Bacterial lactoperoxidases and products and methods related thereto.

Abstract

The invention relates to bacterial lactoperoxidase enzymes and their application as antimicrobial agent, for example as component in the lactoperoxidase system. Provided is a method for the catalytic oxidation of an organic or inorganic substrate, comprising contacting the substrate with a source of in the presence of a polypeptide having lactoperoxidase activity, wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No. 1- 4 of Figure 1, and comprising the following heme-coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, iii) RXGH[TS]; wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has lactoperoxidase activity.

Description

[0001]P137145PC00 Title: Bacterial lactoperoxidases and products and methods related thereto. The invention relates to the field of enzymes and biocatalysis. More specifically, it relates to bacterial lactoperoxidase enzymes and their application as antimicrobial agent, for example as component in the lactoperoxidase system. Lactoperoxidase is an enzyme secreted from mammary, salivary and other mucosal glands which represents a natural antibacterial agent. Lactoperoxidase is a member of the heme peroxidase family of enzymes. In humans, lactoperoxidase is encoded by the LPO gene. Lactoperoxidase catalyzes the oxidation of inorganic and organic substrates by hydrogen peroxide. These substrates include bromide and iodide and therefore lactoperoxidase can be categorised as a haloperoxidase. Another important substrate is thiocyanate. The oxidized products produced through the action of this enzyme have potent and non-specific bactericidal and antiviral activities. Lactoperoxidase, thiocyanate, hydrogen peroxide and hypothiocyanite (product) are known all together as the lactoperoxidase system. This system is of commercial interest as it is widely used in the conservation of dairy products. It is used in remote farms for a quick sterilization of milk and milk products, such mozzarella and cottage cheese (see GRAS Notice 665). Further known uses include oral care products (e.g. toothpaste and mouthwash) for the treatment of gingivitis and periodontitis (Int. J. Mol. Sci.2019, 20, 1443; doi:10.3390 / ijms20061443). Still further, lactoperoxidase, glucose and glucose oxidase, together with iodide or thiocyanate, are commonly used for the preservation of cosmetics (see US 5,607,681). Mammalian peroxidases are notoriously difficult to express as recombinant protein. This may be due to the structural complexity of glycosylation and calcium binding, preventing proper folding in commonly used microbial expression hosts. This has hampered the industrial application of mammalian LPOs as bactericidal agents. To overcome these difficulties, efforts have been undertaken to obtain lactoperoxidases from bacterial sources. For example, a heme peroxidase which has high similarity to bovine lactoperoxidases (LpoPOX) was identified from the cyanobacterium Lyngbya sp. PCC 8106 and termed LspPOX. However, this bacterial enzyme showed only marginal, if any, improvements over LpoPOX when enzymatic activities were compared (Auer et al., J Biol Chem, 288, 2013). WO2024 / 017883A1 relates to means and methods comprising a bacterial heme peroxidase and / or a functional fragment thereof for medical and non- medical use in preventing and / or controlling a pathogen on food, plants, or in or on the human or animal body. Disclosed are bacterial heme peroxidases heme peroxidase that is obtained and / or derived from cyanobacteria, in particular from Hydrocoleum sp. or Okeania sp. (HydPOX, HydPOXs and OkePOXs). Also these enzymes show a relatively low catalytic activity. Therefore, the present inventors set out to identify further bacterial LPO homologs. In particular, they aimed at providing novel lactoperoxidase enzymes that allow for high expression yields in bacterial host cells, have an acceptable stability (thermal and pH) and display a desirable activity towards relevant substrates. To that end, bovine LPO was used in a pBLAST search (NCBI). This resulted in the identification of several predicted protein sequences, including a protein of 556 residues encoded by a gene of Cyanobacterium sp.TDX16 that shared only 37% sequence identity to bovine LPO, 48%sequence identity with HydPOX and OkePOX, and 55% sequence identity with DeltaPOX. Compared to these bacterial enzymes, the novel bacterial lactoperoxidases of the present invention demonstrate higher turnover numbers (kcat) when catalyzing substrates such as ABTS or KI. The bacterial enzymes could be produced at high yield in bacterial host cells and displayed a wide substrate specificity. Furthermore, it was found that the novel enzymes could be protected against e.g. proteolytic degradation or thermal inactivation upon encapsulation in a protein nanocage. Consequently, due to its higher catalytic efficiency the utilization of the present enzymes in industrial processes holds the promise of heightened productivity, reduced reaction times, and / or improved cost-effectiveness. Accordingly, in one aspect the invention provides a method for the catalytic oxidation of an organic or inorganic substrate, comprising contacting the substrate with a source of peroxide in the presence of a polypeptide having lactoperoxidase activity, wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1- 4 of Figure 1 , and comprising the following heme-coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S; iii) RXGH[TS], preferably R[VF]GH[TS]; wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has lactoperoxidase activity. Lactoperoxidases In some embodiments, the invention provides novel polypeptides having lactoperoxidase activity. As used herein, the expression ‘’a polypeptide having lactoperoxidase activity’’ refers to any polypeptide capable of oxidizing of halides (such as fluorine, chlorine, bromine, iodine) and pseudohalides (such as thiocyanate) to the corresponding hypohalous (hypofluorous acid, hypochlorous acid, hypobromous acid, hypoiodous acid) and hypothiocyanous acid in the presence of a peroxide source. Exemplary polypeptides having lactoperoxidase activity display one or more of the following characteristics: - a KMvalue for H2O2of up to 0.6 mM, preferably up to 0.5 mM; - a melting temperature (Tm) of at least 45ºC when assayed at a pH in the range of about 5.5 to 7.5; - a pH optimum in the range of 6 to 7. The lactoperoxidase may comprise an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1- 4 of Figure 1. The term "pairwise sequence identity percentage" generally means the coefficient between amino acid residue positions that have the same amino acid in two aligned sequences over all positions when the two protein sequences are aligned. Percent (%) sequence identity with respect to amino acid sequences disclosed herein is defined as the percentage of amino acid residues in a candidate sequence that are pair-wise identical with the amino acid residues in a reference sequence, i.e. a protein molecule or fragment of the present disclosure, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using public available computer software such as pairwise sequence identity when aligned using the Global alignment with free end gaps method, BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The term "amino acid" or "amino acid residue" refers to an a- or β-amino carboxylic acid. When used in connection with a protein or peptide, the term "amino acid" or "amino acid residue" typically refers to an α-amino carboxylic acid having its art recognized definition such as an amino acid selected from the group consisting of: L-alanine (Ala or A); L-arginine (Arg or R); L-asparagine (Asn or N); L-aspartic acid (Asp or D); L-cysteine (Cys or C); L-glutamine (Gln or Q); L-glutamic acid (Glu or E); glycine (Gly or G); L- histidine (His or H); L-isoleucine (ILE or I): L-leucine (Leu or L); L-lysine (Lys or K); L-methionine (Met or M); L-phenylalanine (Phe or F); L-proline (Pro or P); L-serine (Ser or S); L-threonine (Thr or T); L-tryptophan (Trp or W); L-tyrosine (Tyr or Y); and L- valine (Val or V), although modified, synthetic, or rare amino acids such as e.g. taurine, ornithine, selenocysteine, homocystine, hydroxyproline, thioproline, iodotyrosine, 3-nitro-tyrosine, ornithine, citrulline, canavanine, 5 -hydroxytryptophane, carnosine, cycloleucine, 3,4-dihydroxy phenylalanine, N-acetylcysteine, prolino 1, allylglycine or acetidine-2-carboxylic acid may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, GIu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr). A "fragment" as used herein refers to a portion of a parental protein which portion has lactoperoxidase activity. Such a fragment can comprise consecutive amino acids of the parental protein. A "fragment" can also refer to a protein in which fragments of a parental protein are fused together. A fragment can also comprise modifications such as amino acid substitutions, amino acid deletions or amino acid insertions compared to the parental protein. Motif (i) may comprise the amino acid sequence HDLDLS or HDLDLT. See the sequence H129DLDLS134in Seq No.1 or the sequence H81DLDLT86in Seg. No.3. Motif (ii) comprises the amino acid sequence TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S. Exemplary sequences of motif (ii) include TAWIDGS, TAWIDAS, TSWIDAS and TSYLDAS. Motif (iii) comprises the amino acid sequence RXGH[TS], preferably R[VF]GH[TS. Exemplary sequences of motif (iii) include RVGHT, RFGHS, and RVGHT. Preferably, the polypeptide having lactoperoxidase activity comprises one or more conserved residues corresponding to His81, His294, Leu372, Asn376 and Arg291 of the amino acid sequence of Seq. no.3. In some aspects, the polypeptide comprises a calcium binding site, preferably wherein said calcium binding site is formed by residues corresponding to Asp82, Thr140, Trp142, Asp144 and Ser146 of the aminoacid sequence of Seq. no. 3 as shown in Figure 1. Another non-conservedcalcium ion (10 Å to His294) is bound by the sidechain of Asp350 , the carbonyl oxygen and OG1 of Thr295 (adjacent to His294), the carbonyl oxygens of Glu348 and Lys352 , and two water molecules. Hence, in some embodiments the polypeptide comprises a second calcium binding site formed by residues Asp350, Thr295, Glu348 and Lys352. The polypeptide may comprise or consist of a sequence that has at least 68%, at least 70%, at least 72%, at least 75%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% pairwise sequence identity with any one of Seq. no.1-4 of Figure 1, or a fragment thereof that has lactoperoxidase activity. In one embodiment, the polypeptide may comprise or consist of a sequence that has at least 65%, at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% pairwise sequence identity with Seq. no.1 (DeltaproteobacteriaPOX), or a fragment thereof that has lactoperoxidase activity. In a specific aspect, the polypeptide comprises or consists of a sequence that that has at least 96, 97, 98 or 99 % pairwise sequence identity with Seq. no.1 of Figure 1, or a fragment thereof that has lactoperoxidase activity. In another embodiment, the polypeptide may comprise or consist of a sequence that has at least 65%, at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% pairwise sequence identity with Seq. no.2 (DadabacteriaPOX), or a fragment thereof that has lactoperoxidase activity. In a specific aspect, the polypeptide comprises or consists of a sequence that that has at least 96, 97, 98 or 99 % pairwise sequence identity with Seq. no.2 or a fragment thereof that has lactoperoxidase activity. In yet another embodiment, the polypeptide may comprise or consist of a sequence that has at least 65%, or at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% pairwise sequence identity with Seq. no.3 (CyanoPOX), or a fragment thereof that has lactoperoxidase activity. In a specific aspect, the polypeptide comprises or consists of a sequence that that has at least 96, 97, 98 or 99 % pairwise sequence identity with Seq. no.3 or a fragment thereof that has lactoperoxidase activity. In a still further embodiment, the polypeptide may comprise or consist of a sequence that has at least 65%, or at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% pairwise sequence identity with Seq. no.4 (PlanctomycetesPOX), or a fragment thereof that has lactoperoxidase activity. In a specific aspect, the polypeptide comprises or consists of a sequence that