Artificial bifunctional enzyme with myeloperoxidase activity and glucose oxidase activity and application thereof

Abstract

The invention relates to the field of enzymology. More specifically, the invention relates to an artificial bifunctional enzyme and application thereof.

Description

Technical Field

[0001] The present invention relates to the field of enzymology. More specifically, the present invention relates to an artificial bifunctional enzyme and its applications. Background Art

[0002] Heme peroxidases are heme-containing enzymes present in all living organisms, capable of catalyzing the formation of antimicrobial compounds and participating in innate immunity. These peroxidases are divided into two main superfamilies: the first family is present in plants, fungi, and bacteria and may have originated from gene duplication of a single common ancestor gene; the second family is present in mammals and differs from the first family in its primary and tertiary structures as well as its prosthetic group. Regardless of their origin, heme peroxidases can exhibit microbicidal activity, thanks to their ability to halogenate a series of organic compounds in the presence of hydrogen peroxide, which can be used in biomedicine, biotechnology, or the food industry. As an illustrative example, heme peroxidases may be a valuable therapy against bacterial infections (such as those resistant to antibiotics). This is because these enzymes are able to catalyze halides or pseudohalides into (pseudo)hypohalous acids known to exhibit strong bactericidal and antiviral activities.

[0003] Among heme peroxidases, mammalian peroxidases (MMPs) have been found to play an important role in the innate immune system's destruction of invading pathogens. To date, four main types of mammalian peroxidases have been discovered: myeloperoxidase (MPO) expressed in neutrophils, eosinophil peroxidase (EPO) located in eosinophils, lactoperoxidase (LPO) expressed in the mammary gland, salivary gland, and other mucous glands, and thyroid peroxidase (TPO) present in the thyroid gland. The structure of the active site of heme peroxidases is highly conserved. Heme is held in the protein by two covalent bonds through the autocatalytic formation of two ester bonds with aspartic and glutamic acid residues. Myeloperoxidase (MPO) has a third covalent sulfonium bond, which provides unique spectral properties (the Soret band at 428 nm), thereby distinguishing it from its counterparts. This bond is the main cause of MPO's chlorinating activity.

[0004] In nature, peroxidases are usually co-expressed with oxidases, simply because oxidases produce hydrogen peroxide - the substrate of peroxidases. A typical example can be found in fungi, where peroxidases that contribute to biomass degradation are secreted together with oxidases that produce hydrogen peroxide, thus fueling the peroxidases (Abdel-Hamid et al., Adv. Appl. Microbiol. 2013, 82: 1-28; Ander et al., J. Biotechnol. 1997, 53: 115-131).

[0005] From an industrial perspective, a wide range of applications have been achieved based on the combination of peroxidases and oxidases.

[0006] For example, the E-101 solution, which mainly contains porcine MPO as well as glucose oxidase and sodium chloride, has a microbicidal effect upon contact with glucose, thus allowing the disinfection of human or animal injuries (Denys et al., Infect Immun., 2019;87(7):e00261-19). When combined with glucose oxidase in a composition, LPO has been considered as an additive suitable for the preservation of foods such as milk, or as an oral disinfectant (WO2008105113A1; WO2011116052). It has also been reported that hMPO, together with glucose oxidase, glucose, and halides, acts as an anti-viral agent against HIV (Moguilevsky et al., FEBS Lett. 1992;302(3):209–212).

[0007] Assays and biosensors based on the combination of these enzymes have also been developed in order to apply their complementary activities to the measurement of, for example, the concentration of glucose or uric acid in human serum samples (Barham et al., Analyst 1972, 97:142-145; Chun et al., Biochip J. 2014, 8: 218–226; Mundaca-Uribe et al., Sens. Actuators B2014, 195, 58-62).

[0008] However, these types of artificial systems (where the enzymes are independent of each other) may be relatively complex to implement, because they require the determination of experimental conditions that are optimal for both enzymes. In the case of assays and biosensors, the co-immobilization of the two enzymes on a biocompatible surface is also necessary to ensure that they are spatially close enough for sequential catalytic reactions to occur.

[0009] Therefore, there is a need in the art to provide a simplified bifunctional peroxidase-oxidase system that does not compromise the stability, selectivity, and catalytic reactions of the enzymes.

[0010] The present invention meets the above needs in the art by providing an enzyme chimera that, due to its bifunctional myeloperoxidase-glucose oxidase activity, is capable of catalyzing the formation of hydrogen peroxide and (pseudo)halide compounds in a cascade reaction.

[0011] In fact, the inventors herein report for the first time the successful fusion of the open reading frames of glucose oxidase (GOx from Penicillium amagasakiens) and myeloperoxidase (MPO from Rhodopirellula baltica or Homo sapiens). To this end, they genetically engineered various chimeric constructs in which the enzymes were either fused directly end-to-end with each other or via short bridging amino acid sequences. Even without a bridging sequence, the two enzymes expressed in a single open reading frame remained stable and catalytically active. This result was quite unexpected considering that these proteins are both very large components and that enzymes whose catalytic activity is derived from a quaternary structure (such is the case for MPO and GO, each of which requires dimerization to be active) can be easily inactivated by fusion with another enzyme (Ellis et al., ACS Catal. 2019, 9(12):10812-10869). Even more surprising is the fact that the presence of a peptide bridge (referred to herein as a peptide linker) increased the catalytic activity of the chimera, both at the level of glucose oxidase activity and at the level of myeloperoxidase activity. The chimeras of the present invention have also been shown to be microbicidal. Summary of the Invention

[0012] In a first aspect, the present invention relates to a non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

[0013] In a preferred embodiment, the polypeptide of the present invention is a fusion polypeptide comprising myeloperoxidase coupled, preferably covalently coupled, to glucose oxidase.

[0014] In a preferred embodiment, the C-terminus of the myeloperoxidase is coupled, preferably covalently coupled, to the N-terminus of the glucose oxidase.

[0015] In a preferred embodiment, the myeloperoxidase is coupled, preferably covalently coupled, to the glucose oxidase via a linker, preferably via a peptide linker.

[0016] In a preferred embodiment, the linker is a peptide linker comprising or consisting of the following amino acid sequence: (LX1X2X3X4 X5AX6A)m where X1 is glutamic acid or glycine, X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty, X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, and m is an integer from 1 to 2, preferably 1; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0017] In a preferred embodiment, the peptide linker comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8); or a peptide that is substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

[0018] In another preferred embodiment, the peptide linker comprises a polyglycine amino acid sequence (such as those comprising (G) m where m is an integer from 2 to 10), especially GGGGGGGG (SEQ ID NO: 9)) or consists of the same; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0019] In a preferred embodiment, the myeloperoxidase is a microbial myeloperoxidase, such as myeloperoxidase from Rhodopirellula baltica, or a mammalian myeloperoxidase, such as mammalian myeloperoxidase from Homo sapiens.

[0020] In a preferred embodiment, the glucose oxidase is a microbial glucose oxidase, such as glucose oxidase from Penicillium nisikiense.

[0021] In a preferred embodiment, the polypeptide of the present invention is in the form of a functional oligomer or a mixture of functional oligomers.

[0022] On the other hand, the present invention relates to a nucleic acid encoding the polypeptide of the present invention.

[0023] On the other hand, the present invention relates to a vector comprising the nucleic acid of the present invention.

[0024] On the other hand, the present invention relates to a host cell comprising the vector of the present invention.

[0025] On the other hand, a method for obtaining the polypeptide of the present invention is provided, said method comprising at least the following steps: a) culturing the host cell of the present invention in a culture medium under conditions suitable for expressing said polypeptide; and b) recovering said polypeptide.

[0026] On the other hand, an antimicrobial composition is provided, which comprises the non-naturally occurring polypeptide of the present invention.

[0027] In a preferred embodiment, said antimicrobial composition further comprises glucose or a glucose source, and / or a halide or pseudohalide.

[0028] On the other hand, an in vitro use of the non-naturally occurring polypeptide or composition of the present invention is involved, which is for halogenating non-halogenated organic compounds.

[0029] On the other hand, an in vitro or ex vivo use of the non-naturally occurring polypeptide or composition of the present invention is involved, which is for killing microorganisms or inhibiting the growth of microorganisms.

[0030] On the other hand, a non-naturally occurring polypeptide or composition of the present invention is involved, which is used as a drug, preferably for treating microbial infections.

[0031] On the other hand, (i) the non-naturally occurring polypeptide of the present invention and (ii) glucose or a glucose source and / or a halide or pseudohalide are involved, which are used as a combined preparation for simultaneous, separate or sequential use as a drug, preferably for treating microbial infections.

[0032] Another aspect of the present invention relates to a peptide linker, which comprises the following amino acid sequence or consists of the following amino acid sequence: (LX1X2X3X4 X5AX6A)m wherein X1 is glutamic acid or glycine, X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty, X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, and m is an integer from 1 to 2, preferably 1; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0033] In a preferred embodiment, the peptide linker of the present invention comprises any one of the following amino acid sequences or consists of the following: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8); or a peptide that is substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 . Schematic diagram of the reaction catalyzed by the bifunctional MPO-GO polypeptide according to the present invention.

[0035] Figure 2. Catalytic efficiency of the RbMPO-GO chimera (coupled active site) according to the present invention. (A) Catalytic efficiency of the coupled active site for glucose: All chimeras catalyze glucose in vitro, although the efficiency varies depending on the nature of the peptide linker (if present) and the oligomeric state (n≥1). (B) Catalytic efficiency of the coupled active site for chloride: All chimeras catalyze NaCl in vitro, although the efficiency varies depending on the nature of the peptide linker (if present) and the oligomeric state (n≥1).

[0036] Figure 3 . Bactericidal activity and stability of the RbMPO-GO chimera according to the present invention. (A to N). At different concentrations of the chimera (as shown in each figure) and a fixed concentration of glucose (14 mM) and SCN - (25 mM) or Cl - (80 mM), the A values at 400 minutes and 800 minutes were measured under different kinetics. 620nm For all experiments, the kinetics were run at 37°C for 16 hours. After storing the enzyme at 4°C for several days (d), the kinetic experiments were repeated. The data at 400 minutes and 800 minutes were taken from the kinetic curves. The bactericidal activity was measured at 37°C and evaluated in triplicate. DETAILED DESCRIPTION OF THE INVENTION

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, the terms and molecular biology techniques used herein, such as protein chimerization techniques or enzymology techniques, are well known and commonly used in the art.

