ ID NO: 3) as described in the
Change the ScaI site of the ampicillin gene on lo29 to a MluI site. At the same time, the desired mutation is introduced into the haloperoxidase gene by adding an appropriate primer containing the desired mutation.

Construction of plasmid pElo29 Plasmid pElo29 was constructed from the following fragments: a) a 4.1 kb fragment from the vector pCip (WO 93/34618) cut with BamHI and BglII;
b) Plasmid pAJ014-1 as template (WO 97/04102), primers 146063 and 1460 to introduce BamHI and BglII sites at 5 ′ and 3 ′ of haloperoxidase gene, respectively
62 (SEQ ID NOS: 4 and 5), and the haloperoxidase gene of Curvularia verruculosa amplified by PCR using Pwo polymerase. After ligation of fragments a and b, the entire sequence of the haloperoxidase gene was determined to confirm that no unwanted mutations were introduced during PCR amplification.

Site-directed mutagenesis Site-directed mutagenesis was performed as described above to construct a plasmid carrying a gene encoding a mutant 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. To introduce R487E, primer 139078 (SEQ ID NO: 8) was used. Primer 139085 (SEQ ID NO: 9) was used to introduce V492S. Each mutation was confirmed by determining the entire sequence of each gene. The resulting plasmids were named pElo30, pElo31, pElo34 and pElo37, respectively.

Transformation of JaL228 with pElo30, pElo31, pElo34 and pElo37 Aspergillus oryzae JaL228 (WO 97/27221) is Aspergillus oryzae IFO4177 deficient in alkaline protease and neutral metalloprotease I. This cell was pElo30, together with the plasmid pToC90 (WO 91/17243) carrying the amdS gene.
Co-transformation was carried out with pElo31, pElo34 or pElo37. Transformants were selected using acetamide as described in EP 0 531 372 B1. Transformant spores were repeatedly isolated twice. Spores of each transformant after the second isolation were tested for haloperoxidase production by small-scale fermentation (flask shaking and microtiter dishes).

[0070] The isolates fermented said variant mutant of Curvularia Verruculosa haloperoxidase in Aspergillus oryzae, sucrose per 1L 12 g, 50% yeast extract 20g, MgSO ₄ * 7H ₂ 0
Culture was performed in a tank medium consisting of ₂ g, ₂ g of KH ₂ PO ₄ , ₃ g of K ₂ SO ₄ , ₄ g of citric acid, 1 ml of Pluronic, 182 mg of V ₂ O ₅ and 0.5 ml of a trace metal solution. During fermentation, 1 g of 250% maltose solution 250 g, 50% yeast extract 20 g, citric acid 5 g, pluronic 5 ml, V ₂ O ₅ 18 per liter
A medium consisting of 2 mg and 0.5 ml of trace metal solution was supplied. After fermentation at 34 ° C., pH> 6.0 for 5 days, the fermentation broth was collected. The above trace metal solution: ZnSO ₄ * 7H ₂ O 14.3 g, CuSO ₄ * 5H ₂ O 2.5 g, NiCl ₂ * 6 per liter
_{H 2 O 0.5g, FeSO 4 *} 7H 2 O 13.8g, MnSO 4 * H 2 O 8.5g and citric acid 3.0 g.

[Sequence List] SEQ ID NO: 3: Primer 117996; modified nucleotides are underlined. This removes the Sca site of pElo29 and introduces a MluI site: 5'-GA ATG ACT TGG TTG A CGCG T CAC CAG TCA C-3 'SEQ ID NO: 4: Primer 146063; for amplifying the Curvularia verruculosa haloperoxidase gene PCR primer. BamHI site is introduced by underlined nucleotides: 5'-CGC GGATCC TCT ATA TAC ACA ACT GG-3 'SEQ ID NO: 5: primer 146062; PCR primer for amplifying Curvularia verruculosa haloperoxidase gene. Introduce a BglII site by underlined nucleotides: 5'-GAA GAT CT C GAG TTA ATT AAT CAC TGG-3 'SEQ ID NO: 6: Primer 147293; Introduce D289E mutation to haloperoxidase enzyme by underlined nucleotide : 5'-GG TCT GTA TTG GGC CTA C CT TGG GTC AAA CC-3 'SEQ ID NO: 7: Primer 147295; Introduces D289L mutation to haloperoxidase enzyme by underlined nucleotide: 5'-GG TCT GTA TTG GGC CTA C GA G GG GTC AAA CC-3 'SEQ ID NO: 8: primer 139078; introduces R487E mutation into haloperoxidase enzyme by underlined nucleotide: 5'-CG CCA TTT CT G AG A TCT TCC TGG GC-3 'SEQ ID NO: 9: primer 139085; introduces V492S mutation into haloperoxidase enzyme by underlined nucleotides: 5'-GC ATC TTC CTC GGC AG C CAC TGG CGA TTC GAT GCC G-3'

Example 3 pH Curves of Various Haloperoxidase Mutants Haloperoxidase mutants (derived from Curvularia verruculosa) were prepared as described in Example 2. Experiment: Phenol red test to determine pH characteristics: In a 96-well microtiter plate, 100 μl each of 60 mM Bri, pH 4-8.5
A 0.008% phenol red solution in tton-Robinson buffer was added. In this solution,
50 μl of the diluted enzyme solution containing 40 μl of 0.5 M KBr and 1 mM of orthovanadate were added. The reaction is started by addition of 10 μl of 0.3% hydrogen peroxide and 5% at 595 nm.
The reaction rate was measured for a minute.