that has at least 96, 97, 98 or 99 % pairwise sequence identity with Seq. no.4 or a fragment thereof that has lactoperoxidase activity. In a preferred aspect, the polypeptide comprises the sequence of Seq. no.3, or a fragment thereof that has lactoperoxidase activity. A polypeptide according to the invention may comprise (by genetic fusion) one or more additional amino acid sequences or protein tag(s) at its N- and / or C-terminus. In one embodiment, the polypeptide comprises an N- terminal tag. In another embodiment, the polypeptide comprises a C- terminal tag. In a further embodiment, the polypeptide comprises both an N- and a C-terminal tag. The additional tag sequence(s) may aid in the expression yield, folding, solubilization, purification, encapsulation and / or immobilization of the polypeptide. Such sequences are well known in the art. Exemplary fusion tags include maltose binding protein, N-utilization substance A (NusA), glutathione S-transferase (GST), biotin carboxyl carrier protein, thioredoxin, and cellulose binding domain, short peptide tags such as oligohistidine (6xHis; His-tag), oligolysine, S-peptide, and the FLAGpeptide. Exemplary solubility tag includes SUMO (Small Ubiquitin-likeModifier) or MBP (maltose-binding protein). In a specific aspect, the enzyme contains an N-terminal His-tag. Alternatively, or additionally, it is provided with a SUMO tag. The tag sequence(s) may be (proteolytically) removed from the polypeptide prior to their application to catalyze a lactoperoxidase reaction. For example, SUMO fusion proteins can be cleaved to remove the SUMO moiety using SUMO-specific proteases such as Ulp1. In one aspect, the polypeptide comprises a C-terminal sorting motif, preferably wherein said sorting motif comprises a Pro-Glu-Pro (PEP) triad followed by a hydrophobic alpha-helix and a terminal positively charged segment. For example, the polypeptide contains a C-terminal PEP-CTERM domain comprising a 25-residue domain including an invariant Pro-Glu-Pro (PEP) motif, a thirteen residue strongly hydrophobic sequence likely to span the membrane, and a five-residue strongly basic motif that often contains four Arg residues (see Haft et al., BMC Biol.4, 29, (2006)). In another aspect, the polypeptide comprises an (external) C-terminal tag for targeting the enzyme to the interior of a protein nanocage. The C- terminal sequence may span between 15 to 30 amino acids in length. See Cassidy-Amstutz et al. (Biochemistry 2016, 55, 24, 3461–346). For example, the polypeptide comprises a C-terminal tag for targeting to an encapsulin such as NCBI Reference Sequence: WP_141866254.1 and WP_092074293.1, or a sequence that has at least 70%, preferably at least 80%, more preferably at least 90% or 95% or 99% sequence identity thereto. In one embodiment, the polypeptide comprises C-terminal tag for targeting the enzyme to encapsulin originating from the mesothermophile Mycolicibacterium hassiacum (EncMh). In a specific aspect, the polypeptide has a C-terminal sequence that has at least 70%, preferably at least 80%, more preferably at least 90% or 95% or 99% sequence identity to PPPLPDSEPDREIPADDGSLGIGSLKGTRS. The invention also relates to a composition comprising one or more polypeptide(s) according to the invention. For example, the composition comprises whole cells, permeabilized cells, a cell extract or a cell-free extract comprising a recombinantly expressed enzyme of the invention. In a preferred aspect, the composition is a bacterial cell culture comprising a bacterial host cell expressing one or more polypeptides of the invention as heterologous enzyme. In another embodiment, the composition comprises the enzyme(s) in a soluble or immobilized form. The composition may be a reaction mixture comprising one or more lactoperoxidases, one or more substrates, a source of H2O2, and / or products. Enzyme encapsulation In a specific aspect, the polypeptide having lactoperoxidase activity is used in a form wherein it is stabilized and / or protected e.g. against proteolytic degradation or thermal inactivation. To that end, it is suitably encapsulated in a protein nanocage, a polymer capsule, or the like. Encapsulation may comprise enclosing a substance in a "capsule", to isolate it from the external environment and / or transport it to the desired place where it will be released to act. This technology is used in particular to encapsulate proteins, which can be sensitive to certain environmental factors and degrade (for example, under the action of enzymes or under certain pH conditions), and thus preserve their biological activity and / or prolong their therapeutic effect. It can also be used to delay or prolong the release of proteins through the capsule. Several techniques have been described in the literature for the encapsulation of proteins, leading to systems of microparticle or nanoparticle type, for example based on polymers (Amidi M et al., Advanced Drug Delivery Reviews (2010); 62: 59-82 ), lipid-based systems, such as emulsions, solid lipid nanoparticles or liposomes (Martins S et al., Int J Nanomedicine (2007); 2 (4): 595-607), or virosomes type (Bungener L. et al., Vaccine (2002); 20: 2287-2295) or using virus-like-articles (Patterson DP etal., ACS Chem Biol (2014); 9 (2): 359-365 ). Recently, Sutter et al. havedescribed a family of bacterial proteins, encapsulins, having the ability to self-assemble to form a nano-compartment (or nanoparticle or nanocapsule or "nanocage") In a preferred embodiment, the polypeptide having lactoperoxidase activity is encapsulated in a protein nanocage, more preferably in an encapsulin. The encapsulins are a family of bacterial proteins having the ability to self-assemble to form a nano-compartment .There are several different encapsulin proteins, including EncA, which forms the shell, and EncB, EncC, and EncD, which form the core. Typically, a C-terminal tag is efficient in loading encapsulin with foreign cargo proteins. Encapsulating enzymes within encapsulins provides several advantages. Encapsulins have the ability to enhance the activity and stability of encapsulated enzymes in comparison to their free, non- encapsulated forms. Encapsulation protects the enzymes from proteolytic degradation. Within the encapsulin, the local concentration of substrates is increased, thereby potentially increasing the catalytic rate of encapsulated enzymes. Furthermore, encapsulins have the capability to undergo engineering for precise delivery of encapsulated enzymes by displaying targeting peptides on their outer surface. In one aspect, a polypeptide having lactoperoxidase activity as herein disclosed is encapsulated in an encapsulin such as family 1 encapsulin nanocompartment shell protein from Arthrobacter sp. SLBN-53 (NCBI Reference Sequence: WP_141866254.1; ArthroEnc) or from Dendrosporobacter quercicolus (NCBI Reference Sequence WP_092074293.1; DendroEnc). Other preferred encapsulins include encapsulin originating from the mesothermophile Mycolicibacterium hassiacum (EncMh) (Loncar et al. BBRC, Vol.529, Issue 3, 27 August 2020, Pages 548-553). To that end, the lactoperoxidase polypeptide is provided with a suitable targeting motif as described herein above to allow for targeting to an encapsulin. Polynucleotides and expression in host cells Also disclosed is an isolated polynucleotide encoding a polypeptide according to the invention. The polynucleotide may be comprised in a nucleic acid construct or expression vector, preferably wherein the polynucleotide is operably linked to one or more control sequence(s) that direct the production of the polypeptide in an expression host. Exemplary expression vectors areknown in the art. The vector preferably contains one or more selectablemarkers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. In one embodiment, the vector is an E. coli expression vector. For example, polypeptides can be expressed using a pET- based (IPTG-inducible) vector or a pBAD-based (arabinose inducible) vector. The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous orheterologous to the host cell. The control sequence may also be a leader, anon-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used. The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease {aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB). The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene. Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene and a Bacillus subtilis SP82 gene. A further embodiment of the invention relates to a recombinant host cell comprising the nucleic acid construct or expression vector of the invention encoding a polypeptide as herein disclosed. The recombinant host cell may be a bacterial or fungal host cell. Preferably, the polynucleotide sequence comprised in the host cell is operably linked to one or more control sequence(s) that direct the production of the polypeptide in a bacterial or fungal expression host. In one aspect, the encoding nucleic acid sequence is part of anexpression vector. In another embodiment, the encoding nucleic acidsequence is integrated in the genome of the host cell. For example, it is possible to integrate the encoding gene into the genome of a host organism by methods known in the art, including genome editing methods, homologous recombination, and methods involving the CRISPR Cas system. The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g. a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. In a specific aspect, the host cell is E. coli. For expression with pET- based vectors E. coli BL21, E.coli C41 / C43 or E.coli BL21AI strains can be used, while for pBAD-based vectors E.coli NEB10beta, E.coli TOP10, E.coli, BL21AI and other standard strains be used. In other aspects, the host cell is a fungal host cell, preferably a filamentous fungus, such as Aspergillus sp. In a preferred embodiment, the polypeptide of the present invention is obtained using a host cell allowing for post- translational processing such as glycosylation and / or secretion of the enzyme. For example, an Aspergillus expression system is used. Host cells may be genetically modified to have characteristics that improve genetic manipulation, protein secretion, protein stability and / or other properties desirable for expression or secretion of a lactoperoxidase enzyme. For example, host cells may be modified to contain an enzyme capable of removing a tag sequence that is fused to a polypeptide of the invention. For example, the host cell comprises a vector that encodes not only a SUMO- and His-tagged lactoperoxidase of interest, but also SUMO-tagged Ulp1 protease. Co-expression of these two proteins results in the in vivo cleavage of the enzyme of interest from the SUMO tag, while still leaving the enzyme of interest in a form that can be purified from a soluble cell lysate by nickel affinity chromatography. Also provided is a method of producing a polypeptide having lactoperoxidase activity, comprising (a) cultivating said host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. Suitable media for growing the host of the invention are well known in the art, for example, see Sambrook et al., Molecular Cloning (1989), supra. In general, a suitable media contains all the essential nutrients for the growth of the host system. The media can be supplemented with antibiotics that are selected for host-vector system. An expressed polypeptide can be used in the form of whole cells, permeabilized cells, a cell extract or a cell-free extract comprising an enzyme of the invention. In another embodiment, the enzyme is used in a soluble, immobilized or encapsulated form. Expressed enzyme(s) may be recovered from cells using methods known in the art. Optionally, a protein can be enriched for (e.g., purified or partially purified) using methods well known in the art. For example, the polypeptide may be isolated by conventional procedures including centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, solid phase binding, affinity, hydrophobic interaction, chromatofocusing, and size exclusion chromatography) and / or filtration, or precipitation. Protein refolding steps can be used, as desired, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps. Catalytic oxidation A polypeptide as herein disclosed is suitably used for the catalytic oxidation of an organic or inorganic substrate in the presence of a source of hydrogen peroxide. In one aspect, the catalytic oxidation comprises the oxidation of a halide or pseudohalide