[0038] The present invention can be more readily understood by reference to the following detailed description, the preferred embodiments of the invention included herein, and the examples included herein.

[0039] The present invention provides an artificial polypeptide that can catalyze the formation of hydrogen peroxide in a cascade reaction, thereby driving the formation of (pseudo)halogenated compounds.

[0040] To this end, in a first aspect, the present invention relates to a non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

[0041] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to an exact amino acid sequence, also known as an amino acid sequence. Thus, these terms include polypeptides of any size, preferably polypeptides of at least 50, 100, 250, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 amino acids, and / or polypeptides that have undergone post-translational modification.

[0042] A "non-naturally occurring" polypeptide as used herein refers to a polypeptide or protein that does not exist in nature (i.e., a non-wild polypeptide). Such polypeptides can typically be the product of human agency (e.g., protein engineering).

[0043] The terms "activity", "function", "biological activity" and "biological function" are equivalent and must be understood as is well known in the art. Preferably, such activity is enzymatic activity. In the context of the present invention, the polypeptide is bifunctional because it exhibits at least two functions or activities, which are myeloperoxidase activity and glucose oxidase activity herein.

[0044] The typical characteristic of "myeloperoxidase activity" is that in the presence of hydrogen peroxide, a halide or pseudohalide is oxidized to (pseudo)hypohalous acid according to the following reaction: where X - represents a halide or pseudohalide.

[0045] The term "halide" refers to an ion of a halogen, which includes chloride (Cl - ), bromide (Br - ), or iodide (I - ), and any combination thereof. The term "pseudohalide" refers to a polyatomic anion that is analogous to a halide in acid-base and redox chemistry, which includes thiocyanate (SCN - ). Halides and pseudohalides are collectively referred to as (pseudo)halides herein.

[0046] Myeloperoxidase activity can be detected according to the protocol described in Section 1.8 of Example 1 below, and / or measured according to the protocols described by Tenovuo et al. (Biochim Biophys Acta, 1986; 870(3): 377-84), Auer et al. (J Biol Chem., 2013; 288(38): 27181-27199) and / or Flemmig et al. (J Biol Chem., 2012; 287(33):27913-23).

[0047] Myeloperoxidase activity can generally be provided by myeloperoxidase. "Myeloperoxidase" refers to an enzyme having myeloperoxidase activity, especially when the enzyme is in the form of a haloenzyme (or holoenzyme), i.e., when the enzyme is bound or complexed with at least its cofactor and optionally an ion. Myeloperoxidase in the form of a haloenzyme is generally bound or complexed with at least heme and optionally calcium. Preferred myeloperoxidases according to the present invention are further described below.

[0048] The typical characteristic of "glucose oxidase activity" is the production of hydrogen peroxide after the oxidation of glucose or a glucose source (such as dextrose or sucrose) according to the following reaction:

[0049] Glucose oxidase activity can be detected according to the protocol described in Section 1.7 of Example 1 below, and / or measured according to the protocols described by Roth et al. (Proc Natl Acad Sci U S A., 2003;100(1):62-7), Courjean et al. (J Biotechnol., 2011; 151(1):122-9) and / or Ciaurriz et al. (J Colloid Interface Sci.,2014; 15;414:73-81).

[0050] Glucose oxidase activity can generally be provided by glucose oxidase. "Glucose oxidase" refers to an enzyme having glucose oxidase activity, especially when the enzyme is in the form of a haloenzyme (or holoenzyme), i.e., when the enzyme is bound or complexed with at least its cofactor and optionally a carbohydrate chain or glycan. Glucose oxidase in the form of a haloenzyme is generally bound or complexed with at least flavin adenine dinucleotide (FAD) and optionally glycosylated (e.g., at the 93rd amino acid position with reference to the numbering of amino acid sequence SEQ ID NO: 12, and the glycosylation is preferably GlucNac). Preferred glucose oxidases according to the present invention are further described below.

[0051] In a preferred embodiment, the polypeptide according to the invention is a fusion polypeptide comprising myeloperoxidase conjugated to glucose oxidase.

[0052] As used herein, the term "fusion polypeptide" means a polypeptide produced by linking together two or more (poly)peptides. To this end, the two or more (poly)peptides are directly or indirectly conjugated to each other. Such conjugation can be carried out by biological or physicochemical means. For example, for biological conjugation, the fusion polypeptide can be the translation product of a chimeric gene construct that links a first DNA sequence encoding a first (poly)peptide to a second DNA sequence encoding a second (poly)peptide so as to form a single open reading frame.

[0053] Here, when the C-terminal end of the myeloperoxidase is conjugated to the N-terminal end of the glucose oxidase, both the myeloperoxidase and the glucose oxidase of the fusion polypeptide according to the invention retain catalytic activity.

[0054] Thus, in a preferred embodiment, the C-terminal of the myeloperoxidase is conjugated to the N-terminal of the glucose oxidase.

[0055] In the context of the present invention, the conjugation between the two enzymes is preferably a stable conjugation for in vitro, ex vivo or even in vivo applications, typically by means of covalent conjugation.

[0056] In a preferred embodiment, the myeloperoxidase, preferably the C-terminal of the myeloperoxidase, is covalently conjugated to the glucose oxidase, preferably the N-terminal of the glucose oxidase.

[0057] As used herein, the terms "covalently conjugated", "covalently bound", "covalently linked", "covalent conjugation", "covalent binding" or "covalent linkage" refer to an interatomic linkage resulting from the sharing of one or more pairs of electrons between two atoms, such as between two or more (poly)peptides. Typical examples of covalent bonds between two or more (poly)peptides include, but are not limited to, peptide bonds (the covalent bonds that typically link amino acids to each other) and bridges (disulfide bridges formed between cysteine chains; or bridges covalently linked through an oxygen atom between lysine and cysteine). Two or more (poly)peptides can be covalently conjugated to each other directly or indirectly. Indirect conjugation means that the two or more (poly)peptides are linked to each other through one or more intermediate binding moieties (such as linkers). Direct conjugation means that two or more (poly)peptides are linked to each other without any intermediate binding moiety (such as a linker).

[0058] Surprisingly, the inventors have demonstrated herein that the direct fusion of said myeloperoxidase with glucose oxidase does not abolish the catalytic activity of each of said enzymes in the resulting polypeptide. Without being bound by theory, the inventors believe that after such fusion, the individual enzymes retain their ability to fold independently of the remainder of the polypeptide chain.

[0059] Thus, in a preferred embodiment, said myeloperoxidase is directly covalently coupled to said glucose oxidase.

[0060] The inventors have further demonstrated herein that the indirect fusion of said myeloperoxidase with glucose oxidase via a linker can enhance the catalytic activity of said enzymes in the resulting polypeptide - this enhancement being compared to the direct fusion of said two enzymes. Without being bound by theory, the inventors believe that after such fusion, the individual enzymes enhance their ability to fold independently of the remainder of the polypeptide chain.

[0061] Thus, in a preferred embodiment, said myeloperoxidase is covalently coupled to said glucose oxidase via a linker.

[0062] "Linker" or "spacer" as used herein refers to a chemical or biological moiety (whether synthetic or natural) capable of coupling two molecules to each other and which can create a spatial separation between said molecules. Chemical linkers are well known in the art and are typically made of polymer chains of different lengths, which can be homobifunctional or heterobifunctional with the same or different reactive groups and contain at least one atom, preferably at least one carbon atom. In contrast, biological linkers are typically made of nucleic acids and / or amino acid sequences of different lengths and contain at least one nucleic acid and / or amino acid. Such linkers and methods for coupling molecules (especially proteins) have been widely described in the literature, particularly in Chen et al. (Adv Drug Deliv Rev, 2013; 65(10):1357 - 1369) and Thermo Scientific: Bionconjugation and crosslinking technical handbook (2018), and thus those skilled in the art can easily make a selection and design.

[0063] In the context of the present invention, biological linkers are particularly preferred, especially peptide linkers.

[0064] "Peptide linker" or "peptide-based linker" refers to a biological linker as defined above and composed of amino acid sequences of different lengths. As a non-limiting indicative range, the length of the peptide linker can be from about 2 amino acids to about 50 amino acids. Preferred peptide linkers according to the present invention are those peptide linkers whose sequence length comprises from about 3 to about 35 amino acids, preferably from about 3 to 35 amino acids, more preferably from about 8 to about 18 amino acids. Peptide linkers are well known in the art (Chen et al., Adv Drug Deliv Rev, 2013; 65(10):1357-1369, which is incorporated herein by reference in its entirety, especially Table 3), and can generally be characterized as rigid, semi-rigid, flexible or cleavable. Rigid peptide linkers exhibit a relatively rigid structure, usually having a helical structure or being rich in proline. Amino acids such as proline, arginine, phenylalanine, glutamic acid and glutamine are usually present in rigid linkers. Proline may also contribute to the formation of a hinge in the structure. Examples of rigid peptide linkers include, but are not limited to, peptide linkers forming an α-helix, such as A(EAAAK)mA (m is an integer from 2 to 5) (SEQ ID NO: 1), the proline-alanine linker PAPAAP (SEQ ID NO: 2) and the polyproline linker (P)m (m is an integer from 2 to 8). Semi-rigid linkers are peptide linkers having limited flexibility, i.e., having a structure that is not rigid but not completely flexible. Examples of semi-rigid linkers are described in WO2010080424A1 (which is incorporated herein by reference in its entirety). Flexible peptide linkers allow the coupled molecules to move freely relative to each other; such linkers are usually rich in small, non-polar or polar amino acids, such as glycine and / or serine. Examples of flexible peptide linkers include, but are not limited to, polyglycine (G)m (m is an integer from 2 to 10) and the glycine-serine linker (GGGGS)m (m is an integer from 2 to 5) (SEQ ID NO: 3).

[0065] Rigid, semi-rigid and flexible linkers are stable in vivo and thus do not allow the coupled (poly)peptides to separate. On the other hand, cleavable peptide linkers are susceptible to reduction or enzymatic cleavage; examples of such linkers include disulfide bridges or protease cleavage sites. Those skilled in the art will readily understand that cleavable peptide linkers are not suitable for the present invention. In a preferred embodiment, the linker used in the present invention is not a cleavable peptide linker. Thus, the peptide linker can be a rigid, semi-rigid or flexible peptide linker.