Results: The activity for each enzyme was calculated for its maximum value. ─────────────────────────────────── pH pH pH pH pH pH pH pH pH pH mutant 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 ─────────────────────────────────── wt rCv 0 0.01 0.14 nd 0.16 0.34 1.00 0.89 0.51 0.18 D289E 0.01 0.10 0.63 0.98 1.00 0.85 0.52 0.25 0.11 0.03 D289L 0 0 0 0.01 0.12 0.39 0.97 1.00 0.55 0.14 R487E 0 0 0.31 0.84 1.00 0.83 0.48 0.20 0.09 0.03 V492S 0 0.01 0.53 0.95 1.00 0.98 0.77 0.42 0.19 0.06 ──分 か る As can be seen from this table, the optimal pH of the wild type is 7.0, The optimal pH for mutant D289E, R487E and V492S is 6.0, and the optimal pH for mutant D289L is 7.5.

Example 4 pH Curve of Purified Variants of Haloperoxidase (V492S) The haloperoxidase variant (V492S) described above and the wild-type haloperoxidase of Curvularia verruculosa were purified and tested with Chicago Sky Blue. Purification of haloperoxidase: Fermentation broth with haloperoxidase activity was purified using GF / F (Whatmann) and 0.22 μl.
m (GS, Millipore) and then concentrated on Filtron (cut off 10 kDa). The pH of the sample was adjusted to 7.5 and Q-Seph was equilibrated in 50 mM Tris-HCl, pH 7.5.
An arose column (Pharmacia) was applied. Haloperoxidase was eluted with a linear gradient of 0-1 M NaCl / 50 mM Tris-HCl, pH 7.5. Fractions containing haloperoxidase were collected and Superdex equilibrated in 50 mM sodium acetate, 0.1 M NaCl, pH 5.5.
Further purification was performed on a 75 column 16/60 (Pharmacia).

Experiment: In a 96-well microtiter plate, 100 μl of 60 mM Britton, pH 4-8
-Robinson buffer, 50 µl of enzyme solution, 25 µl of 0.4 M NaCl, and 25 µl of Chicago Sky Blue diluted to OD610 = 5 were added. The reaction was started by the addition of 10 μl of 2 mM H ₂ O ₂ . The activity was measured as a linear decrease in 595 nm absorbance.

Specific 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 8 0 0.01 ────────────Result : The activity of V492S is clearly increased at low pH compared to the wild-type enzyme.

[Sequence list]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12N 9/08 4L031 9/08 C12S 3/04 C12S 3/04 D06L 3/11 D06L 3 / 11 D06M 16/00 Z D06M 16/00 C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, M, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Invention Jergensen, Luise Denmark, Denmark-2880 Bagsbaerto, Novo Ale, Novo Nordisk Actiesel Skabu F-term (reference) CA28 CA57 CA60 4H 003 BA10 BA12 DA01 DA02 DA05 EC01 EC02 EC03 EE11 FA34 FA42 FA47 4J038 BA182 EA011 MA08 MA10 NA02 4L031 BA39

Claims (22)

[Claims] Claims 1. A parental vanadium-containing haloperoxidase having haloperoxidase activity, having an optimal pH changed, and having at least 1
Mutants having mutations at positions corresponding to the following three 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 With the proviso that the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 1 or has at least 80% homology to SEQ ID NO: 1. 2. The mutant according to claim 1, which is derived from the parent vanadium-containing haloperoxidase and has 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; provided that the parent haloperoxidase has the amino acid sequence of SEQ ID NO: 2. 3. The mutant according to claim 1, wherein the parental haloperoxidase is derived from Curvularia. 4. The method of claim 1 wherein the parental haloperoxidase is Curbularia inenaquaris.
The mutant according to claim 1, which is derived from (Curvularia inaequalis). 5. The method of claim 1 wherein the parental haloperoxidase is Curbularia velcrosa.
vularia verruculosa). 6. A DNA construct comprising a DNA sequence encoding the haloperoxidase variant according to any one of claims 1 to 5. 7. A recombinant expression vector carrying the DNA construct according to claim 6. 8. A cell transformed with the DNA construct according to claim 6 or the vector according to claim 7. 9. The cell according to claim 8, which is a microorganism. 10. The cell according to claim 9, which is a bacterium or a fungus. 11. The cell of claim 10, which is a cell of Aspergillus niger or Aspergillus oryzae. 12. Use of a haloperoxidase variant according to any of claims 1 to 5 for bleaching. 13. Use according to claim 12, for bleaching soil. 14. Use according to claim 12, for bleaching cotton. 15. Use according to claim 12, for bleaching dyes. 16. Use of a haloperoxidase mutant according to any one of claims 1 to 5 for controlling microorganisms. 17. Use of a haloperoxidase variant according to any of claims 1 to 5 for cleaning hard surfaces. 18. A detergent additive comprising the haloperoxidase variant according to any one of claims 1 to 5 in the form of a non-scattering granule, a stabilized liquid, or a protected enzyme. 19. One or more other enzymes, such as proteases, lipases,
19. The additive for a detergent according to claim 18, further comprising an amylase and / or a cellulase. 20. A detergent composition comprising the haloperoxidase variant according to claim 1. 21. One or more other enzymes, such as proteases, lipases,
The cleaning composition according to claim 20, further comprising amylase and / or cellulase. 22. A paint containing the haloperoxidase variant according to claim 1.