substrate, to obtain the corresponding hypohalous acid or pseudohalous acid having antibacterial activity. A hypohalous acid is an oxyacid consisting of a hydroxyl group single-bonded to any halogen. Examples include hypofluorous acid, hypochlorous acid, hypobromous acid, and hypoiodous acid. In some embodiments, fluorine, chlorine, bromine or iodine is oxidized to yield, respectively, hypofluorous acid, hypochlorous acid, hypobromous acid or hypoiodous acid. In another embodiment, thiocyanate is oxidized to hypothiocyanous acid. The source of hydrogen peroxide required by the lactoperoxidase may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide. Any solid entity which liberates upon dissolution a peroxide which is useable by lactoperoxidase can serve as the source of hydrogen peroxide. Compounds which yield hydrogen peroxide upon dissolution in water or an appropriate aqueous based medium include metal peroxides, percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea peroxide, perborates and peroxycarboxylic acids or salts thereof. Any compound which generates a peroxide that lactoperoxidase can use to oxidize a substrate, e.g. halides, is an acceptable source of hydrogen peroxide for this invention. Mixtures of two or more of these compounds can also be used. Another source of hydrogen peroxide is a hydrogen peroxide generating enzyme system, such as an oxidase together with a substrate for the oxidase. Examples of combinations of oxidase and substrate compriseamino acid oxidase (see e. g. US 6,248, 575) and a suitable amino acid,glucose oxidase (see e. g. WO 95 / 29996) and glucose, lactate oxidase and lactate, galactose oxidase (see e.g. WO00 / 50606) and galactose, and aldose oxidase (see e.g. WO 99 / 31990) and a suitable aldose. The source of hydrogen peroxide may be added at the beginning of or during the catalytic reaction process, e. g., typically in an amount corresponding to levels of from 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM, and particularly to levels of from 0.01 to 1 mM. Hygienic compositions and uses thereof Since oxidized products produced by lactoperoxidase have potent bactericidal activities, a polypeptide disclosed herein is advantageously included in or added to a composition or formulation which may benefit from enhanced preservation or conservation. In a further embodiment, the invention provides the use of a polypeptide as herein disclosed as hygienic and / or antibacterial agent. Still further embodiments relate to the use of a polypeptide of the invention as a biocatalyst, preferably as a biocatalyst for the manufacture of a bactericidal or hygenienic agent. Exemplary compositions comprising one or more lactoperoxidase polypeptides as herein disclosed include a personal care product, a cosmetic composition, an oral care composition, a detergent composition, a dairy product, a (vegan) food supplement. One of the advantages of using a bacterial lactoperoxidase of the present invention is that there is no need for product labeling of 'biocide', as the active agent would be classified as an enzyme ingredient, rather than biocide. This facilitates the regulatory route since for biocides the regulation is much more stringent. The composition typically comprises one or more lactoperoxidase(s), a source of hydrogen peroxide, together with one or more substrate(s) to beoxidized in situ into a bactericidal product, such as iodide or thiocyanate. Ina specific aspect, the polypeptide is comprised in a protein cage, preferably in an encapsulin-based protein cage as described herein above. The polypeptide may be formulated in a powder, liquid, spray, tonic, paste, lotion and / or an ointment. The polypeptide can be co- formulated with at least one further agent selected from the group consisting of oils, spreading agents, emulsifiers, ionic compounds, sugars, inorganic compounds, organic compounds, nonionic compounds, amino acids, peptides, lipids, and / or proteins. In one embodiment, the invention provides a toothpaste comprising components forming a lactoperoxidase system and which have inhibitory action against cariogenic oral microflora. For example, it comprises a lactoperoxidase enzyme as herein disclosed, e.g. (encapsulated) CyanoPOX, thiocyanate ions, and hydrogen peroxide generating enzymes like glucose oxidase and amyloglucosidase. This allows the generation of hydrogen peroxide from glucose present in saliva. Also provided is a method for preventing and / or controlling the growth and / or the spreading of a pathogen, said method comprising contacting the pathogen with a polypeptide having lactoperoxidase activity or with a composition as defined herein above. It was found that a bacterial lactoperoxidase as disclosed in the present invention is an effective biofilm inhibitor. In one aspect, the invention provides a method for the prevention, inhibition or suppression of (bacterial) biofilm formation. A biofilm is a structure formed by a community of microorganisms such as bacteria and mold attached to a solid or liquid surface together with secretions. Biofilms generated in household or factory drainage facilities, water circulation systems, and the like cause slimming of pipes, and foul odors. Biofilms also cause equipment degradation, such as sewer pipe corrosion. In the reverse osmosis membranes used in seawater desalination plants, piping facilities in paper mills, etc., adverse effects due to biofilm formation are regarded as problems. In addition, biofilms cause microbial contamination. Biofilm generated in hot spring facilities may cause infections. In the medical field, biofilms formed on medical instruments such as dialysis tubes, endoscopes, and contact lenses may be the source of infection. Biofilm formation in the skin and oral cavity can cause disease. The primary pathogen in all forms of caries has been recognized for many years as S. mutans, a Gram-positive, alpha-haemolytic, facultative anaerobe. In the food field, biofilms formed on food or cooking utensils can cause rot and food poisoning. The biofilm formation inhibitor of the present invention can suppress biofilm formation, e.g. biofilm formation by the bacterium S. downei which is known to play a role in dental plaque formation. According to the present invention, biofilm formation can be safely suppressed with respect to the human body. In addition, according to the present invention, it is possible to prevent biofilm formation without the risk of causing bacterial resistance as in the case of using conventional bactericidal agent or an antibacterial agents. Hence, biofilm formation can be effectively suppressed even when used over a long period of time. In a preferred aspect, the bacterial lactoperoxidase such as the CyanoPOX enzyme or a homolog thereof is used as biofilm inhibitor. More preferably, the biofilm inhibitor e.g. for use in the household or personal care area, is obtained using an Aspergillus sp. expression system. In a specific aspect, the invention provides a (pourable) liquid detergent composition comprising a bacterial lactoperoxidase as herein disclosed. The lactoperoxidase can enhance the hygienization effect of laundry detergents, particularly during wash cycles at lower temperatures (e.g., 20-40°C). The lactoperoxidase may be incorporated in any type of conventional detergent formulation in a hygienically effective amount. Suitably, the enzyme is encapsulated, e.g. through traditional microencapsulation methods or using encapsulin technologies as detailed herein below. Herewith, the invention provides a liquid detergent formulation that maintains a high level of sanitation and prevents biofilm accumulation under energy-efficient, low-temperature washing conditions. It also allows to reduce or eliminate the need for conventional bleach activators such as tetraacetylethylenediamine (TAED). Therefore, in one embodiment the invention provides a (low temperature) liquid laundry detergent composition comprising a bacterial lactoperoxidase as herein disclosed, e.g. CyanoPOX or a functional fragment thereof. A low-temperature laundry care formulation may comprise surfactants, builders, enzymes, and other additives to maintain cleaning power at lower wash temperatures (e.g., 30°C or 40°C). These detergents aim to leverage the benefits of lower energy consumption without compromising stain removal or fabric care. Formulations may include anionic, non-ionic, and potentially cationic surfactants to effectively lift dirt and grime from fabrics. Suitable builders like sodium carbonate, sodium silicate, and sodium citrate help to soften hard water, enhance surfactant performance, and contribute to overall detergency. In addition to the lactoperoxidase(s) of the invention, the laundry care composition may comprise further enzymes. Proteases, amylases, and cellulases, play a crucial role in breaking down protein, starch, and cellulose stains, respectively. They are particularly effective at lower temperatures and can help to combat the reduced cleaning power often seen with lower wash temperatures. In one embodiment, the detergent formulation comprises an enzyme cocktail comprising e.g. a protease, amylase, lipase, mannanase and / or cellulase, and the like. Other additives, such as brighteners, fragrances, and preservatives, can be included to enhance the aesthetics and functionality of the product. The pH of the detergent can affect the effectiveness of enzymes and other cleaning agents. Formulations often include citric acid or other pH adjusters to maintain an optimal range. The detergent composition can be a conventional surfactant-based formulation. For example, it is a formulation designed to avoid Linear Alkylbenzene Sulfonate (LAS) to create a milder formulation for enzyme stability. In some embodiments, the low- temperature laundry detergent is specifically formulated to be environmentally friendly, using biodegradable (biobased) surfactants and / or builders. For example, the lactoperoxidase is included in a biosurfactant- based laundry detergent formulation. LEGEND TO THE FIGURES Figure 1: sequence alignment of the four bacterial sequences selected as putative bacterial lactoperoxidases based on their sequence similarity to thebovine lactoperoxidase. A non-covalently bound b-type heme cofactor issituated in the central core of the CyanoPOX structure (see also Figures 6 and 7). His81 is identified as the distal heme ligand (i) and His294 as the proximal heme ligand (iii). One of the propionate chains of the heme has salt bridges with the side chains of Arg291 (iii) and Arg379 (v). The other propionate chain interacts with Asp84 (i) and backbone nitrogen of Thr86 (i). A conserved calcium ion is observed on the distal site of the heme). Its ligands are sidechains and carbonyl oxygens of Asp82 (i) (adjacent to His81) and Thr140 (ii), side chains of Asp144 (ii), Ser146 (ii) and the carbonyl of Trp142 (ii). Another non-conserved calcium ion (10 Å to His294) is liganded by the sidechain of Asp350 (iv), the carbonyl oxygen and OG1 of Thr295 (iii) (adjacent to His294), the carbonyl oxygens of Glu348(iv) and Lys352 (iv), and two water molecules (Fig.5). Having these calcium ions close to the active site indicates they are structurally and catalytically important. Figure 2: UV-visible spectrum of CyanoPOX with and without H2O2 Figure 3: pH dependence of CyanoPOX activity and thermal stability. 