[0066] The following peptide linkers are particularly suitable for the fusion polypeptides according to the present invention.

[0067] In a preferred embodiment, the peptide linker comprises or consists of the following amino acid sequence: (LX1X2X3X4 X5AX6A)m wherein X1 is glutamic acid or glycine, X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty, X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, and m is an integer from 1 to 2, preferably 1; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0068] Two amino acid sequences are "homologous", "substantially homologous" or "substantially similar" when one or more amino acids are replaced by one or more biologically similar amino acids, or when at least about 80% of the amino acids are identical between two sequences, or at least about 90%, preferably at least about 95%, still preferably at least about 96% or 97%, more preferably at least about 98% or 99% of the amino acids are identical, but the two sequences still exhibit the same (or substantially the same) essential structure (e.g., tertiary, quaternary, rigid, semi-rigid, flexible, cleavable, etc.) and / or the same (or substantially the same) essential biological activity. In other words, two homologous amino acid sequences are said to be functional. Compared to a reference amino acid sequence, a homologous amino acid sequence may typically contain, for example, silent mutations, conservative substitutions or minor deletions of genetic material that do not affect (or do not substantially affect) the structure or biological activity of the reference sequence. Similar or homologous sequences can be identified by alignment using algorithms well known in the art. For example, the best alignment of sequences can be performed by a global homology alignment algorithm, such as the algorithm described by Needleman and Wunsch (Journal of Molecular Biology, 1970, 48(3): 443–53) or by a computer implementation of this algorithm. Global homology alignment may be preferred if sequences of the same or similar length are used for alignment. Preferably, the percentage identity can be calculated over the entire length of the reference sequence.

[0069] As used herein, "conservative substitution" means replacing one amino acid (or corresponding codon) with another amino acid (or corresponding codon) without changing the overall conformation and / or function of the reference (poly)peptide (or corresponding nucleic acid), including but not limited to replacing one amino acid (or corresponding codon) with an amino acid (or corresponding codon) having similar properties (such as polarity, hydrogen bond-forming potential, acidity, basicity, shape, hydrophobicity, aromaticity, etc.). Amino acids having similar properties are well known in the art. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and may be interchangeable. Similarly, the hydrophobic amino acid isoleucine can be replaced with leucine, methionine, or valine. Neutral hydrophilic amino acids that can be replaced with each other include asparagine, glutamine, serine, and threonine.

[0070] By "substitution" or "modification", the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

[0071] Therefore, it should be understood that in the context of the present invention, conservative substitution is considered in the art to be replacing one amino acid with another amino acid having similar properties. Examples of conservative substitutions are set forth in Table 1 below.

[0072]

[0073] Alternatively, conservative amino acids can be grouped as described in Lehninger, 1975, as set forth in Table 2 below.

[0074]

[0075] As another alternative, exemplary conservative substitutions are set forth in Table 3 below.

[0076]

[0077] In another preferred embodiment, the peptide linker of the present invention comprises any one or consists of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7), or LEKRPEAEA (SEQ ID NO: 8); or a peptide that is substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

[0078] The peptide linkers (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7), and LEKRPEAEA (SEQ ID NO: 8) can be substantially characterized as rigid peptide linkers, while the peptide linkers LEGGEAEA (SEQ ID NO: 4) and LGKRGAGA (SEQ ID NO: 5) can be substantially characterized as semi-rigid peptide linkers.

[0079] In another preferred embodiment, the peptide linker of the present invention comprises or consists of a polyglycine amino acid sequence, such as comprising (G)m (where m is an integer from 2 to 10), particularly GGGGGGGG (SEQ ID NO: 9); or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0080] The peptide linkers (G)m (where m is an integer from 2 to 10) and GGGGGGGG (SEQ ID NO: 9) can be substantially characterized as flexible peptide linkers.

[0081] In the context of the present invention, rigid and semi-rigid peptide linkers are particularly preferred. Particularly preferred examples of such peptide linkers are LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), and (LEKREAEA)2 (SEQ ID NO: 6).

[0082] In a preferred embodiment, the myeloperoxidase is a microbial myeloperoxidase, such as myeloperoxidase from Rhodopirellula baltica, or a mammalian myeloperoxidase, such as mammalian myeloperoxidase from Homo sapiens.

[0083] Natural myeloperoxidase and polypeptides that are substantially homologous thereto are covered herein.

[0084] For example, a particularly preferred myeloperoxidase according to the present invention comprises or consists of the native amino acid sequence SEQ ID NO: 10 of Rhodopirellula baltica, or a polypeptide that is substantially homologous thereto, preferably derived from SEQ ID NO: 10 by one or more conservative substitutions.

[0085] As another example, a particularly preferred myeloperoxidase according to the present invention comprises or consists of the native amino acid sequence SEQ ID NO: 11 of Homo sapiens, or a polypeptide that is substantially homologous thereto, preferably derived from SEQ ID NO: 11 by one or more conservative substitutions.

[0086] Conservative substitutions can in particular be introduced into non-critical amino acids or non-critical regions.

[0087] In fact, amino acids crucial for the biological activity of the preferred myeloperoxidase of the present invention have been identified. They include the amino acids at positions 199, 202, 203, 316, 317, and 407 of the reference amino acid sequence SEQ ID NO: 10, or the amino acids at positions 257, 260, 261, 408, and 502 of the reference amino acid sequence SEQ ID NO: 11. More particularly, these amino acids are Q199, D202, H203, E316, N317, and H407 in SEQ ID NO: 13, or Q257, D260, H261, E408, M409 in SEQ ID NO: 11.

[0088] Thus, in a preferred embodiment, the myeloperoxidase comprises or consists of the native amino acid sequence SEQ ID NO: 10 of Pycnoporus sanguineus; or is a polypeptide substantially homologous thereto, preferably derived from SEQ ID NO: 10 by one or more conservative substitutions, provided that the following amino acids are conserved: Q199, D202, H203, E316, N317, and H407.

[0089] In another preferred embodiment, the myeloperoxidase comprises or consists of the native amino acid sequence SEQ ID NO: 11 of Homo sapiens; or is a polypeptide substantially homologous thereto, preferably derived from SEQ ID NO: 11 by one or more conservative substitutions, provided that the following amino acids are conserved: Q257, D260, H261, E408, M409.

[0090] In a preferred embodiment, the glucose oxidase is a microbial glucose oxidase, such as the glucose oxidase from Penicillium nisikaze.

[0091] This document encompasses native glucose oxidase and polypeptides substantially homologous thereto.

[0092] For example, a particularly preferred glucose oxidase according to the present invention comprises or consists of the native amino acid sequence SEQ ID NO: 12 of Penicillium nisikaze; or is a polypeptide substantially homologous thereto, preferably derived from SEQ ID NO: 12 by one or more conservative substitutions.

[0093] As explained above, myeloperoxidase and glucose oxidase are each known in the art to require oligomerization to have catalytic activity. This is why the inventors have evaluated different oligomeric states of the polypeptides according to the present invention herein.

[0094] In a preferred embodiment, the polypeptide according to the present invention is in the form of a functional oligomer (n≥1) or a mixture of functional oligomers (n≥1).

[0095] More specifically, the polypeptide according to the present invention can be in the form of a functional monomer, a functional multimer, or a mixture thereof.

[0096] "Oligomer" or "oligomeric state" as used herein refers to the structural units that make up an oligomeric polypeptide. The number of these structural units (n) (also referred to as the degree of oligomerization) can be equal to or greater than 1 (n≥1). When n is greater than 1, the structural units are typically linked together covalently or non-covalently. n is usually less than one hundred, and typically less than thirty. An oligomer with n = 1 is referred to as a monomer or a single unit, while an oligomer with n>1 can be referred to as a multimer or a multi-unit. A monomer or a single unit usually consists of one polypeptide (or polypeptide chain) herein, while a multimer or a multi-unit usually consists of at least two polypeptides (or polypeptide chains). Oligomers with gradually increasing lengths are referred to as dimers (n = 2), trimers (n = 3), tetramers (n = 4), pentamers (n = 5), hexamers (n = 6), heptamers (n = 7), octamers (n = 2), nonamers (n = 9), decamers (n = 10), etc.

[0097] Although different oligomers of the polypeptide according to the present invention may exhibit different catalytic efficiencies, they all basically retain their functions because they all exhibit myeloperoxidase activity and glucose oxidase activity. If desired, those skilled in the art can select oligomers with the desired levels of myeloperoxidase and glucose oxidase activities. To reduce production time and cost and improve production yield, it may also be desirable to make the polypeptide according to the present invention preferably in the form of a mixture of functional oligomers.

[0098] The method for preparing the polypeptide according to the present invention is described below.

[0099] The polypeptide according to the present invention can be encoded by nucleic acid.

[0100] "Nucleic acid" or "nucleotide sequence" as used herein refers to the exact sequence of natural nucleotides (i.e., A, T, G, C, and U) or unnatural nucleotides. These terms cover single-stranded or double-stranded DNA, as well as the transcription products of said DNA, such as RNA.

[0101] Thus, in another aspect, the present invention relates to a nucleic acid encoding the polypeptide described herein.

[0102] Like the polypeptide it encodes, the nucleic acid is non-naturally occurring.

[0103] The nucleic acids of the present invention can be prepared by methods well known in the art, including but not limited to any synthetic and / or recombinant methods. The nucleotide sequences of the nucleic acids are designed to allow for efficient production of functional polypeptides, for example, by codon optimization based on the desired expression system (such as the host cell), within the skill of those in the art.

[0104] The nucleic acids according to the present invention can advantageously be incorporated into a vector for amplifying the nucleic acid or expressing the polypeptides of the present invention in a host cell.

[0105] Accordingly, another aspect of the present invention is to provide a vector comprising the nucleic acid disclosed herein.

[0106] The vector can advantageously be incorporated into a host cell such as a prokaryotic or eukaryotic cell. Thus, the vector can be a prokaryotic or eukaryotic vector.

[0107] Accordingly, the present invention also relates to a host cell comprising the vector of the present invention.

[0108] The term "vector" generally refers to a tool that can be used to perform molecular biology and gene recombination procedures. Such tools are commonly used and well known in the art. The term encompasses vectors that are capable of replication for amplifying a nucleic acid of interest (i.e., cloning vectors), or expressing a polypeptide encoded by the nucleic acid in a host cell (i.e., expression vectors). These types of vectors are publicly available and include but are not limited to plasmids, cosmids, YACs, BACs, viral vectors (adenovirus, AAV, retroviruses such as lentivirus, EBV episomes, etc.), and phage vectors. Since the vector does not exist in nature in association with the nucleic acid of the present invention (i.e., it is not naturally occurring), it is referred to herein as being recombinant.

[0109] Methods for inserting nucleic acids into vectors are known to those skilled in the art. Generally, techniques well-known in the art can be used to insert nucleic acids into one or more restriction endonuclease sites (see, for example, the techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, 2012). Preferably, the vector also contains nucleotide sequences that allow transcription of the nucleic acid, expression of the protein encoded by the nucleic acid, and / or purification. These sequences include (but are not limited to) at least one sequence selected from the following: one or more signal peptide sequences, origin of replication, one or more gene marker selections, enhancer elements, promoters, transcription terminators, and sequences that may allow purification of the protein. Insertion of such sequences into the vector can be carried out by standard ligation techniques known to those skilled in the art, such as the techniques mentioned above. Those skilled in the art also know that these nucleotide sequences can be selected according to the host cell in which the vector is to be replicated and / or in which the polypeptide encoded by the nucleic acid is to be expressed.

[0110] For example, depending on the origin of replication selected, the vector can replicate in one or more host cells: the origin of replication of plasmid pBR322 is generally suitable for most Gram-negative bacteria, the origin of replication of plasmid 2μ is generally specific for yeast, and various viral origins of replication (SV40, polyomavirus, adenovirus, VSV, or BPV) are particularly useful for cloning vectors in mammalian cells.

[0111] As another example, depending on the promoter selected, the nucleic acid can be transcribed and the corresponding polypeptide expressed in one or more host cells: the promoters T7, Lac, trp, tac, λPL are generally specific for Escherichia coli; the promoters PHO5, GAP, TPI1, ADH are generally suitable for yeast; the polyhedrin and P10 promoters and their equivalents are generally used for insect cells; and finally, the promoters CMV, MT1, SV40, SRα, retroviral promoters, and heat shock protein gene promoters are particularly suitable for mammalian cells.

[0112] Non-exhaustive examples of selectable marker genes commonly included in vectors are genes that confer resistance to antibiotics or toxins (such as ampicillin, neomycin, G418, hygromycin, kanamycin, tetracycline, chloramphenicol, or combinations thereof), and genes that allow compensation for auxotrophic defects (such as the gene encoding dihydrofolate reductase DHFR that allows resistance to methotrexate, or the TPI gene of Schizosaccharomyces pombe).

[0113] Non-exhaustive examples of nucleotide sequences that allow purification of polypeptides are histidine sequences (histidine tags or Hisx6), FLAG sequences, and GST sequences. A cleavage sequence for a protease (such as VTE) may further be present in order to subsequently remove the purification sequence.

[0114] Non-exhaustive examples of prokaryotic vectors are: pET (Novagen), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, and pRIT5 (Pharmacia).

[0115] Non-exhaustive examples of eukaryotic vectors are pWLNEO, pSV2CAT, pPICZ, pcDNA3.1(+)-Hyg (Invitrogen), pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pCI-neo (Stratagene), pMSG, pSVL (Pharmacia), and pQE-30 (QLAexpress).

[0116] In a preferred embodiment, the vector of the present invention is a prokaryotic vector, preferably a pET vector, such as the pET21a vector.

[0117] The terms "host cell", "cell", and "cell line" as used herein may be used interchangeably and refer to a prokaryotic or eukaryotic cell into which the vector of the present invention can be introduced, for example, to amplify the nucleic acid as described above and / or to express the polypeptide encoded by the nucleic acid. For this purpose, the host cell can be "transfected" or "transformed" by methods known in the art for transferring or introducing the vector into the host cell. Examples of such methods include, but are not limited to, electroporation, lipofection, calcium phosphate transfection, transfection using DEAE-dextran, microinjection, and particle bombardment.

[0118] The choice of the host cell generally depends on the selected use, i.e., cloning of the nucleic acid or expression of the polypeptide encoded by the nucleic acid. A person skilled in the art will be able to select a suitable host cell from among many cell lines that are publicly available (especially through the American Type Culture Collection (ATCC)).

[0119] Examples of prokaryotic cells include, but are not limited to, bacteria such as Gram-negative bacteria of the genera Escherichia (e.g., Escherichia coli BL21, C41, RR1, LE392, B, X1776, W3110, DH5α, JM109, KC8), Serratia, Pseudomonas, Erwinia, Methylobacterium, Rhodobacter, Salmonella, and Zymomonas, and Gram-positive bacteria of the genera Corynebacterium, Brevibacterium, Bacillus, Arthrobacter, and Streptomyces.

[0120] Examples of eukaryotic cells include, but are not limited to, cells isolated from fungi, plants, and animals. Such cells include, in particular, but are not limited to, yeasts such as yeasts of the genus Saccharomyces, cells from fungi such as cells of the genera Aspergillus, Neurospora, Fusarium, or Trichoderma, animal cells such as HEK293 cells, NIH3T3, Jurkat, MEF, Vero, HeLa, CHO, W138, BHK, COS, COS-7, MDCK, C127, Saos, PC12, HKG, and insect cells such as Sf9, Sf21, Hi Five™, or cells of the silkworm (Bombyx mori).

[0121] In a preferred embodiment, the host cell of the present invention is a prokaryotic cell, preferably a cell of the genus Escherichia, more preferably Escherichia coli, such as Escherichia coli BL21 (e.g., BL21 Star (DE3)).

[0122] In the present invention, the inventors have recombinantly produced the polypeptide of the present invention in high yield while still allowing proper refolding of the polypeptide.

[0123] According to another aspect, the present invention relates to a method for obtaining the polypeptide of the present invention, the method comprising at least the following steps: a) culturing the host cell of the present invention in a medium under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide.

[0124] The host cell used in the method is preferably as described above.

[0125] In a preferred embodiment, the host cell is a prokaryotic cell, preferably a cell of the genus Escherichia, more preferably Escherichia coli, such as Escherichia coli BL21 (e.g., BL21 Star (DE3)).

[0126] In step b), if the polypeptide is expressed intracellularly, the polypeptide can be recovered from the host cell, and / or if the polypeptide is expressed extracellularly, the polypeptide can be recovered from the culture medium of the host cell.

[0127] One skilled in the art can use any conventional method that allows the recovery of the polypeptide. For example, if the polypeptide is expressed in a soluble form in the host cell, the host cell can be recovered by centrifugation and suspended in a buffer, and then the cell can be disrupted, for example, by an ultrasonic homogenizer (sonication) or a cell disruptor (optionally in combination with urea treatment) to obtain a cell-free extract.

[0128] In the context of the present invention, it may be necessary to refold the solution to ensure that the recovered polypeptide is fully folded and functional, i.e., in the form of a haloenzyme. To this end, the polypeptide recovered in step b) can be advantageously dissolved in a solution containing heme and FAD (or a functional derivative of heme or FAD) and optionally calcium. Heme and FAD are in fact cofactors of myeloperoxidase and oxidase, respectively.

[0129] The polypeptide recovered in step b) can be advantageously purified in another step of the method (defined as step c)). Preferably, the purification step allows the obtaining of a 100% pure or almost 100% pure polypeptide.

[0130] One skilled in the art can use any conventional method that allows the purification of the polypeptide. For example, if the polypeptide is recovered in the cell-free extract as detailed above, a purified sample can be obtained from the supernatant obtained by centrifuging the extract using conventional methods or a combination of conventional methods to isolate and purify the polypeptide of the present invention. These methods include, but are not limited to, solvent extraction, ammonium sulfate salting out, desalting, dialysis precipitation, filtration, ultrafiltration, organic solvents, preparative electrophoresis, isoelectric focusing, various chromatographic methods such as ion exchange chromatography (anion, using resins such as diethylaminoethyl (DEAE) agarose; or cation, by using resins such as S-Sepharose (Pharmacia)), hydrophobic chromatography (using resins such as butyl agarose or phenyl agarose), size exclusion chromatography, affinity chromatography using antibodies, adsorption chromatography, chromatofocusing, high performance liquid chromatography (HPLC) and reverse phase HPLC, and any combination thereof.

[0131] In the case of using chromatography in step c), one or more sub-steps can be carried out, including but not limited to binding the obtained polypeptide to a solid support such as a chromatography column, a washing step, and an elution step. The sub-steps can be repeated multiple times as needed to achieve the desired degree of purification of the polypeptide.

[0132] To separate different oligomeric forms of the polypeptide, a size exclusion chromatography column may be preferred. On the other hand, if a person skilled in the art wishes to reduce production time and cost, a desalting column can be used to ensure the separation of the polypeptide in the form of a mixture of all functional oligomers.

[0133] Then the polypeptide recovered in step b) or purified in step c) can be dissolved in a suitable buffer. According to the present invention, a particularly preferred dissolution buffer is Tris buffer at pH 7.5, preferably Tris pH 7.5 CaCl2.

[0134] Examples of methods for obtaining the polypeptide of the present invention are described in section 1.4 of Example 1 below and in Eggenreich et al. (Biotechnology Reports, 2016, 10: 75 - 83). Such methods can especially allow the production of the polypeptide in high yields. As reported in the examples below, the inventors of the present invention obtained from about 40 mg to about 600 mg of the polypeptide of the present invention per liter of cultured host cells.

[0135] Due to the ability of the polypeptide cascade of the present invention to catalyze the formation of (pseudo)hypohalous acid from hydrogen peroxide and (pseudo)halide (the latter is known to exhibit strong bactericidal and antiviral activities), the polypeptide of the present invention can be used as an antimicrobial agent.

[0136] Therefore, another aspect of the present invention is to provide an antimicrobial composition comprising the non-naturally occurring polypeptide described herein.

[0137] The term "antimicrobial" as used herein refers to killing microorganisms or inhibiting the growth of microorganisms, and thus encompasses the terms "bactericidal", "bacteriostatic", "virucidal", "virustatic", "fungicidal", "fungistatic", "parasiticidal", and "parasitostatic". Examples of microorganisms that can be killed or whose growth can be inhibited according to the present invention will be further detailed below.