40 mM KI, 0.5 mM H2O2 and 0.4 nM of purified CyanoPOX were used to measure CyanoPOX activity. The melting temperatures of CyanoPOX were measured in different 100 mM citrate and KPi buffers. No Tm values were obtained for the conditions in the moderately acidic pH range (3-4.5). Figure 4: Effect of pre-incubation temperature on CyanoPOX activity.40 mM KI, 0.5 mM H2O2 and 0.4 nM of purified CyanoPOX in 50 mM KPi, pH 7.0 were used to measure CyanoPOX activity after 15 min pre- incubations at defined temperatures. Figure 5: Michaelis-Menten kinetics for CyanoPOX on different substrates Figure 6: Three-dimensional crystal structure of CyanoPOX. Ribbon diagram of the CyanoPOX molecule (single arrow is N-terminus and two arrows represent C-terminus). The heme cofactor is shown in sticks (iron atom is represented as a ball and depicted with a double headed arrow). Ca2+ions are depicted with curved arrows and the Mg2+ion with three arrows. Figure 7: Zoomed view of the active site of CyanoPOX and the calcium binding sites. Active site residues are shown as sticks and are depicted with a single arrow. Other important residues are depicted with double headed arrows. The inset zooms into the calcium binding sites showing ligating residues and metal-ligand bonds as black dashed lines. Figure 8: Encapsulin proteolytic resistance SDS PAGE gels. Figure 9: SDS-PAGE analysis of proteolytic stability of empty DendroEnc, CyanoPOX-loaded DendroEnc and CyanoPOX. Lanes: M - marker. Lane 1 – chymotrypsin (white arrow). Lane 2 – DendroEnc (black arrow). Lane 3 - DendroEnc incubated with chymotrypsin. Lane 4 - DendroEnc CyanoPOX. Lane 5 - DendroEnc CyanoPOX incubated with chymotrypsin. Lane 6 – CyanoPOX (grey arrow). Lane 7 - CyanoPOX incubated with chymotrypsin. EXPERIMENTAL SECTION Example 1: Identification of putative bacterial lactoperoxidase homologs. The bovine lactoperoxidase sequence ((XP_024835317.1) was used as a query and BLAST search against known sequences was used with filtering of bacterial genomes. A selection of 25 sequences were aligned and analyzed, of which four sequences were selected for experimental testing: CyanobacteriumPOX hypothetical protein B7486_19210 [cyanobacterium TDX16] DadabacteriaPOX peroxiredoxin [Candidatus Dadabacteria bacterium] DeltaproteobacteriaPOX peroxidase [Deltaproteobacteria bacterium] PlanctomycetesPOX peroxiredoxin [Planctomycetes bacterium Pla144] Below are the original sequences with underlined parts that were removed as they were identified as a secretion signals using SignalP tool. Figure 1 shows a sequence alignment of the four bacterial sequences. >CyanoPOX MNRRTCSGHSFVRALGVIVVVASSTTAL AQNRTIDGTDNNLSHNVWGSTNQHLDRAGPAAYADGMSMPAGGSRPSARAVSNGIAAQTG SMLNDRMLSDWVWQWGQFLDHDLDLTDAASPAESFPIPVPMGDPFFDPFNTGTQTIGLSR SAYDPATGSVDARQQMNQITSWIDASNVYGSDMTRANALRTMSGGRLATSAGDLLPFNTG GLPNAGGTSPSLFLAGDVRSNEQSGLAAVHTLFVREHNRLADQIAAANPGMGDEDIYQQA RKIVGAQMQIITYNEFLPALLGSAAPSPMSIGYDDSINPNIMNEFANACYRVGHTMLSPT ILRLDNAGNVIPHGNLALQDAFFNPNRIINEGGIAPILKGLASQAMQEIDNKIVDDVRNF LFGPPGSGGLDLASLNIQRGRDHGLPDYNSTRVMMGLTSVSSFADISSDPAVQAALMSLY GTVNDIDLWVGALAEDHLAGSSVGELIAAVLGEQFTRLRDGDRYWYERDDFFVNNPSLLA ELQATRLSDIIRRNSDITNIQDNVFLIPEPATLGLLMFGAAFLRKRRS >DadabacteriaPOX MMNILACFRFALPLLLIGFLVV GCHEDSNSNNDETRSIDGSGNNLQDPLMGATFIELLRLVFSDYADGISEIPEAGLPSARV VSNIVSSQDELIPNTLNASDYVWQWGQFVDHDIDLTDGVNPPEPADIPVPAGDPFFDPLD TGTQVIAFNRSVFDTSTGTGIDNPRQQINKITAWIDASNVYGSDVERAIALRTNDGTGRL NTSAGDLLPFNTEGLPNDGGPDPSLFLAGDVRSNEQVGLTSMHTLFVREHNRYVEELAAE RPGLSGDRLYERGRRFVGALMQAITYNEFLPALLGEGTIPAYNGYNPNVNASIANIFSAA AYRFGHSMLSPEILRLDQNLNVIPEGNLPLLDAFFTPETITDEGGIDPILRGLAKQITQR VDPFIIDAVRNFLFGPPGSGGLDLAALNIQRGRDHGLPKYNDTREQMGLTRVESFQDISS DPEIQMRLEDAFGNVDDIDIWTGGLSEDLVPGSHLGEVFHLIIKIQFEFLRDGDRFWYER KLSGAELQEVQVTQLSDVIRRNTSIGFELQDNVFLVP >DeltaproteobacteriaPOX MKRLISKGNLWFRGLGALLLLFV ANCGNSPAESAAAGEESAAALSGQDSAGAGEGRENAGPEAEAADKTEAPEARSIDGSGNN RHQTLWGSAGIRLERLAPPAYADGVSEPGGAERPNPRAVSNAIVAQDESIPNARGLSAFM FVWGQFLDHDLDLSMTDPNDPFPIEIPLGDPFFDPSGSGEATMAFSRSVFDPETGSSSDK PRQQINALTAWIDGSQVYGSDAARAAWLRSGVGGRLKTSEGDLLPLNDGSQANAPSNSSD FFVAGDLRVNEQTALAAIHTLFVREHNRLAVELQERHPDWDDERLYQEARRWVGAFLQSI TFHEFLPALLGKEAIGPYRGYDPTLNPNILNEFSTAFFRVGHTMLTSEIPLLDENGQSLPSGDLSLQDAFFNVELLKEQGLDPLLRGLMAQAMEEIDSHVVAEVRNFLFGAPGSGGLDLPSLNLQRGRDHGLPDYNTLRVALGLEAVSDFSELSSDPEVQAAFREVYASIDDVDPWIGAL SEDHLEGAGVGPTLHAALLRQFEALRHGDRFWYENDPAFSREDRRRIAATQLSDIIRRNS GVTEAQAKAFEVPR >PlanctomycetesPOX MFSAIHQSPRFPFVASFFVVFFLGSTIAL GENRSIDGTGNNLLNSTWGAAGTNLARMSAPAYDDGISTPRGSSLTLPNPRDVSNMVVAQ AASQPNAHSMTGWVFQWGQFVDHDLDLTGSASPSEPYHISIPAGDPIFDPGDTGTQTMSF NRSNYDTTTGTSVANPRQQINQITSYLDASNVYGSDGARAAALRTLSGGRLKTSAGDLLP LNTLGLANDTGGPADPTQFYVAGDVRANEQVGLTAIHTLFMREHNRLADEIAVANPLWDD EQIYQRARKLVGAQIQAITYQEFLPALLGSAAPDINSVYDPGLNASILNEFSTALYRVGH TMLPQSLMRMQNDGSEAPGGAMELRDAFFLPQNLAATNELEYHLKGLASEVQQDVDMHMV DDVRNFLFGIPVPGGFDLASLNIQRGRDHGLPDYNSMRVAFGLSPKLTFADISSDLTVQT GLQSLYGTVDEIDAWVGALSEDHVVGCQVGELIAAGLVEQFTRARDGDRFWYTRDEELSG DLAWLSSLRLSDIIRLNSGITNLQDHVFFMAVPEPGSLALALLAAVIVPISVRRR Example 2: Cloning, protein expression and purification of CyanoPOX. The CyanoPOX gene of Cyanobacterium sp. TDX16 encoding 556 residues shared 37% sequence identity with bovine LPO was selected for further analysis. The synthetic gene encoding for CyanoPOX was ordered at IDT (Integrated DNA Technologies) and chemicals were purchased from Sigma- Aldrich and Fluorochem. With the assistance of IDT tools, the gene was codon optimized (Table 1) to enable its expression in E. coli.Table 1. Sequence of E. coli codon optimized CyanoPOX gene.Name of the gene Sequence ‘3 - ‘5CyanoPOX-His ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCCTGGTGCCGC GCGGCAGCGCTGCACAGAATCGCACGATCGATGGGACGGATAACAATTT GAGTCATAACGTTTGGGGTTCAACAAACCAACATTTGGATCGTGCAGGA CCCGCGGCTTACGCGGATGGCATGTCCATGCCTGCCGGCGGATCTCGCC CTTCCGCTCGCGCCGTATCGAATGGAATCGCCGCCCAGACTGGTTCAATG CTGAACGATCGCATGTTAAGCGATTGGGTTTGGCAATGGGGGCAGTTCC TGGATCATGACTTAGATCTTACAGATGCGGCGTCCCCCGCTGAATCATTC CCGATCCCTGTCCCCATGGGGGACCCCTTCTTCGACCCCTTCAACACAGG AACCCAGACCATCGGGCTTTCGCGCAGCGCTTATGATCCTGCGACCGGC TCCGTAGATGCGCGCCAGCAAATGAATCAGATCACGTCCTGGATTGATG CTTCGAATGTGTATGGCAGCGACATGACGCGTGCCAACGCATTGCGTAC TATGTCCGGAGGCCGTTTAGCTACTAGTGCTGGTGATCTTTTACCCTTTA ACACCGGTGGTCTTCCCAATGCTGGCGGCACAAGTCCTTCGTTATTCCTG GCTGGTGATGTACGTAGCAATGAGCAATCTGGTTTGGCAGCGGTACACA CATTATTTGTGCGCGAGCACAACCGTCTTGCTGATCAGATTGCCGCCGCC AACCCGGGCATGGGAGATGAAGATATCTATCAGCAGGCGCGTAAGATCG TCGGAGCTCAGATGCAGATTATTACATACAATGAATTTTTGCCAGCCCTG TTAGGTAGCGCTGCGCCTTCGCCGATGAGTATTGGGTACGACGATAGCA TTAATCCAAACATTATGAACGAGTTCGCTAATGCGTGCTATCGCGTAGG ACATACAATGCTTAGCCCAACGATCCTGCGCTTAGATAACGCGGGCAAT GTGATCCCCCACGGCAATTTGGCCTTACAGGATGCATTTTTTAATCCAAA CCGCATTATCAACGAAGGTGGGATTGCTCCTATCTTGAAGGGTTTGGCA AGTCAAGCGATGCAAGAGATCGATAATAAAATCGTCGACGATGTACGCA ATTTCTTATTCGGCCCTCCCGGTAGCGGAGGCTTAGATTTGGCTTCCTTG AATATCCAGCGTGGTCGCGACCACGGGTTGCCCGATTATAATTCAACGC GCGTCATGATGGGATTAACCAGCGTCTCCAGCTTTGCGGATATTTCAAGC GACCCCGCAGTACAAGCAGCATTGATGTCACTTTACGGGACTGTGAATG ATATCGACCTGTGGGTAGGAGCCCTTGCTGAAGATCATTTAGCAGGTTC ATCAGTTGGGGAGTTAATTGCGGCGGTTTTGGGCGAACAGTTCACCCGTT TGCGTGATGGCGATCGCTATTGGTATGAACGTGATGATTTTTTCGTCAAC AATCCATCGTTACTGGCTGAATTGCAGGCCACGCGTCTGAGTGATATTAT TCGTCGTAACTCGGATATTACGAATATTCAGGACAACGTATTCCTTATTC CGGAGCCGGCTACTTTGGGCCTTTTAATGTTCGGAGCAGCGTTTTTGCGT AAACGTCGCTCG CyanoPOX-His-SUMO ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCCTGGTGCCGC GCGGCAGCGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCC AGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTG TCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTT AAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGA CTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCC CTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGA ACAGATTGGTGGTGCACAGAATCGCACGATCGATGGGACGGATAACAAT TTGAGTCATAACGTTTGGGGTTCAACAAACCAACATTTGGATCGTGCAG GACCCGCGGCTTACGCGGATGGCATGTCCATGCCTGCCGGCGGATCTCG CCCTTCCGCTCGCGCCGTATCGAATGGAATCGCCGCCCAGACTGGTTCAA TGCTGAACGATCGCATGTTAAGCGATTGGGTTTGGCAATGGGGGCAGTT CCTGGATCATGACTTAGATCTTACAGATGCGGCGTCCCCCGCTGAATCAT TCCCGATCCCTGTCCCCATGGGGGACCCCTTCTTCGACCCCTTCAACACA GGAACCCAGACCATCGGGCTTTCGCGCAGCGCTTATGATCCTGCGACCG GCTCCGTAGATGCGCGCCAGCAAATGAATCAGATCACGTCCTGGATTGA TGCTTCGAATGTGTATGGCAGCGACATGACGCGTGCCAACGCATTGCGT ACTATGTCCGGAGGCCGTTTAGCTACTAGTGCTGGTGATCTTTTACCCTT TAACACCGGTGGTCTTCCCAATGCTGGCGGCACAAGTCCTTCGTTATTCC TGGCTGGTGATGTACGTAGCAATGAGCAATCTGGTTTGGCAGCGGTACA CACATTATTTGTGCGCGAGCACAACCGTCTTGCTGATCAGATTGCCGCCG CCAACCCGGGCATGGGAGATGAAGATATCTATCAGCAGGCGCGTAAGAT CGTCGGAGCTCAGATGCAGATTATTACATACAATGAATTTTTGCCAGCCC TGTTAGGTAGCGCTGCGCCTTCGCCGATGAGTATTGGGTACGACGATAG CATTAATCCAAACATTATGAACGAGTTCGCTAATGCGTGCTATCGCGTAG GACATACAATGCTTAGCCCAACGATCCTGCGCTTAGATAACGCGGGCAA TGTGATCCCCCACGGCAATTTGGCCTTACAGGATGCATTTTTTAATCCAA ACCGCATTATCAACGAAGGTGGGATTGCTCCTATCTTGAAGGGTTTGGC AAGTCAAGCGATGCAAGAGATCGATAATAAAATCGTCGACGATGTACGC AATTTCTTATTCGGCCCTCCCGGTAGCGGAGGCTTAGATTTGGCTTCCTT GAATATCCAGCGTGGTCGCGACCACGGGTTGCCCGATTATAATTCAACG CGCGTCATGATGGGATTAACCAGCGTCTCCAGCTTTGCGGATATTTCAAG CGACCCCGCAGTACAAGCAGCATTGATGTCACTTTACGGGACTGTGAAT GATATCGACCTGTGGGTAGGAGCCCTTGCTGAAGATCATTTAGCAGGTT CATCAGTTGGGGAGTTAATTGCGGCGGTTTTGGGCGAACAGTTCACCCG TTTGCGTGATGGCGATCGCTATTGGTATGAACGTGATGATTTTTTCGTCA ACAATCCATCGTTACTGGCTGAATTGCAGGCCACGCGTCTGAGTGATATT ATTCGTCGTAACTCGGATATTACGAATATTCAGGACAACGTATTCCTTAT TCCGGAGCCGGCTACTTTGGGCCTTTTAATGTTCGGAGCAGCGTTTTTGC GTAAACGTCGCTCGTAA Golden Gate methodology (Engler et al.2014) was used for cloning the synthetic gene in pBAD His vector (ampicillin resistance). pBAD SUMO vector was created similarly and used only for crystallization purposes.5 µg of the obtained PCR mixture was added to 50 µL RbCl2competent NEB10β E. coli cells. Following an incubation period of 30 min on ice, the cells were heat shocked for 45 seconds at 42 ⁰C and placed on ice once more for another 3 minutes.500 µL of SOC media was used to recover the cells at 37 ⁰C for 45 min. Overnight cultures were prepared in 5 mL LB medium containing 100 µg / mL amp and incubated at 37 ⁰C while being agitated at 135 rpm. 500 mL Terrific Broth medium supplemented with 100 µg / mL amp was used for the expression. Cultures were grown at 37 ⁰C while being agitated at 135 rpm in a baffled flask until an OD600reached ~0.6. Expression of CyanoPOX was commenced by adding L-arabinose (0.02% final concentration). Additionally, 5-aminolevulinic acid (1 mM, final concentration) and iron sulfate (1 mM, final concentration) were introduced into the cultures to facilitate the biosynthesis of heme. Cultures were incubated at 17 ⁰C, 135 rpm for 72 hours and then harvested by centrifugation (3700 rpm, 40 minutes, 4 ⁰C).33 mL lysis buffer (50 mM KPi, 150 mM NaCl, pH 7.5) was used to resuspend cell pellets which were subsequently disrupted by sonication (5 s on 5 s off, 10 minutes, 70 % amplitude). Supernatants were obtained by centrifuging at 12,000 rpm for 45 minutes at 4 ⁰C and afterwards loaded onto Ni Sepharose gravity columns containing 3 mL resin. Columns were washed with a wash buffer (50 mM Kpi, 150 mM NaCl, 20 mM imidazole, pH 7.5) equivalent to five times the column volume. The elution of proteins was performed using 3 mL of elution buffer (50 mM KPi, 150 mM NaCl, 500 mM imidazole, pH 7.5). PD10 columns were employed to substitute the elution buffer for a storage solution buffer (50 mM KPi, 150 mM NaCl, pH 7.5). Concentrations of proteins were measured using the NanoDrop ND 1000 UV-Visible spectrophotometer (ε412 = 58.44 mM–1cm–1) (Deniau et al.2003). The UV-Vis spectra of CyanoPOX were acquired in the range from 250-750 nm at 25 ⁰C using a JASCO V-660 spectrophotometer following the necessary enzyme dilution. Example 3: Characterization of CyanoPOX. Overexpression of the CyanoPOX protein in E. coli NEB10β and subsequent purification yielded about 80 mg of red-colored enzyme from 1 L of culture. SDS-PAGE analysis revealed a distinct protein band at around 55 kDa. The observed molecular weight of this protein corresponded to the expected molecular weight of His-CyanoPOX protein (calculated: 58 kDa). The UV- visible absorbance spectrum of purified CyanoPOX (Fig.2) exhibited maxima at 412 and 280 nm which are characteristic for heme-containing proteins. Also, the so-called visible bands typical for hemoproteins were present in the 500 - 700 nm range. The intensity of the Soret band at 412 nm was slightly lower compared with the absorbance at 280 nm resulting in a Rz value of 0.75 (A412 / A280). Rz values for LPOs are typically close to 1 which may suggest that the CyanoPOX was not fully loaded with the heme cofactor. Incubation of CyanoPOX with 4.0 mM H2O2 resulted in a change in a shift of the Soret band to a higher wavelength and a decrease in intensity (Fig. 2). It showed that CyanoPOX is reactive with hydrogen peroxide. Example 4: Stability of CyanoPOX. The ThermoFluor method (Cummings et al.2006) was used to investigate the thermostability of CyanoPOX. Enzyme concentrations of 1 mg / mL were diluted 5-fold in 100 mM citrate and KPi buffers with a range of pH values from 3.0 to 8.0. ThermoFluor assay was conducted starting at 20 ⁰C and gradually increasing to 95 ⁰C in 1 ⁰C increments every 60 seconds, while monitoring the process with an RT-PCR thermocycler (CFX96, Bio-Rad). Retained activities upon thermal incubation of CyanoPOX were evaluated in a temperature range from 25-90 ⁰C. The enzyme was incubated for 15 minutes in BioRAD T100 thermal cycler, followed by centrifugation using an Eppendorf microcentrifuge 5425 at 12,000 rpm for 2 minutes. Supernatants were collected for enzymatic activity analysis. Data was processed using MS office 2019. As is shown in Figure 3, CyanoPOX demonstrated the highest