[0138] Other components may be included as needed. These components may be provided in a single composition or may be split into binary compositions according to the needs of a particular application for mixing at a later time before use. In fact, those skilled in the art will understand that one of these components may be left out and provided separately from the polypeptide (thus as a binary composition) to prevent premature reaction and depletion of the components.

[0139] Those skilled in the art will readily understand that the antimicrobial composition (whether single or binary) will preferably contain a suitable substrate for the glucose oxidase such as glucose or a glucose source (e.g., dextrin or sucrose) and / or a suitable substrate for the myeloperoxidase such as a (pseudo)halide.

[0140] Thus, in a preferred embodiment, the antimicrobial composition (whether single or binary) further comprises glucose or a glucose source and / or a halide or a pseudo halide.

[0141] The halide or pseudo halide may be selected from iodide, thiocyanate, bromide, chloride and any combination thereof, especially when the myeloperoxidase is from Rhodococcus erythropolis balticus or myeloperoxidase from Homo sapiens. According to the invention, particularly preferred (pseudo)halides are thiocyanate and chloride, especially thiocyanate.

[0142] For the purposes of the present invention, the antimicrobial composition may be in a form suitable for in vitro, ex vivo or in vivo use, depending on the location of the microorganism to be targeted. The form of the composition is preferably selected so as to obtain direct contact with the microorganism of interest. For example, if the antimicrobial composition is intended to be administered to a subject, depending on the location of the microorganism to be targeted, the composition may be in a form suitable for oral, nasal, topical, transdermal or parenteral administration.

[0143] The antimicrobial composition of the present invention may additionally comprise a pharmaceutically acceptable excipient.

[0144] As used herein, the term "pharmaceutically acceptable excipient" refers to an inactive or inert and thus non-toxic pharmaceutical grade compound which has no pharmacological action per se. Such excipients may be used to improve the performance of the composition, such as shelf life, retention time at the site of application, consumer acceptance, etc. It includes, but is not limited to, surfactants (cationic, anionic or neutral), surface stabilizers, other enhancers such as preservatives, wetting agents or emulsifiers. Solvents, buffers, salt solutions, dispersion media, isotonic agents and absorption delaying agents, etc.; they are physiologically compatible.

[0145] A professional technician may also wish to combine the antimicrobial composition of the present invention with one or more therapeutic agents, either within the composition (single composition) or separately (binary composition). Such therapeutic agents include, for example, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, and any combination thereof. For illustrative purposes, examples of therapeutic agents suitable for the present invention include, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, fluoroquinolones, silver, copper, chlorhexidine, polyhexamethylene biguanide, biguanides, chitosan, and / or acetic acid.

[0146] The polypeptide or composition of the present invention can be widely used in industrial, pharmaceutical, medical, cosmetic, and ecological applications, as well as in the food industry. It is worth noting that it can be used for any purpose for which the free combination of myeloperoxidase and glucose oxidase has been reported.

[0147] For example, due to its myeloperoxidase activity, the polypeptide or composition of the present invention can be used to obtain halogenated organic compounds of interest.

[0148] Therefore, another aspect of the present invention is to provide an in vitro use of the non-naturally occurring polypeptide or composition described herein for halogenating non-halogenated organic compounds.

[0149] In other words, the present invention relates to an in vitro method for halogenating non-halogenated organic compounds, the method comprising the step of contacting the polypeptide or composition described herein with a non-halogenated organic compound in vitro.

[0150] As used herein, the term "organic compound" refers to a gaseous, liquid, or solid compound containing carbon in its molecule.

[0151] Examples of the halogenation of non-halogenated organic compounds (RH) using the polypeptide of the present invention are as follows: where RI represents a halogenated organic compound, where H2O2 is provided by the oxidation of glucose (or a glucose source) by glucose oxidase.

[0152] Particularly preferred halogenated organic compounds of interest include, but are not limited to, reactive organic compounds and chemical intermediates used in organic chemical synthesis, such as disinfectants, nutrients, pesticides, drugs, antibiotics (advantageously plant antibiotics), antioxidants, adhesives, and radiopaque contrast agents.

[0153] For illustrative purposes, when the halide is an iodide, the iodinated compounds of interest can include, but are not limited to, phenolic compounds (such as mono-, di-, tri-, tetraiodoresorcinol, dibromoiodophenol and its polymers, and iodinated phlorotannins such as iodinated fuhalols, phlorethols, fucols, fucophlorethols, eckols and carmalols), volatile hydrocarbon compounds (such as iodoform, iodomethane, diiodomethane, bromoiodomethane, iodoethane, iodopropane, iodobutane, etc.), terpenes, amino acid derivatives (such as mono- and diiodotyrosine, which are precursors of thyroxine) and fatty acid derivatives (such as eiseniaiodides). Iodomethane, diiodomethane and iodoform can be used as disinfectants or pesticides. Iodomethane, also known as methyl iodide, can also be used as a chemical intermediate in organic chemical synthesis, especially for methylating other compounds such as phenols, carboxylic acids, ammonia and derived amines, and for the industrial-scale production of acetic acid and acetic anhydride.

[0154] As another example, when the halide is an iodide, the radiopaque agents obtainable by the present invention can include, but are not limited to, 1,3,5-triiodobenzene and its derivatives, such as the ionizing agents diatrizoate, metrizamide and ioxaglic acid, and the non-ionizing agents ioversol, iopamidol, iohexol, ioxilan, iopromide and iodixanol. Such agents can be used for X-ray imaging, such as fluoroscopy.

[0155] Due to its myeloperoxidase activity, the polypeptide or composition of the present invention can also be used to inhibit the growth of various microorganisms, especially those pathogenic microorganisms, such as microorganisms resistant to conventional therapies, in in vitro, ex vivo or in vivo applications.

[0156] Accordingly, another aspect of the present invention is to provide an in vitro or ex vivo use of a non-naturally occurring polypeptide or composition as described herein for killing microorganisms or inhibiting the growth of microorganisms.

[0157] In other words, the present invention relates to an in vitro or ex vivo method for killing microorganisms or inhibiting the growth of microorganisms, which method comprises the step of contacting in vitro or ex vivo the polypeptide or composition as described herein with a material or surface contaminated or at risk of being contaminated with microorganisms.

[0158] In fact, such applications are particularly suitable for treating materials or surfaces contaminated or liable to be contaminated with microorganisms in order to disinfect them before or after use. The material or surface can be the surface of any device, laboratory material, surgical material, etc., such as medical devices, contact lenses, etc., especially those materials intended for use in contact with a subject (such as sutures, bandages, gauzes, staples, zippers, etc.).

[0159] "Microorganism" as used herein refers to bacteria, but also includes viruses, fungi, and parasites. For illustrative purposes, examples of bacteria that can be effectively inhibited by this aspect of the present invention include, but are not limited to, various Gram-negative or Gram-positive bacteria such as Escherichia spp. (e.g., Escherichia coli), Enterococcus spp., Staphylococcus spp., Streptococcus spp., Citrobacter spp., Enterobacter spp., Klebsiella spp., Proteus spp., Acinetobacter spp., Pseudomonas spp., Aeromonas spp., and Pasteurella spp., to name a few, as well as Bacillus spp., Clostridium spp., etc. Examples of fungi that can be effectively inhibited by this aspect of the present invention include, but are not limited to, Aspergillus spp., Fusarium spp., Trichophyton spp., etc. According to the present invention, a particularly preferred microorganism is Escherichia, such as Escherichia coli.

[0160] Furthermore, since the myeloperoxidase in the polypeptide operates by a mechanism completely different from that of the molecules involved in traditional therapies such as antibiotics, in certain cases, this aspect of the present invention can be used to eliminate drug-resistant, multi-drug-resistant, or antibiotic-resistant microorganisms. For illustrative purposes, examples of drug-resistant microorganisms include, but are not limited to, the pathogenic bacteria MRSA (methicillin-resistant Staphylococcus aureus), VRSA (vancomycin-resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus), penicillin-resistant Enterococcus, PRSP (penicillin-resistant Streptococcus pneumoniae), isoniazid / rifampicin-resistant Mycobacterium tuberculosis, and other antibiotic-resistant strains of Escherichia coli, Salmonella, Campylobacter, and Streptococcus.

[0161] In another aspect, the present invention relates to an unnatural polypeptide or composition as described herein for use as a medicament, preferably for the treatment of microbial infections.

[0162] In particular, the present invention relates to the use of the polypeptide or composition as described herein for the preparation of a medicament, preferably for the treatment of microbial infections.

[0163] The present invention also provides a method for treating a microbial infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide or composition described herein.

[0164] The present invention also relates to (i) the non-naturally occurring polypeptides of the present invention and (ii) glucose or a glucose source and / or a halide or pseudohalide as described above, as a combined preparation for use simultaneously, separately or sequentially as a medicament, preferably for treating a microbial infection.

[0165] Generally, the term "treatment" means obtaining the desired physiological or pharmacological effect, depending on the severity of the symptom or disorder of interest or the risk thereof, i.e., herein depending on the severity of the microbial infection or the risk of occurrence of such symptom or disorder. The effect can be prophylactic in terms of partial or total prevention of the symptom or disorder, and / or therapeutic in terms of partial or total cure of the symptom or disorder. The term "prophylactic" describes the ability to avoid or minimize the onset or development of a symptom or disorder before its onset (e.g., after exposure to a microbe but before the onset of the relevant symptom). The term "therapeutic" refers to the ability to inhibit the symptom or disorder (i.e., prevent its development) and / or alleviate the symptom or disorder (i.e., cause the regression of the improvement). In the context of the present invention, a prophylactic effect is generally said to have been achieved when, for example, an asymptomatic subject exposed to a microbe remains asymptomatic or quasi-asymptomatic (e.g., without the development of an infection) after treatment according to the present invention, while a therapeutic effect is generally said to have been achieved when, for example, a symptomatic subject infected with a microbe recovers (e.g., partial or complete remission of the infection) after treatment according to the present invention.

[0166] Due to the broad activity spectrum of the polypeptides or compositions of the present invention, the polypeptides or compositions of the present invention can be advantageously used for treating polymicrobial infections. Polymicrobial diseases involve multiple infectious agents and may include complex, complicated, mixed, dual, secondary, synergistic, concurrent, polymicrobial or co-infections. Polymicrobial diseases include, for example, infections associated with abscesses, AIDS-related opportunistic infections, conjunctivitis, gastroenteritis, hepatitis, multiple sclerosis, otitis media, periodontal diseases, respiratory diseases and genital infections.

[0167] The polypeptides or compositions of the present invention can also be advantageously used for treating microbial infections resistant to conventional therapies. Examples of such infections include those associated with the resistant microbes as described above.

[0168] As described above, treatment according to the present invention can be achieved by administering a therapeutically effective amount of the polypeptides or compositions described herein to a subject in need thereof using any suitable administration regimen. For example, depending on the type of infection affecting the subject, the administration can be oral, nasal, topical, transdermal, parenteral, or any combination thereof. The route of administration is preferably designed to bring the polypeptide or composition into direct contact with the infecting microorganism. A person skilled in the art can readily determine and adjust the dosage and / or regimen of administration based on the age, weight, and / or severity of the infection of the subject.

[0169] It should be further understood that according to the present invention, the "subject" to be treated is preferably a human or an animal, more preferably a human.

[0170] In another aspect, the present invention relates to a peptide linker comprising or consisting of the following amino acid sequence: (LX1X2X3X4 X5AX6A)m wherein X1 is glutamic acid or glycine, X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty, X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, and m is an integer from 1 to 2, preferably 1; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0171] In a preferred embodiment, the peptide linker of the present invention comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7), or LEKRPEAEA (SEQ ID NO: 8); or a peptide that is substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions. More particularly preferred peptide linkers of the present invention are LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), and (LEKREAEA)2 (SEQ ID NO: 6).

[0172] Conservative substitutions are as described above.

[0173] The above peptide linker can be used in vitro to couple molecules to each other, preferably by covalent coupling. Examples of such molecules are myeloperoxidase and glucose oxidase as described herein.

[0174] The present invention will be better understood from the following detailed experiments. However, those skilled in the art will understand that the presented examples are not restrictive and various modifications, substitutions, omissions, and changes can be made without departing from the scope of the present invention.

[0175] Examples

[0176] The aim of this study was to produce an artificial bifunctional enzyme capable of catalyzing the formation of (pseudo)hypohalite compounds from glucose and (pseudo)halides. For this purpose, the present inventors selected the open reading frame of myeloperoxidase from Rhodococcus erythropolis balticus (Example 1) or from Homo sapiens (Example 2) and fused it with glucose oxidase from Penicillium nisikiae. Two types of chimeras were obtained by genetic engineering: one with the native peptide linker between the two enzymes and the other without such a linker between the two enzymes. The results showed that the presence of this native linker was crucial for activating glucose oxidase within the chimera and for being able to form sufficient microbicidal compounds to kill the Escherichia coli strain used for antimicrobial resistance analysis. To understand the key properties of this native linker, several mutations were made: the sequence and length of the linker seemed to be important for the topology, oligomerization, kinetics, and microbicidal properties of the chimera.

[0177] Example 1

[0178] 1. Materials and Methods

[0179] 1.1. Construction of the chimera RbMPO-Gox without a peptide linker

[0180] The GOx penag ORF from the pPiczα-GOx penag vector (Zhang, Biotechnology Advances, 2011; 29(6):715 - 725) was PCR amplified to introduce two restriction sites, namely NdeI and XhoI. Then the amplified DNA was digested at these two sites and the digested fragment containing the GOx penag ORF was ligated into the pET21a plasmid (digested with NdeI and XhoI).

[0181] The pEt21a-RbMPO-6His vector was digested with XhoI and the pET21aGOx penagDigest with NdeI. Use these two linearized vectors as large primers and amplify using the following primers: RbMPO C-Ter and GOx penag (Table 4). Subsequently, digest the PCR product with DpnI at 37 °C for 2 h and purify. Perform the final ligation step and then transform the new construct into DH5α. To verify the correctness of the chimeric construct, identify the clone by colony PCR using two primers, GOx65rc and RbMPOH407A (Table 4), which amplify the fusion region.

[0182] The plasmid pET21a-RbMPO-Gox was obtained.

[0183]

[0184] 1.2. Construction of the chimeric RbMPO-Gox with a peptide linker

[0185] Digest the pPiczαGOx penag vector with the restriction enzyme XhoI at two positions flanking the enzyme ORF.

[0186] Digest the pET21aRbMPO-6His plasmid with XhoI at a site immediately before the 6His tag at the C-Ter of the enzyme. Ligate the linearized plasmid with the ORF of GOx penag to obtain the final chimeric construct. In this construct, the nucleic acid sequence encoding the peptide linker LEKREAEA (SEQ ID NO: 7) is directly present at the 5'-end of the RbMPO ORF. After transformation into DH5α bacteria, the construct was verified by colony PCR using the oligonucleotides GOx 65rc and RbMPOH407A.

[0187] The plasmid pET21a-RbMPO-LEKREAEA-Gox was obtained.

[0188] 1.3. Construction of the chimeric RbMPO-GOx with an alternative peptide linker

[0189] Modify the chimeric using the primers in Table 5 with the Quick-Change kit from Stratagene or the Q5 site-directed mutagenesis kit from NEB Biolabs.

[0190] Use the pET21a-RbMPO-GOx vector (without linker) as a substrate to add the sequence encoding 8 Gly (SEQ ID NO: 9) or LGKRGAGA (SEQ ID NO: 5).

[0191] The pET21a-RbMPO-LEKREAEA-GOx vector was used as a matrix to replace the linker with sequence i) LEKREAEALEKREAEA (SEQ ID NO: 6), ii) LEGGEAEA (SEQ ID NO: 4), or iii) LEKRPEAEA (SEQ ID NO: 8).

[0192] Thus, the following five plasmids with modified linkers were obtained, namely: pET21a-RbMPO-GGGGGGGG-Gox, pET21a-RbMPO-LGKRGAGA-Gox, pET21a-RbMPO-LEKREAEALEKREAEA-Gox, pET21a-RbMPO-LEGGEAEA-Gox, and pET21a-RbMPO-LEKRPEAEA-Gox.

[0193]

[0194] 1.4. Generation and purification of chimeras

[0195] Each pET21a vector expressing the chimera was transformed into Escherichia coli BL21Star (DE3), and bacterial clones were grown on LB agar ampicillin and chloramphenicol nutrient media. 1 L was incubated with a 10 mL preculture performed the previous day and the OD was measured 600nm, until the bacterial growth reaches the logarithmic growth phase (0.5 - 0.8). Use 500 µM IPTG induction to trigger the transcription and translation of the chimeras. After 24 hours at 22 °C, centrifuge to collect the bacterial pellet. Crush the bacteria at 2200 bar (4 °C) and wash the bacterial pellet with a solution of 2 M urea, 50 mM Tris, 5.5 mM CaCl2 pH 7.5. The chimera with a molecular weight of 142088.88 Da is produced in bacterial inclusion bodies, so the pellet needs to be treated with a solution of 8 M urea, 50 mM Tris, 5.5 mM CaCl2 pH 7.5 at 4 °C for 4 hours to redissolve all the proteins. Drop the supernatant recovered after centrifugation into the refolding solution (20 mM Tris pH 7.5, 5.5 mM CaCl2, 5 µM hemin, 200 µM FAD, 1 mM oxidized glutathione, 1 mM reduced glutathione, and 10% v / v glycerol) for 5, 8, or 15 days. First, concentrate the mixture with a concentrator cartridge (Sartorius PES Cassettes Vivaflow 200, molecular weight cut-off 30 kDa), and then concentrate it with a 30 kDa amicon (Merck-Millipore amicon Ultra-15, PLHK, membrane Ultracel PL) to obtain a volume of less than 13 mL (first set of experiments) or 7.5 mL (second set of experiments).

[0196] For the first set of experiments, inject the resulting protein solution onto a size exclusion chromatography (Hiload 26 / 600 200pn Cytiva: fractionation range 10000 - 600000 Da). After isocratic elution (50 mM Tris, 5.5 mM CaCl2 pH 7.5), concentrate the peak containing the chimera. The column is calibrated with gel filtration standards from Biorad. This step achieves the separation of different oligomeric states (n ≥ 1) of each chimera for further characterization of each oligomeric state.

[0197] For the second set of experiments, desalt the resulting protein solution on a PD10 column equilibrated with 50 mM Tris pH 7.5, then lyophilize and store in the lyophilized form at 4 °C. This allows the separation of all different oligomeric states of the chimera in a single mixture for further characterization of the mixture.

[0198] 1.5. UV Spectroscopy

[0199] Evaluate the UV spectra of each chimera using a spectrophotometer that scans the protein sample (diluted 100-fold) from 800 to 200 nm. Based on 144285 M -1.cm -1 The theoretical molar extinction coefficient ε was used to determine the concentration of the purified enzyme by applying the Beer-Lambert law.

[0200] 1.6. SDS-PAGE

[0201] Mini-PROTEAN® precast gels (Bio-rad) were used. These gels are characterized by a gradient of acrylamide of 4 - 15%. The protein samples to be analyzed were mixed with Filler Blue 4X (containing SDS and β-mercaptoethanol) and then heated to 95 °C for 5 minutes. The proteins were electrophoretically separated according to their molecular weights using a denaturing migration buffer (SDS) and an amperage of 35 mM per gel. After electrophoresis, the gels were stained in Coomassie blue solution for 3 hours and then underwent successive decolorization steps in a 10% acetic acid 30% ethanol solution.

[0202] 1.7. Glucose oxidase activity of the chimeras

[0203] The glucose oxidase activity of the chimeras was evaluated using a D-gluconic acid / D-glucono-δ-lactone kit from Megazyme. The day before, a solution containing 5 μM chimeras, 250 mM glucose, and 50 mM NaPi pH6 was incubated at 37 °C. The next day, 15.7 μL of this solution was taken and the activity of GOx penag in the chimeras was measured at 340 nm, which corresponds to the wavelength of the final product NADPH of the kit. After reacting overnight, the concentration of D-gluconic acid produced by the chimeras was calculated using the following relationship: where ε 葡萄糖酸 = 6300 M -1 .cm -1 , and the molecular weight of the same compound is 196.1 g / mol.