thermostability under slightly acidic conditions. The highest Tmvalues were obtained at pH 6.0-6.5. Highly acidic conditions (pH < 4.5) appeared to be too harsh for the enzyme as no Tmvalues could be obtained. These results indicate that CyanoPOX exhibits lower thermostability compared to LPO, as the mammalian heme peroxidase representative has the highest Tm value (74 ⁰C) at pH 6 (Ozer 2014). A comparable pH-dependent profile was observed for enzymatic activity (Fig.3). CyanoPOX exhibited the highest activity at pH 6.5. These properties depict resemblance with the data reported for bovine LPO (Ozer 2014). CyanoPOX showed to retain activity upon 15 min incubations up to 50 ⁰C (Fig.4). This is in line with the observed melting temperature and shows that CyanoPOX is a moderately thermostable enzyme. The apparent melting temperatures determined at pH 7.5 and 5.5-6 using ThermoFluor method were as follows: Tm app (CyanoPOX) = 49⁰C – pH 7.5 and 47.5⁰C – pH 5 Example 5: Steady-state kinetics.Reactions using CyanoPOX in 50 mM KPi , 150 mM NaCl, pH 7.0 werecarried out at 25 ⁰C using either ABTS, KI, guaiacol or 2,6-DMP as substrates at different concentrations. Enzyme concentration was adjusted to fit a specific substrate in order to be able to follow the enzyme kinetic in the linear range, while 0.5 mM H2O2 was used in all of the reactions. The pH optimum for CyanoPOX and activity measurements upon thermal incubation were determined through enzymatic activity analysis with KI at 25 ⁰C. Reactions were performed with 40 mM KI, 0.5 mM H2O2 and 0.4 nM of purified CyanoPOX. Kinetic parameters were determined using 50 mM KPi, pH 7.0 and 0.4 nM of purified CyanoPOX. Substrate concentrations were varied and used in combination of 0.5 mM of H2O2(when varying the substrate concentration) or 40 mM KI (when varying the hydrogen peroxide). Formation of oxidized products from the substrates was monitored (ABTS ε420= 36.0 mM–1cm–1(Kenzom et al.2014); KI ε350= 26.0 mM–1cm–1(Ferrari et al.1997); guaiacol ε470= 26.6 mM–1cm–1(Koduri et al. 1995) and 2,6-DMP ε469 = 53.2 mM–1cm–1(Breslmayr et al.2019)) on the BioTek Synergy HTX multi-mode microplate reader. Initial rates of the reactions were obtained from the linear regions of the reaction curves. Data was processed using GraphPad Prism 6.05 (La Jolla, CA, USA). The CyanoPOX substrate profile was evaluated to examine whether it shares similarities to mammalian LPOs. Steady state kinetic parameters were successfully obtained for several substrates (Table 2 and Fig. 5). Initial rates of the reactions were acquired and were fitted using the Michaelis-Menten formula. CyanoPOX demonstrated the highest activity with KI (kcat= 910 s-1) and a relatively low KM value (7.0 mM). A bright yellow color appeared when using this substrate, indicating the formation of hypoiodite (IO-). Furtmüller et al. (2002) in their work report a second order rate constant (1.2 x 108M-1s-1) for bovine LPO with I-. CyanoPOX also showed good activity with other substrates that are typically accepted by mammalian LPOs (Ozer 2014; Kalluri et al.2019; Okazaki et al.2000). Guaiacol and ABTS showed somewhat lower but still high kcatvalues (380 and 570 s-1). However, the catalytic efficiencies for these two substrates are relatively low due to high KM values (>100 mM). High KM value for ABTS seems to be specific for CyanoPOX as this result shows discrepancy with the findings reported by Ozdemir et al. (2001) for bovine LPO. Furthermore, 2,6-DMP exhibited a somewhat low kcat value (620 s-1) when compared to KI. Due to the low KMvalue (35 mM) for 2,6-DMP compared to ABTS and guaiacol, 2,6- DMP has emerged as the second best performing substrate after KI. Having an insight into the kinetic parameters of H2O2for peroxidases is crucial for understanding its enzymatic activity and substrate affinity. CyanoPOX displays a KM value of 0.48 mM for H2O2 which closely resembles the value (0.41 mM) reported for the bovine LPO by Burec et al. (1963). Table 2. Steady-state kinetic parameters of CyanoPOX. Reactions were performed at different enzyme concentrations, adjusted to fit a specific substrate. Activity was assessed by monitoring the formation of oxidized products from the substrates at different wavelengths.Substrate kcat (s-1) KM (mM) kcat / KM (mM-1 s-1) KIb910 7.0 130 2,6-DMPb620 35 18 guaiacolb380 170 2.3 ABTSa570 150 3.7 H2O2c1390 0.48 2900a Reaction was performed in 50 mM KPi , 150 mM NaCl, pH 5.0.b Reactionwas performed in 50 mM KPi, 150 mM NaCl, pH 7.0.cKinetic parameters for H2O2 were obtained through enzymatic activity analysis with KI at different H2O2concentrations. Example 6: Structural elucidation of CyanoPOX. This example describes the elucidation of the crystal structure of CyanoPOX. For crystallization experiments, a SUMO-tagged CyanoPOX expression construct was prepared. The same expression and purification methods as described herein above were used. Purified His-tagged SUMO- CyanoPOX was concentrated with Amicon Ultra 30k 0.5 mL centrifuge filters to an adequate volume. The thus obtained sample was incubated overnight with His-SUMO protease (1 mg / mL). CyanoPOX obtained after SUMO cleavage was further purified by gel filtration using a Superdex 200 HR10 / 30 column (Cytiva), equilibrated with 20 mM HEPES buffer, 150 mM NaCl, pH 7.3 on an Äkta explorer system with wavelengths set at 280, 254 and 412 nm. Tawny brown colored CyanoPOX fractions were pooled and concentrated to 13.7 mg / mL using an Ultracel-30K filter unit (Millipore). Dynamic light scattering (DLS) experiments were performed using a DynaPro MS800TC instrument (Wyatt Technology Corporation) at 294 K. DLS data were processed and analyzed with Dynamics software. Initial sitting-drop crystallization screening was performed using a Mosquito crystallization robot (STP Labtech) in 96-well MRC2 plates (Swissci). Crystals were grown from 0.1 M MES buffer pH 6.0 and 20% PEG6000 supplied with 0.2 M magnesium chloride or 0.2 M calcium chloride. Prior to data collection, crystals were briefly soaked in a cryoprotectant solution containing the crystallization solution supported with 20% glycerol, and flash-cooled in liquid nitrogen. X-ray diffraction data were recorded at the MASSIF-1 beamline at the ESRF, Grenoble (Bowler et al.2015). Automatic data processing, using the program autoPROC with anisotropic analysis (Vornheim et al.2011), was performed at the ESRF. The crystals belonged to space group P21 with cell dimensions of a = 52.7, b = 72.5, c=56.2 Å and β=95.9°. The VM is 1.9 Å3 / Da (Matthews 1968) with a solvent content of 34%, indicating a tightly packed crystal. The structure of CyanoPOX could be determined by molecular replacement using Phaser (Agirre et al.2023) with an AlphaFold2 (Jumper et al.2021) (ColabFold) model. The asymmetric unit contained 1 monomer of 57.9 kDa. Refinement and model building was done using the programs Coot (Emsley et al.2010) and REFMAC5 (Agirre et al.2023). The heme cofactor was built in the Fo – Fcelectron density map and occupancy refinement was done with phenix.refine (Liebschner et al.2019). The quality of the model was analyzed with PDB_REDO and MolProbity (Agirre et al.2023). PyMOL (Schrödinger) was used for figure preparation. Data collection statistics and refinement details are recorded in (Table 3). Atomic coordinates and experimental structure factor amplitudes were deposited in the Protein Data Bank PDB number 8S6C. Table 3. Crystallographic data collection and refinement statistics. CyanoPOX (8S6C) Resolution range (Å)a 55.9 – 1.74 (1.78 - 1.67)Cell dimensions (Å) a, b, c, α, β, γ 52.7, 72.5, 56.2, 90.0, 95.8, 90.0Number of observationsa261877 (12136) Number of unique reflectionsa38817 (1941) Completeness (spherical)(%)a79.4 (22.9) Completeness (ellipsoidal) (%)a91.4 (54.9) Multiplicitya6.7 (6.3) CC1 / 2a0.987 (0.561)Overall I / σ (I)a 7.6 (1.3)Rmerge (%)a0.106 (1.237) Rpim (%)a0.044 (0.534) R / Rfree (%) 14.6 / 18.1Protein residues 525Protein B value (Å2) 24.8Heme molecules 1Heme B values (Å2) 29.7Glycerol molecules 1Ca2+ / Mg2+ ions 2 / 1Water molecules 364r.m.s.d. bonds (Å) 0.007r.m.s.d. angles (°) 1.61Ramachandran outliers 0.4favored 95.6Clashscore 5.3Molprobity score 1.50PDB accession ID 8S6CaValues in parentheses are for the highest resolution shell. The crystal structure of CyanoPOX with a bound heme cofactor was determined at 1.67 Å resolution in the monoclinic space group P21 with one molecule in the asymmetric unit (Fig.6). Size exclusion chromatography with subsequent DLS analysis indicated that CyanoPOX is monomeric in solution with estimated molecular weights of 50 kDa and 34 kDa, respectively. Also, CyanoPOX in the crystal structure seems monomeric, with a size of 55 x 50 x 44 Å (Fig.6). The closest structural homologues of CyanoPOX determined by a DALI search is LPO from Dictyostelium discoideum (Zscore = 48.9, 36% identical on residue level, root-mean-square deviation of 1.8 Å, (PDB 6ERC (Nicolussi et al.2018)). There is also structural homology with LPOs from yak (7DE5) (Viswanathan et al.2021), water buffalo (3ERH) (Sheikh et al.2009), sheep (7VE3) (Singh et al.2022), goat (5FF1) (Singh et al. 2016), bovine (7DN6) (Singh et al.2021) and the Human Myeloperoxidase (MPO) (1CXP) (Fiedler et al.2000), all with ~40% sequence identity, Zscores of 34-46 and rmsd values of ~2.0 Å. The CyanoPOX structure includes residues 1–525. The three C-terminal residues were not visible in electron density. The secondary structure of CyanoPOX is largely α-helical and starts (according to DSSP) with H1(Ala48-Val51), H2(Trp71-Leu83), H2b(Ser146- Tyr149), H3(Met153-Leu159), H5(Ser204-Ala227), H6(Gly233-Leu261, Pro258 conserved), distorted H8(Asn283-Met296), H9(Asn326-Asn330), H10(Ala335-Leu341), H11(Asp355-Asn359), H12(Leu372-Asp382), H13(Tyr388-Met394), 310 H14(Phe403-Asp405), H15 (Pro410-Tyr420), H16(Leu428-Ala434), H17(Glu445-Asp460), H18(Asp470-Val473), H19(Pro476-Gln483), H20(Leu487-Arg493) and hydrophobic H21(Thr512- Leu516). The secondary structural α-helix numbering is according to Sharma et al. (2013) for LPOs. H2a (in LPO) and H2b (in CyanoPOX) are situated at a slightly different position and are therefore called different. The short H4, situated on the surface of LPO, is missing in CyanoPOX. Furthermore, six short β-strands, B1(Ile97-Val99), B2(Thr115-Ile116), B3(Thr300-Ile301), B4(Leu316-Ala317), B5(Phe360-Leu361) and B6(Gly369- Leu370) are present. The sequence of CyanoPOX is shorter than that of mammalian peroxidases. These enzymes contain a ~20 residues longer N- terminus, an insert before H1, the inserted H4 and a ~10 residue longer C- terminus. The cores of the enzymes overlap very well. The Dictyostelium peroxidase has an excursion in the H2a / H2b region. Disulfide bridges and N-glycosylation sites as observed in the eukaryotic peroxidases are absent in CyanoPOX. Interestingly, the last 22 residues at the C-terminus of the CyanoPOX structure have a PEP-CTERM protein-sorting domain motif (Haft et al. 2006). It consists of a highly conserved Pro-Glu-Pro triad (residues 508-510) followed by a hydrophobic α-helix (Thr512-Leu513-Gly514-Leu515-Leu516- Met517) and finally a positively charged segment (524 till C-terminus). It is believed that PEP-CTERM forms a protein export sorting system and may be the recognition sequence for protein-processing functions that could include protein modification, cleavage, sorting, and attachment (Haft et al. 2012). The helical part of the motif is predicted to be transmembrane. However, the six residue α-helix is too short to cross the membrane, usually 18 or more residues are needed for that. To our knowledge this is the first time this motif has been observed in a crystal structure. A non-covalently bound b-type heme cofactor is situated in the central core of the CyanoPOX structure. The occupancy of the heme group was refined to 75%. On the proximal heme site the conserved residues, His294, Leu372, Asn376 and Arg291 are situated. His81 is identified as the distal heme ligand (Figure 7). Other residues close to the distal site of the porphyrin ring are Asn77 and Arg199. One of the propionate chains of the heme has salt bridges with the side chains of Arg291 and Arg379. The other propionate chain interacts with Asp84 and backbone nitrogen of Thr86. The covalent bonds with the methyl groups of the heme observed in mammalian peroxidases to Asp80 and Glu202 (CyanoPOX numbering) are absent, although these residues are conserved. Possibly, the two heme to protein ester bonds have not been formed as they are derived from a hydrogen peroxide-mediated post- translational modification (Singh et al.2008). CyanoPOX did not undergo a peroxide treatment during purification / crystallization. The active site located in the distal heme cavity is easily accessible from the solvent. The hydrophobic entrance is large, about 11 Å in diameter (Fig.7. A conserved calcium ion is observed on the distal site of the heme (11 Å to His81). Its ligands are sidechains and carbonyl oxygens of Asp82 (adjacent to His81) and Thr140, side chains of Asp144 and Ser146 and the carbonyl of Trp142. Another non-conserved calcium ion (10 Å to His294) is liganded by the sidechain of Asp350, the carbonyl oxygen and OG1 of Thr295 (adjacent to His294), the carbonyl oxygens of Glu348 and Lys352, and two water molecules (Fig. 7). Having these calcium ions close to the active site indicates they are structurally and catalytically important. Furthermore, a potential magnesium ion is located close to Met162 and surrounded by 4 water molecules at short distances (2.1-2.2 Å). Example 7: Kinetic parameters and thermal stability of bacterial lactoperoxidases This example demonstrates the catalytic performance of the bacterial lactoperoxidase CyanoPOX (Seq. nr.3) and its homologs DeltaproteobacteriaPOX (Seq. nr.1), DadabacteriaPOX (Seq. nr.2) and PlanctomycetesPOX (Seq. nr.4). Enzyme activities were assessed using potassium iodide (KI) and sodiumthiocyanate (NaSCN) as substrates at pH 5.0 and pH 7.0 (Table 4 ).Across all enzymes and substrates, a higher activity was consistently observed at pH 5.0, indicating a general preference for more acidicconditions. Among the tested lactoperoxidases , CyanoPOX exhibitedthe highest asp80activity with NaSCN, while DeltaproteobacteriaPOX showed better performance with KI, highlighting distinct substrate preferences among these related enzymes. Table 4. Kinetic parameters of bacterial lactoperoxidases Substrate KI (2 mM) NaSCN (0.2 mM)pH 5.0 7.0 5.0 7.0kobs(s-1) CyanoPOX 400 ± 24 118 ± 10 178 ± 10 84 ± 26DeltaproteobacteriaPOX 576 ± 28 160 ± 24 94 ± 9 77 ± 9DadabacteriaPOX 32 ± 6 11 ± 0.5 44 ± 9 10 ± 1PlanctomycetesPOX 304 ± 38 72 ± 4 78 ± 11 41 ± 8Apparent melting temperatures (TM) of the bacterial lactoperoxidases were determined using the ThermoFluor method to assess thermal stability under different pH conditions. All enzymes exhibited TM values around 50^°C, indicating comparable overall thermal stabilities (see table 5). For CyanoPOX, no significant difference in TM was observed between pH 5.0 andpH 7.0, indicating stability across this pH range. In contrast, theCyanoPOX homologs DeltaproteobacteriaPOX, DadabacteriaPOX andPlanctomycetesPOX showed a slight increase in melting temperatures at pH 7.0 relative to pH 5.0, indicating enhanced stability under neutral conditions. Table 5. Thermal stability of bacterial lactoperoxidases Tm(°C) pH 5.0 7.0CyanoPOX 46.0 ± 0 46.0 ± 0DeltaproteobacteriaPOX 47.5 ± 0 54.5 ± 0 DadabacteriaPOX 50 ± 0 52.5 ± 0.7PlanctomycetesPOX 51.5 ± 0.7 52.5 ± 0.7Example 8 : Lactoperoxidase Stabilization y encapsulation inEncMh. This example describes that encapsulation of a bacterial lactoperoxidase in a protein cage shows enhanced resistance to proteolytic degradation. Exemplary enzyme CyanoPOX was encapsulated in an encapsulin originating from the mesothermophile Mycolicibacterium hassiacum (EncMh) essentially according to previously published methods (Loncar et al. BBRC, Vol.529, Issue 3, 27 August 2020, Pages 548-553). CyanoPOX cargo protein was expressed with a N-terminal 6xHis-tag and a C-terminal tag (PPPLPDSEPDREIPADDGSLGIGSLKGTRS) for targeting the enzyme to the interior of EncMh. This C-terminal 30 residues peptide is used by the native DyP of M. hassiacum. Co-expression of EncMh and target enzyme was accomplished by using a two-plasmid system. Co-precipitation of EncMh with cargo protein during PEG precipitation and co-elution upon gelpermeation was taken as proof of successful loading of EncMh. Purified encapsulated CyanoPOX (1 mg / mL) was incubated for 2 or 24 h with bovine trypsin or Proteinase K to investigate the resistance to proteolytic degradation. Trypsin from bovine pancreas (1 mg / mL) was in 50 mM Tris-HCl with 2 mM DTT, while Proteinase K from T. album (10 mg / mL) was in 33.3 mM HEPES pH 7.5 with 1 mM CaCl2. Reactions contained 25 µL of 1 mg / mL encapsulated CyanoPOX and 475 µL of protease solution. Trypsin samples were incubated at 37°C, while Proteinase K samples were incubated at 55 °C. Samples were then analysed by SDS PAGE and Commassie staining (Figure 8). CyanoPOX control and an encapsulin construct without CyanoPOX were also subjected to proteolytic resistance testing in accordance with experimental protocols to serve as a comparative control within the experimental framework. Incubating encapsulated CyanoPOX with proteases and subsequent analysis using SDS-PAGE revealed that encapsulated CyanoPOX resists trypsin but is susceptible to Proteinase K (Figure 8). This is supported by the observation of distinct lower molecular weight bands, approximately 25 kDa in size, which are indicative of trypsin presence. A notable finding is evident from lanes 12 and 13, where the cargo within the encapsulated CyanoPOX construct demonstrates resilience to trypsin treatment, as indicated by the persistent visibility of CyanoPOX bands even after 24 hours of protease exposure. This finding provides further validation of the effective encapsulation achieved within the encapsulated CyanoPOX construct. Example 9: Stabilization of lactoperoxidase by encapsulation in DendroEnc. Chemicals and materials The synthetic gene encoding for DendroEnc was cloned in the pBAD vector (ampicillin resistance) from Twist Bioscience. The synthetic gene encoding for cargo protein (CyanoPOX-cargo was ordered at IDT (Integrated DNA Technologies). Superdex 200 Increase 10 / 300 GL column was purchased from Cytiva. Chymotrypsin from bovine pancreas and other chemicals were purchased from Sigma-Aldrich. Cloning, protein co-expression and purification IDT tools were used to codon optimize the sequence encoding the cargo enzyme CyanoPOX-cargo harboring a N-terminal 6xHis-tag and the C- terminal targeting peptide PPPLPDSEPDREIPADDGSLGIGSLKGTRS), derived from a native dye-decolorizing peroxidase (DyP) of Mycolicibacterium hassiacum to enable their expression in E. coli. The synthetic gene was cloned in pET vector (kanamycin resistance) using the Golden Gate methodology.26 5 µg of the cargo enzyme and encapsulin PCR mixtures were added to 50 µL RbCl2 competent E. coli BL21-AI cells. The cells were left to incubate on ice for 30 min followed by the heat shock for 45 seconds at 42 ⁰C. After the heatshock, the cells were placed on ice for another 3 minutes. 500 µL of SOCmedia was used to recover the cells. They were then placed in the thermal shaker at 37 ⁰C for 45 min, 700 rpm.100 µL of the cell mixture was spread onto the LB agar plates containing 100 µg / mL ampicillin and 50 µg / mL kanamycin. LB agar plates were placed into an incubator at 37 ⁰C overnight. Overnight cultures were grown in 5 mL LB medium that was supplemented with 100 µg / mL ampicillin and 50 µg / mL kanamycin and were incubated at 37 ⁰C, 135 rpm. Terrific Broth medium (500 mL), supplemented with 100 µg / mL ampicillin and 50 µg / mL kanamycin was used for the expression. Cultures were incubated at 37 ⁰C, 135 rpm in baffled flasks until the OD600 reached ~ 0.75. Expression of cargo enzyme and encapsulin was induced by adding IPTG (1 mM final concentration) and L-arabinose (0.02% final concentration).5- aminolevulinic acid (1 mM, final concentration) and iron sulfate (1 mM, final concentration) were added to the cultures containing CyanoPOX cargo in order to facilitate the biosynthesis of heme. Cultures were incubated at 24 °C at 135 rpm for 48 hours. They were then harvested by centrifugation (3700 rpm, 40 minutes, 4 ⁰C). Cell pellets were resuspended in 35 mL lysis buffer (50 mM KPi, 150 mM NaCl, pH 7.5) and were sonicated on ice (5 s on 5 s off, 10 minutes, 70 % amplitude). Cleared cell free extract was obtained by centrifugation at 12,000 rpm for 45 minutes at 4 ⁰C. For precipitation of encapsulin-cargo constructs, equal volumes of cell free extract were mixed on ice with equal volume of 8% PEG-8000, 50 mM KPi pH 7.5, 2 M NaCl (used for DendroEnc constructs). The obtained solution was incubated for 4 hours at 4 ⁰C followed by centrifugation at 12,000 rpm for 45 minutes at 4 ⁰C. The obtained pellet was resuspended in 50 mM KPi, 150 mM NaCl, pH 7.5. The resuspended mixture was selectively loaded onto Ni Sepharose gravity columns containing 3 mL resin in order to remove the excess unencapsulated cargo-enzyme. 3 µl of encapsulins at concentrations 3 to 6 mg / ml, were applied onto a freshly glow-discharged copper R2 / 1300 mesh grid (Quantifoil), blotted for 3 s on both sides with blotting force 0 and plunge-frozen in liquid ethane using the Vitrobot Mark IV system (Thermo Fisher Scientific) at 13 °C and 100% humidity. Datasets were collected using a Talos Arctica transmission electron microscope (Thermo Fisher Scientific) equipped with an XFEG at 200 kV using the automated data-collection software EPU version 2.7 (Thermo Fisher Scientific).2 images per hole with defocus range of -0.5 - -2.5 µm were collected with K3 detector (Gatan) operated in super resolution mode. Image stacks with 50 frames were collected with the total exposure time of 3.5 sec and total dose of 45 e- / Å2. For endroEnc, 4498 micrographs were used for data processing. After motion correction and CTF estimation performed in Relion version 3.1, 108,615 particles were picked in crYOLO28, extracted with box size 440 and pixel size 1.59 in Relion version 3.1, and imported in CryoSPARC.14,526 particles after 2D classification were used for ab-initio 3D refinement with simultaneous sorting into 3 classes. One class with the largest number of particles, 6,765, was used for non-uniform 3D refinement with icosahedral (I) symmetry. These particles were subjected to one more round of 3D refinement with simultaneous sorting into 2 classes. The class with the largest number of particles, 4,549, was used for non-uniform 3D refinement with icosahedral (I) symmetry. The reconstruction and corresponding particle alignments were used for Bayesian polishing in Relion, followed by final non-uniform 3D refinement in CryoSPARC. Proteolytic stability of DendroEnc constructs To assess proteolytic resistance, purified DendroEnc encapsulins (1 mg / mL) obtained after SEC were subjected to a 24 hour incubation with chymotrypsin from bovine pancreas. The protease stock solution was prepared by dissolving bovine chymotrypsin (1 mg / mL) in 100 mM Tris-HCl buffer (pH 7.8) with 10 mM CaCl₂.25 µL of 1 mg / mL encapsulin constructs were mixed with 475 µL of protease solution. Chymotrypsin digestion was performed at 50 °C. Samples were analyzed by SDS PAGE. CyanoPOX was also tested for proteolytic resistance in accordance with experimental protocols in order to serve as a control. Activity of DendroEnc CyanoPOX constructs Reactions with DendroENC CyanoPOX (1 mg / mL) were performed at 25 °C. Enzymatic reaction with guaiacol was tested at 10 mM concentration in a 50 mM KPi, 150 mM NaCl, pH 7.5, while ABTS was tested at 10 mM concentration in 50 mM KPi, 150 mM NaCl, pH 5.0.0.5 mM of H2O2 was used in all of the reactions. Formation of oxidized products was monitored at different wavelengths on the BioTek Synergy HTX multi-mode microplate reader (ABTS ɛ420 = 36.0 mM-1 cm-1 and guaiacol ɛ470 = 26.6 mM-1 cm- 1).22 Reaction rates were determined from the linear regions of the reaction curves. Results This example demonstrates successful packing and folding of CyanoPOX within a 240-mer encapsulin named DendroEnc. Cryo-electron microscopy (CryoEM) was used to obtain 2D class averages of empty and cargo-loaded DendroEnc. This analysis enabled us to visualize the density corresponding to the encapsulated enzyme (Figure 10), providing further confirmation of efficient cargo loading. However, the observed density appeared bulky and unstructured, with no indication of a defined symmetrical positioning of the cargo. Since the symmetry of the cargo might differ from that of the encapsulin shell, we performed shell subtraction to analyse the cargo alone. Further 2D classifications and 3D refinements did not reveal a clear structural organization, suggesting