[0204] 1.8. Myeloperoxidase activity of the chimeras

[0205] Chloride was used as the substrate

[0206] The fluorescent probe aminophenyl fluorescein (APF) (λ 激发 = 485 nm, λ 发射The amount of HOCl produced by the chimera was measured at 525 nm ([[ID=]], as a measure of its chlorination activity. Three tests were conducted: (1) at different glucose concentrations (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 20 mM), using a fixed concentration of chimera (1 µM) and NaCl (20 mM); (2) at different NaCl concentrations (0, 10, 20, 50, 100, 250, 500, 750 µM and 1 mM), using a fixed concentration of chimera (1 µM) and glucose (20 mM); (3) at different H2O2 concentrations (0, 0.01, 0.02, 0.05, 0.079, 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, 200, 500 mM), using a fixed concentration of chimera (1 µM) and NaCl (20 mM). For all experiments, 10 µM APF and 50 mM NaPi buffer pH 7.5 were used in each assay, and the volume was made up to 20 µL (384-well plate) with quantitative replenishment. Each experiment was performed in triplicate. A kinetic analysis was performed for 10 - 30 minutes for each range of values, and the growth rate of the fluorescence intensity was compared with the fluorescein standard curve. Thus, the fluorescence intensity measured per second is proportional to the amount of fluorescein, and thus proportional to the amount of HOCl produced by the chimera.

[0207] Steady-state kinetic parameters were determined by fitting the experimental data to the following equation according to the saturation curve shape: where kss corresponds to the steady-state rate constant (initial velocity divided by the concentration of the chimera); kcat is the catalytic constant (maximum velocity divided by the concentration of the chimera); [S] is the concentration of the substrate (glucose or NaCl); K M is the Michaelis constant, corresponding to the concentration of the substrate (glucose or NaCl) at which 50% of the maximum velocity is observed; Ki is the inhibition constant observed when inhibition is caused by an excess of the substrate (glucose or NaCl); and k2 is the slope when no saturation is observed within the range of substrate concentrations tested.

[0208] Using thiocyanate as the substrate

[0209] Initial rate measurements under steady-state conditions were carried out on a BioLogic SFM400 stopped-flow instrument using single mixing mode, a Xe / Hg lamp, and a TC100 / 10 type reaction cell. One syringe was filled with the chimera and the other with glucose and NaSCN. The concentrations of the chimera and glucose were fixed (300 nM and 50 mM, respectively), and measurements were made at 37 °C and 240 nM. At 37 °C, in 50 mM sodium phosphate buffer at pH 6, measurements were made at 240 nm - for the formation of OSCN (hypothiocyanite). For each concentration point in the NaSCN substrate range, at least five injections of 120 s were made, and the sampling periods were as follows: 20 µs, 20 ms, 20 ms, and 2000 points per period. The slope was calculated from the average of the injections at each concentration point, and k was determined using the slope and the following formula ss (s -1 ): - The molar extinction coefficient of OSCN was ε -OSCN = 951 M -1 .s -1 . The data collected were analyzed using Origin software, and the k ss values were fitted with the Michaëlis–Menten equation

[0210] 1.9. Microbicidal activity and stability of the chimera

[0211] A glycerol stock of the strain Escherichia coli ATCC 25922, which is recommended for performing the antimicrobial spectrum / for evaluating the antimicrobial activity of compounds of interest, was placed on a Petri dish containing tryptic soy broth (TSB) agar medium at 37 °C and cultured for 16 hours. A single colony was picked and inoculated into 10 mL of TSB medium, and the pre-culture was shaken overnight at 190 rpm and 37 °C. The pre-culture was inoculated into 25 mL of TSB medium at an inoculum of OD 620nm = 0.09. When the latter reached the logarithmic phase, a 10 mL bacterial dilution (in TSB) was made, and based on the conversion relationship of OD 620nm = 1.0 corresponding to 1.10 8 CFU / mL, a bacterial amount of 2.10 6 CFU / mL was obtained. The microbicidal test was carried out in a 96-well microplate (Greiner Bio-One™, Cellstar™ µclear™, white flat bottom), with a final volume of 100 µL per well and in triplicate for each experimental condition. In each well, under all conditions, 50 µL of bacteria was plated to 1.106 CFU / mL. OD was measured using a Wallac Victor2 1420 microplate reader or a SpectraMax® Paradigm® microplate reader (Molecular Devices). 620nm Measurements were taken every 15 minutes for 16 h. The samples were shaken before each reading and measurements were carried out in a thermostatic environment at 37 °C. After storing the enzyme at 4 °C for several days, these experiments were repeated under the same conditions to also evaluate the stability of the chimeras.

[0212] 2. Results

[0213] 2.1. Construction of RbMPO-GOx chimeras

[0214] After the bacterial transformation step, several clones were selected and colony PCR was performed to verify the presence of the chimeric construct. For chimeras without a peptide linker, primers GOx65rc and RbMPOH407A were used, or for chimeras containing a peptide linker, the primers described in Table 5 above were used. Sequencing confirmed the presence of a complete chimeric sequence in each construct.

[0215] 2.2. Production and purification of RbMPO-GOx chimeras

[0216] In the first set of experiments, all chimeras were purified 8 days after reconstitution with heme and FAD (the cofactors of RbMPO and GOx, respectively). For each chimera, approximately 40 mg of protein was obtained per liter of bacterial culture. Depending on the linker peptide sequence, one or more peaks were detected during the purification process. Since size exclusion chromatography was performed, the molecules corresponding to the first peak had a higher molecular weight than those in the subsequent peaks. Free FAD molecules and other components of the reconstitution solution were eluted in the total volume of the S200 column. Calibration using standard proteins allowed determination of the oligomeric state (n ≥ 1) of the chimeras based on the elution volume used to calculate the partition coefficient. A total of 7 chimeras with or without a peptide linker were purified, and the different oligomeric states of these chimeras were analyzed and studied based on their elution volume and purity. These oligomeric states are summarized in Table 6 below. At the same time, SDS-PAGE analysis showed that the different peaks corresponded to pure chimeras with a band molecular weight of approximately 150 kDa.

[0217] The UV spectra of each chimera were obtained, including the spectrum of free heme (data not shown). The spectrum of free heme (RbMPO cofactor) showed two main peaks, one at 385 nm and the other at 615 nm. For the chimeras, the fixation of heme at the RbMPO active site could be demonstrated by the shift of these two peaks to 412 nm and 637 nm, respectively. The peak at 280 nm is the characteristic absorption peak of the amino acids tyrosine and tryptophan, which are components of RbMPO and GOx.

[0218] The following information could be deduced from these spectra: , the ratio and the mass percentage of each chimera in each oligomeric state. Rz indicates the presence of heme in the chimera. indicates the purity of the chimera against DNA and / or can indicate the presence of FAD in the active site of GOx. These parameters are also summarized in Table 6.

[0219] The apparent molecular weight (MW app ) of the chimeras was determined from their elution volume (and then their partition coefficient K AV ) and the calibration curve of the S200 column. Their apparent oligomeric state was then obtained by dividing MW app by the monomer MW (MW 单体 ). From the concentration of each chimera and then from the UV-visible spectrum, using Beer-Lambert, the dilution factor of the chimera, and the molecular extinction coefficient, the relative amounts of each oligomeric state of each chimera were determined. See also Table 6.

[0220]

[0221] The most active chimera seems to be RbMPO-LEGGEAEA-GOx, as it has its monomer state, optimal Rz, and highest ratio compared to other chimeras.

[0222] In the second set of experiments, the chimera RbMPO-LEGGEAEA-GOx was produced and purified as a mixture (i.e., without separating the oligomeric states). Approximately 151 mg of protein was obtained from 250 ml of bacterial culture, which is a considerable yield compared to the first set of experiments using the individual oligomeric states of the chimera.

[0223] 2.3. Enzymatic characterization of the individual active sites of the chimeras

[0224] Two enzymatic tests were performed to determine the activities of the two active sites separately.

[0225] 2.3.1. Glucose oxidase activity

[0226] Measuring D-gluconic acid produced by GOx using the Megazyme kit, thus only characterizing the specific glucose oxidase activity of the chimera ( penag ). An increase in absorbance at 340 nm indicates the presence of NADPH and is thus indirect evidence of D-gluconic acid production. The test was carried out at pH 6, which is known to be optimal for GOx Figure 1 ). After incubation for 17 h, A penag was measured as the blank value. Then, as a final step, the production of NADPH was triggered and the increase in A 340nm was observed (data not shown). Based on the ΔA 340nm value, the concentration of D-gluconic acid produced by the chimera could be calculated and its specific glucose oxidase activity determined, as shown in Table 7 below. 340nm

[0227]

[0228] All chimeras showed glucose oxidase activity, although with different efficiencies.

[0229] The chimera with the highest activity was RbMPO-LEKREAEA-GOx (2.463 ± 0.246 IU / mg), followed by the monomer of RbMPO-LEGGEAEA-Gox (0.731 ± 0.031 IU / mg), the trimer of RbMPO-GGGGGGGG-GOx (0.639 ± 0.155 IU / mg), the trimer of RbMPO-LGKRGAGA-GOx (0.443 ± 0.01 IU / mg), and finally the trimer of RbMPO-LEKRPEAEA-GOx (0.268 ± 0.03 IU / mg).

[0230] Similarly, the chimera RbMPO-LEGGEAEA-GOx produced as a mixture also showed glucose oxidase activity, as D-gluconic acid production from glucose was detected using the Megazyme kit at a level of 0.298 ± 0.049 IU / mg.

[0231] 2.3.2. Myeloperoxidase activity

[0232] Using chloride as the substrate

[0233] To determine the RbMPO activity of the chimeras, a range of H2O2 concentrations was assayed at a fixed concentration of NaCl. HOCl reacts with the APF probe and the appearance of fluorescein was monitored. The steady-state kinetic parameters are summarized in Table 8 below.

[0234] ​

[0235] All chimeras showed chlorination activity, although with different efficiencies.