that the encapsulated enzymes do not adopt a fixed symmetrical arrangement within the encapsulin. These findings imply that CyanoPOX may remain in a flexible state inside DendroEnc. To determine whether DendroEnc can protect cargo proteins from proteolytic degradation, we evaluated its ability to shield cargo proteins from protease activity. Incubation of empty DendroEnc and CyanoPOX- loaded DendroEnc with chymotrypsin, followed by SDS-PAGE analysis, revealed no detectable proteolytic cleavage of the encapsulin shell (Figure 11). Additionally, the encapsulated CyanoPOX remained intact after 24 hours of chymotrypsin exposure, whereas non-encapsulated CyanoPOX was completely degraded. These results confirm the protective function of DendroEnc, further validating its ability to effectively encapsulate and safeguard bacterial lactoperoxidase enzymes. Example 10: Inhibition of Biofilm formation This example shows the effect of bacterial lactoperoxidase enzymeon the formation and stability of biofilms. It demonstrates thepotential of bacterial lactoperoxidase enzyme as a biofilm-modulating agent for antimicrobial purposes e.g. in the household care or industrial sector. The capacity of the bacterial lactoperoxidase to prevent biofilm formation by Streptococcus downei DSM5635 (RG1; O84B8N) was evaluated using a 96-well plate assay format. All additions resulted in a final volume of 100 µL per well. 1. Preparation of Control and Enzyme Treatment Conditions: ●Bacterial Inoculum Preparation: A suspension of Streptococcusdownei was prepared in tryptic soy broth (TSB) supplemented with 1% sucrose to achieve a bacterial concentration that, upon addition to the wells, would result in a starting concentration of 1 × 10⁷ CFU / mL in the assay. ●Enzyme Treatment Wells:○ 75 µL of the prepared Streptococcus downei inoculum in TSBwith 1% sucrose was dispensed into designated wells. ○Immediately thereafter, 25 µL of the lactoperoxidase variantsolution was added to these wells to achieve a final enzyme concentration of 0.25 mg / mL. ●Positive Control (Bleach Treatment) Wells:○ 75 µL of the prepared Streptococcus downei inoculum in TSBwith 1% sucrose was dispensed into designated control wells. ○To these wells, 25 µL of a 5% (w / v or v / v, specify if known)sodium hypochlorite (bleach) stock solution was added. This resulted in a final bleach concentration of 1.25% in the well. ●Negative Control / Growth Control Wells:○ wells containing 75 µL of the Streptococcus downei inoculumplus 25 µL of the buffer / vehicle used for the enzyme, without the enzyme itself, to represent maximum biofilm formation 2. Incubation for Biofilm Formation: ●The 96-well plates containing all samples (enzyme treatments andcontrols) were incubated at 37°C for 21 hours in an anaerobic environment to promote biofilm development. 3. Biofilm Quantification and Data Analysis: ●Following incubation, the extent of biofilm formation in each well wasquantified using a suitable staining method (crystal violet staining followed by measurement of absorbance). ●The inhibitory effect of the lactoperoxidase variant was determinedby comparing the biofilm signal in enzyme-treated wells to the signal in the negative control wells. The biofilm inhibition achieved by buffer that was used as a reference point, defined as providing 0% inhibition. MethodsTo assess the efficacy of bacterial lactoperoxidase in preventing biofilmformation within detergent formulations, multiple variants of the exemplary enzyme CyanoPOX were evaluated under several experimentalconditions (Table 6 ). The tested variants included: CyanoPOX expressedin Escherichia coli (CyanoPOX-E. coli), encapsulated CyanoPOX expressed in E. coli (DendroEnc / CyanoPOX-E. coli), and CyanoPOX expressed in Aspergillus (CyanoPOX-Aspergillus).Table 6 . Biofilm prevention (%) by CyanoPOX variants under variousconditions, compared to commercial bleach (89^±^1% inhibition). Biofilm prevention (%) Condition CyanoPOX-E. coli DendroEnc / CyaCyanoPOX- noPOX-E. coli Aspergillus 50 mM HEPES 100 mM NaCl pH 7.0 55.0 ± 1 53.7 ± 0 89.4 ± 350 mM HEPES 52.8 ± 3 58.0 ± 4 88.0 ± 4tap water 50.6 ± 2 63.7 ± 2 88.7 ± 20.2 mM NaSCN 50 mM HEPES 100 mM51.9 ± 12 56.8 ± 5 90.3 ± 4NaCl pH 7.0 0.5 mM H2O250 mM HEPES 100 mM52.7 ± 2 62.1 ± 2 90.0 ± 2NaCl pH 7.0 0.5 mM H2O20.2 mM NaSCN 50 mM55.3 ± 2 58.5 ± 5 90.3 ± 2HEPES 100 mM NaCl pH 7.0 0.2 mM NaSCN tap water 56.9 ± 2 60.8 ± 3 90.4 ± 20.5 mM H2O2 tap water 55.3 ± 9 57.7 ± 10 90.0 ± 4These data demonstrate a notable biofilm prevention efficacy across multiple test systems for (laundry) detergent applications. The enzyme alone exhibited substantial biofilm prevention activity, with the CyanoPOX produced in Aspergillus sp. showing the highest efficacy. This enhanced performance may be attributed to the post-translational modifications inherent to the Aspergillus expression system or auxiliary proteins secreted during production. These results position bacterial lactoperoxidase as herein disclosed as a robust enzymatic system for (e.g. detergent) applications requiring biofilm control. Example 11: Use of Lactoperoxidase in Detergent Formulations for Enhanced Low-Temperature Hygienization and Biofilm Prevention This example demonstrates the utility of CyanoPOX, a representative bacterial lactoperoxidase enzyme as described herein, for enhancing the hygienization effect of laundry detergents, particularly during wash cycles at lower temperatures (e.g., 20-40°C). This addresses the growing consumer demand for energy-saving laundry practices, which reflects a new paradigm of user awareness regarding energy consumption. However, lower wash temperatures can compromise microbial control, leading to poor hygienization and the formation of biofilms within washing machines and on textiles. As demonstrated in Example 10, CyanoPOX exhibits significant biofilm prevention activity. This example further illustrates its application in detergent formulations to maintain high levels of sanitation and prevent biofilm accumulation under energy-efficient, low-temperature washing conditions. The potential to reduce or eliminate the need for bleach activators like tetraacetylethylenediamine (TAED) is also considered. Materials and Methods CyanoPOX Preparation CyanoPOX is prepared according to methods described previously in this application. For certain formulations or to enhance stability against other detergent components like proteases, CyanoPOX may be used in an encapsulated form, either through traditional microencapsulation methods or using encapsulin technologies as detailed elsewhere in this application. Detergent Base Formulations CyanoPOX is incorporated into representative liquid detergent formulations. The following formulations are prepared based on compositions known in the art. Formulation A (Conventional Surfactant-Based) Role in Final % (w / w) Source Component Formulation Example Range Reference Deionized water Solvent To 100 Sodium Laureth Anionic surfactant 9-20Sulfate (LES) Non-ionic (Amidet B-112 Alcohol ethoxylated 1-4 surfactant type as per ) Sodium citrate Chelating agent 2-3 (anhydrous) Foam Coconut Fatty acid 1-2 booster / stabilizer NaOH pH adjustment 0.5-1Protease Enzymatic cocktail 0.15-0.35Amylase Enzymatic cocktail 0.10-0.25Lipase Enzymatic cocktail 0.02-0.07Mannanase Enzymatic cocktail 0.02-0.07Cellulase Enzymatic cocktail 0.02-0.07Preservative (e.g., Preservative 0.2-0.6 Acticide PHE) Note: Formulation A is designed to avoid Linear Alkylbenzene Sulfonate (LAS) to potentially create a milder formulation for enzyme stability. Formulation B (Biosurfactant-Based) Role in Final % (w / w) Source Component Formulation Example Range Reference Deionized water Solvent To 100 Rhamnolipid Anionic 15-20 (Rewoferm RL 100) biosurfactant Sodium citrate Chelating agent 1-3 (anhydrous) Foam Coconut fatty acid 1-2 booster / stabilizer NaOH pH adjustment 1-2 0.10-0.20 (if added Protease Enzymatic cocktail to EC)Amylase Enzymatic cocktail 0.10-0.20Lipase Enzymatic cocktail 0.02-0.06Mannanase Enzymatic cocktail 0.02-0.06Cellulase Enzymatic cocktail 0.02-0.06Preservative (e.g., Preservative 0.2-0.6 Acticide PHE) Note: Formulation B utilizes a biosurfactant known for good compatibility with enzymes and potential for simpler formulations. Incorporation of CyanoPOX and TAED (Optional) ^ CyanoPOX: CyanoPOX (free or encapsulated) is added to Formulation A and Formulation B at a concentration range determined effective for antimicrobial action (e.g., 0.01% - 0.5% w / w). ^ TAED System: o Scenario 1 (TAED combination): CyanoPOX is combined with TAED (e.g., 0.1% w / w) and a source of hydrogen peroxide (either generated in situ by other detergent components or added). o Scenario 2 (Reduced / No TAED): Formulations are tested with reduced levels of TAED or in the complete absence of TAED, relying solely on the CyanoPOX system for hygienization. o Hygienization Efficacy Testing at Low Temperatures Standardized fabric swatches (e.g., cotton, polyester) are inoculated with a representative mixed bacterial culture (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). The swatches are then washed in a laboratory-scale washing machine simulator using a 0.5% dilution of the test detergent (Formulation A or B, with and without CyanoPOX + / - TAED) in water. Wash cycles are conducted at low temperatures (20°C, 30°C, and 40°C) for typical short program durations (e.g., 0.5-1 hour). Control washes are performed with formulations lacking CyanoPOX and / or TAED. After washing, surviving bacteria on fabric swatches are quantified using standard plating techniques (e.g., colony-forming unit counts). Log reduction values are calculated compared to initial inoculum and control washes. Biofilm Prevention Assessment The capacity of the detergent formulations containing CyanoPOX to prevent biofilm formation on typical washing machine surfaces (e.g., plastic, rubber, stainless steel coupons) and on textiles during repeated low-temperature wash cycles is assessed. This assessment is guided by the methodologies and positive biofilm prevention results for CyanoPOX as detailed in Example 10. Coupons are exposed to repeated wash / rinse cycles with inoculated water and the test detergents. Biofilm formation is quantified using methods such as crystal violet staining or viable cell counts from scraped biofilms. Results ^ Enhanced Low-Temperature Hygienization: Detergent formulations containing CyanoPOX (especially in combination with an optimized level of TAED, or potentially without TAED) are expected to show significantly higher log reductions of bacteria on fabric swatches at 20-40°C compared to control formulations lacking CyanoPOX. It is anticipated that complete eradication of bacteria can be achieved even in short laundry programs. ^ Reduced TAED Requirement: It is expected that the inclusion of CyanoPOX will allow for a reduction in the concentration of TAED needed to achieve a target hygienization level, or its complete elimination, thereby reducing cost and chemical load. ^ Biofilm Prevention: Consistent with the findings in Example 10, detergent formulations containing CyanoPOX are expected to significantly reduce or prevent biofilm formation on test surfaces and textiles compared to control detergents during repeated low- temperature wash cycles. ^ Formulation Compatibility: Both conventional (LES-based) and biosurfactant-based (rhamnolipid) formulations can serve as effective matrices for CyanoPOX activity, with the biosurfactant formulation potentially offering enhanced enzyme stability and greener credentials. Encapsulated CyanoPOX may be used to achieve improved stability in the presence of proteases. Conclusion This example demonstrates that the incorporation of CyanoPOX into liquid laundry detergent formulations provides a significant enhancement in hygienization performance, particularly at energy-saving low wash temperatures (20-40°C). Furthermore, CyanoPOX can contribute to the prevention of biofilm formation in washing machines and on textiles. This enzymatic approach allows for the development of effective, sustainable detergents that meet modern consumer demands for energy efficiency without compromising on hygiene, potentially reducing or eliminating the need for conventional bleach activators like TAED and other harsh biocides. The use of tailored formulations, including those based on biosurfactants, can further enhance enzyme performance and the overall sustainability profile of the detergent product.

Claims

Claims 1. A method for the catalytic oxidation of an organic or inorganic substrate, comprising contacting the substrate with a source of peroxide in the presence of a polypeptide having lactoperoxidase activity, wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1- 4 of Figure 1, and comprising the following heme-coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S; iii) RXGH[TS], preferably R[VF]GH[TS]; wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has lactoperoxidase activity.

2. The method of claim 1, wherein the polypeptide comprises a calcium binding site formed by residues corresponding to Asp82, Thr140, Trp142, Asp144 and Ser146 of the amino acid sequence of Seq. no.3 as shown in Figure 1.

3. The method of claim 1 or 2, wherein the polypeptide comprises a C- terminal sorting motif, preferably wherein said sorting motif comprises a Pro-Glu-Pro triad followed by a hydrophobic alpha-helix and a terminal positively charged segment.

4. The method according to any one of claims 1-3, wherein the polypeptide having lactoperoxidase activity displays one or more of the following characteristics: - a KM value for H2O2 of up to 0.6 mM, preferably up to 0.5 mM; - a melting temperature (Tm) of at least 45ºC when assayed at a pH in the range of about 5.5 to 7.5; - a pH optimum in the range of 6 to 7.

5. The method according to any one of claims 1-4, wherein the polypeptide comprises a sequence that has at least 70%, preferably at least 80%, more preferably at least 90% pairwise sequence identity with any one of Seq. no.1-4 of Figure 1, or a fragment thereof that has lactoperoxidase activity.

6. The method according to any one of claims 1-5, wherein the polypeptide comprises a sequence that has at 80%, preferably at least 90%, more preferably at least 95% pairwise sequence identity with the sequence of Seq. no.1 (DeltaproteobacteriaPOX), or a fragment thereof that has lactoperoxidase activity.

7. The method according to any one of claims 1-5, wherein the polypeptide comprises the sequence that has at 80%, preferably at least 90%, more preferably at least 95% pairwise sequence identity with of Seq. no.2 (DadabacteriaPOX), or a fragment thereof that has lactoperoxidase activity.

8. The method according to any one of claims 1-5, wherein the polypeptide comprises a sequence that has at 80%, preferably at least 90%, more preferably at least 95% pairwise sequence identity with the sequence of Seq. no.3 (CyanoPOX), or a fragment thereof that has lactoperoxidase activity.

9. The method according to any one of claims 1-5, wherein the polypeptide comprises a sequence that has at 80%, preferably at least 90%, more preferably at least 95% pairwise sequence identity with the sequence of Seq. no.4 (PlanctomycetesPOX), or a fragment thereof that has lactoperoxidase activity.

10. The method according to any one of the preceding claims, wherein the polypeptide further comprises an N- and / or C-terminal protein tag allowing for enhanced expression, solubilization, purification, encapsulation, targeting, secretion and / or immobilization.

11. The method according to any one of the preceding claims, wherein the polypeptide is a recombinantly produced enzyme, preferably wherein the enzyme is comprised in a cell-free extract, or wherein the enzyme is used as purified, and optionally immobilized.

12. The method according to any one of the preceding claims, wherein the polypeptide is encapsulated in a protein nanocage, preferably in an encapsulin, or in a polymer capsule such as a liposome.

13. The method according to any one of the preceding claims, wherein the catalytic oxidation comprising the oxidation of a halide or pseudohalide to obtain the corresponding hypohalous acid or pseudohalous acid.

14. A composition comprising a polypeptide a polypeptide comprising an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1- 4 of Figure 1, and comprising the following heme- coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S; iii) RXGH[TS], preferably R[VF]GH[TS]; wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has lactoperoxidase activity.

15. The composition of claim 14, wherein the polypeptide comprises a sequence that has at least 90%, preferably at least 95%, more preferably at least 97% pairwise sequence identity with any one of Seq. no.1-4 of Figure 1, or a fragment thereof that has lactoperoxidase activity.

16. The composition according to claim 14 or 15, wherein said composition is a personal care product, a cosmetic composition, an oral care composition, a detergent composition, or a dairy product.

17. The composition of any one of claims 14-16, wherein said polypeptide is co- formulated with at least one further agent selected from the group consisting of oils, spreading agents, emulsifiers, ionic compounds, sugars,inorganic compounds, organic compounds, nonionic compounds, amino acids, peptides, lipids, and / or proteins.

18. The composition of any one of claims 14-17, wherein the polypeptide is formulated in a powder, liquid, spray, tonic, paste, lotion and / or an ointment.

19. The composition according to claim 18, being a liquid laundry care formulation, preferably a low-temperature laundry care formulation.

20. The use of a polypeptide as hygienic and / or antibacterial agent, wherein the polypeptide comprises an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1-4 of Figure 1, and comprising the following heme-coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S; iii) RXGH[TS], preferably R[VF]GH[TS]; wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has lactoperoxidase activity, preferably wherein the polypeptide comprises a sequence that has at least 90%, preferably at least 95%, more preferably at least 97% pairwise sequence identity with any one of Seq. no.1-4 of Figure 1, or a fragment thereof that has lactoperoxidase activity.

21. The use of a polypeptide as a biocatalyst, preferably as a biocatalyst for the manufacture of a bactericidal agent, wherein the polypeptide comprises an amino acid sequence having at least 65% pairwise sequence identity with any one of Seq No.1-4 of Figure 1, and comprising the following heme-coordinating residues / motifs: i) HDLDL[ST]; ii) TX[WY][IL]D[GA]S, preferably T[AS][WY]ID[GA]S; iii) RXGH[TS], preferably R[VF]GH[TS]; wherein X is any amino acid; and(b) a fragment of the polypeptide of (a) that has lactoperoxidase activity, preferably wherein the polypeptide comprises a sequence that has at least 90%, preferably at least 95%, more preferably at least 97% pairwise sequence identity with any one of Seq. no.1-4 of Figure 1, or a fragment thereof that has lactoperoxidase activity.

22. The composition of any one of claims 11-15 or the use of claim 17-21, wherein said polypeptide is comprised in a protein cage, preferably in an encapsulin-based protein cage.

23. A method for preventing and / or controlling the growth and / or thespreading of a pathogen, said method comprising contacting the pathogen with a bacterial polypeptide having lactoperoxidase activity as defined in any one of claims 1-11.

24. The method according to claim 23, comprising the prevention, inhibition or suppression of biofilm formation, preferably biofilm formation on a solid surface.

25. A nucleic acid construct or expression vector comprising apolynucleotide sequence encoding the polypeptide as defined in any one of claims 1-9, the polynucleotide sequence being operably linked to one or more control sequence(s) that direct the production of the polypeptide in a bacterial or fungal expression host.

26. A recombinant host cell, preferably a bacterial or fungal host cell, comprising the nucleic acid construct or expression vector of claim 25.

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