[0236] The chimera with the highest activity was RbMPO-LEKREAEALEKREAEA-GOx in the dimer / monomer state, followed by its trimer state. The dimer / monomer form of this chimera had a high specificity constant (1508 ± 19.4 M -1 .s -1 ) as compared to other chimeras. In terms of catalytic efficiency, the next ones were RbMPO-LEGGEAEA-GOx (34.2 ± 19.4 M -1 .s -1 ) and RbMPO-LEKRPEAEA-GOx (excluded) (6.82 ± 4.27 M -1 .s -1 ), and the latter was slightly more inhibited by H2O2 (Ki = 17.3 ± 10.6 mM). RbMPO-GGGGGGGG-GOx (excluded) had a low specificity constant (1 ± 0.6 M - 1 .s -1 ), but was very weakly inhibited by H2O2 (Ki = 140.5 ± 65.6 mM). Finally, the linker-free chimera RbMPO-Gox had a medium level of specificity constant (4.44 ± 3.15 M -1 .s -1 ).

[0237] Since HOCl reacting with APF was detected, the chimera RbMPO-LEGGEAEA-GOx produced in the form of an oligomeric mixture also showed myeloperoxidase activity. At 15 mM glucose and 500 mM NaCl, its kss was determined to be about 10 -3 s -1 . However, due to the heterogeneity of this protein, the individual kinetic parameters kcat, K M and K i could not be determined.

[0238] 2.4. Enzymatic characterization of the coupled active sites of the chimeras

[0239] Using chloride and glucose as substrates

[0240] Then the activities of the two active sites were evaluated by combining them with each other ( Figure 1 ).

[0241] In short, the aim was to measure the amount of HOCl produced using an APF probe, with glucose and NaCl as substrates for the enzyme, respectively. Two types of kinetics were performed: i) Kinetics at various different concentrations of glucose and a fixed concentration of NaCl, and ii) Kinetics at various different concentrations of NaCl and a fixed concentration of glucose.

[0242] The steady-state kinetic parameters are summarized in Tables 9 and 10 below.

[0243]

[0244]

[0245] All chimeras showed glucose activity and chloride activity, although with different efficiencies. Their catalytic efficiencies for glucose and NaCl in their oligomeric states are summarized in Figure 2A and 2B .

[0246] In short, in terms of glucose, the monomer 2 of RbMPO-LEGGEAEA-GOx had the best catalytic efficiency (2.73 ± 1.5 M -1 .s -1 ). Followed by the dimer / monomer of RbMPO-LEKREAEALEKREAEA-GOx (2.47 ± 1.58 M -1 .s -1 ), the trimer of RbMPO-LEKREAEALEKREAEA-GOx (1.7 ± 1.34 M -1 .s -1 ) and the trimer of RbMPO-LGKRGAGA-GOx (1.1 ± 0.8 M -1 .s -1 ), although the latter had strong inhibition (Ki = 6.1 ± 1.2 mM). The kcat / KM of all other chimeras was below 1 M -1 .s -1 . Overall, within each chimera, the exclusion peak seemed to have the lowest glucose oxidase activity. Most importantly, the presence of the linker improved the catalytic efficiency for glucose.

[0247] In terms of chloride, the dimer / monomer of RbMPO-LGKRGAGA-GOx (2.4 ± 0.9 M -1 .s -1 ) had the highest catalytic efficiency, followed by the dimer / monomer of RbMPO-LEKREAEALEKREAEA-GOx (0.139 ± 0.076 M -1 .s -1). Next is the excluded form of RbMPO-LGKRGAGA-GOx (0.020 ± 0.009 M -1 .s -1 ). Other chimeras are also active but exhibit reactivity below 0.02 M -1 .s -1 . Due to the large error range, the catalytic efficiencies of the dimer / monomer and trimer of RbMPO-GGGGGGGG-GOx are not yet determined. It should be further noted that the presence of the linker increases the catalytic efficiency towards chloride.

[0248] The chimera RbMPO-LEGGEAEA-GOx produced in a mixture form also simultaneously shows glucose oxidase activity and myeloperoxidase activity: when using 15 mM glucose and 500 mM NaCl as the initial substrate concentrations, the k ss / [glucose] value of this protein is 10 -3 M- 1 .s -1 .

[0249] In summary, these results confirm the positive effect of the peptide linker on the enzyme catalytic efficiency. In addition, in order to avoid the differences in catalytic efficiency observed when using different oligomeric states of the chimera, mixtures of these oligomeric states can be used.

[0250] Using thiocyanate and glucose as substrates

[0251] For completeness, since RbMPO can catalyze various (pseudo)halides, including not only chloride but also iodide, bromide, and thiocyanate (to be published), the myeloperoxidase activities of some chimeras were evaluated using thiocyanate as the substrate in the presence of a fixed concentration of glucose.

[0252]

[0253] These results show that the said peptide linker is also suitable for the efficient catalysis of thiocyanate and glucose.

[0254] 2.5. Microbicidal activity and stability of the chimera

[0255] Microbicidal experiments were then carried out with the chimeras that appeared to show the highest catalytic efficiency towards glucose and chloride, namely: RbMPO-LEGGEAEA-Gox, RbMPO-LGKRGAGA-Gox and RbMPO-LEKREAEALEKREAEA-GOx. RbMPO-LEGGEAEA-Gox and RbMPO-LGKRGAGA-GOx were also of interest because they were less inhibited by H2O2. For completeness, the different oligomeric states of these chimeras, as well as mixtures of oligomeric states (only for RbMPO-LEGGEAEA-Gox), were evaluated.

[0256] As Figure 3 shown in A to 3N, in the presence of NaSCN or NaCl, all of these chimeras inhibited the growth of bacteria. Interestingly, this microbicidal effect was observed after storing the chimeras at 4 °C for 4 to 21 days, thus demonstrating the stability of these chimeras.

[0257] 3. Conclusions

[0258] In this study, various fusions of GOx and MPO enzymes were constructed and produced in suitable bacterial strains. For this purpose, a refolding step of 8 days was required in the presence of FAD and heme cofactors. The purification yields were improved, ranging from approximately 40 mg per liter of bacterial culture (for individual oligomers) to approximately 604 mg (for oligomer mixtures) of pure chimeric enzymes.

[0259] The first chimeric protein was produced by directly fusing the C-terminus of the MPO enzyme to the N-terminus of the GOx enzyme.

[0260] Then different peptide linkers were genetically engineered between the two enzymes: these linkers showed different characteristics in terms of amino acid charge, amino acid length, degree of amino acid flexibility, and / or tertiary structure. For this purpose, chimeras were produced using the peptide linker LEKREAEA or its mutant variants (introducing proline in the middle of the peptide; mutating one or more positively or negatively charged amino acids to neutral glycine; mutating all amino acids to neutral glycine; repeating the peptide linker). Several oligomeric states (n≥1) of each of these chimeras were identified and purified for further analysis. One chimera was also produced in the form of a mixture of its oligomeric states in order to reduce production costs and time.

[0261] The presence of the peptide linker increased the enzyme activity, especially at the GOx activity level, and also increased the enzyme activity towards NaCl (chloride activity, which is a myeloperoxidase activity), compared to the chimera without a linker.

[0262] Then, the microbicidal properties of the most active chimeras were studied: this study confirmed their microbicidal activity as well as their stability, whether in the form of isolated oligomers or mixtures thereof.

[0263] Example 2

[0264] Experiments similar to those carried out in Example 1 were performed in order to generate chimeric constructs in which myeloperoxidase from Homo sapiens was fused with glucose oxidase from Penicillium nizukii. Similarly, two types of chimeras were obtained by genetic engineering: one with the original peptide linker and the other without such a linker between the two enzymes.

Claims

1. A non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

2. The polypeptide according to claim 1, wherein the polypeptide is a fusion polypeptide comprising myeloperoxidase coupled, preferably covalently coupled, to glucose oxidase.

3. The polypeptide according to claim 2, wherein the C-terminus of the myeloperoxidase is coupled, preferably covalently coupled, to the N-terminus of the glucose oxidase.

4. The polypeptide according to claim 2 or 3, wherein the myeloperoxidase is coupled, preferably covalently coupled, to the glucose oxidase through a linker, preferably through a peptide linker.

5. The polypeptide according to claim 4, wherein the linker is a peptide linker comprising or consisting of the following amino acid sequence: (LX1X2X3X4 X5AX6A)m where X1 is glutamic acid or glycine, X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty, X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, and m is an integer from 1 to 2; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

6. The polypeptide according to claim 5, wherein the peptide linker comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8); or a peptide that is substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

7. The polypeptide according to claim 4, wherein the linker is a peptide linker comprising or consisting of a polyglycine amino acid sequence; or a peptide that is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

8. The polypeptide according to any one of the preceding claims, wherein the myeloperoxidase is a microbial myeloperoxidase, preferably myeloperoxidase from Rhodopirellula baltica, or a mammalian myeloperoxidase, preferably myeloperoxidase from Homo sapiens.

9. The polypeptide according to any one of the preceding claims, wherein the glucose oxidase is a microbial glucose oxidase, preferably glucose oxidase from Penicillium amagasakiens.

10. The polypeptide according to any one of the preceding claims, wherein the polypeptide is in the form of a functional oligomer or a mixture of functional oligomers.

11. A nucleic acid encoding a polypeptide as defined in any one of the preceding claims.

12. A vector comprising the nucleic acid as defined in claim 11.

13. A host cell comprising the vector as defined in claim 12.

14. A method for obtaining a polypeptide as defined in any one of claims 1 to 10, said method comprising at least the following steps: a) culturing the host cell as defined in claim 13 in a medium under conditions suitable for expressing the polypeptide; and b) recovering the polypeptide.

15. The method according to claim 14, wherein the polypeptide recovered in step b) is further dissolved in a solution comprising heme and flavin adenine dinucleotide (FAD) and optionally calcium.

16. An antibacterial composition comprising a non-naturally occurring polypeptide as defined in any one of claims 1 to 10.

17. The composition according to claim 16, further comprising glucose or a glucose source and / or a halide or pseudohalide.

18. The in vitro use of a non-naturally occurring polypeptide as defined in any one of claims 1 to 10 or a composition as defined in claim 16 or 17 for halogenating a non-halogenated organic compound.

19. The in vitro or ex vivo use of a non-naturally occurring polypeptide as defined in any one of claims 1 to 10 or a composition as defined in claim 16 or 17 for killing microorganisms or inhibiting the growth of microorganisms.

20. A non-naturally occurring polypeptide as defined in any one of claims 1 to 10 or a composition as defined in claim 16 or 17 for use as a medicament, preferably for treating a microbial infection.

21. (i) A non-naturally occurring polypeptide as defined in any one of claims 1 to 10 or a composition as defined in claim 16 or 17 and (ii) glucose or a glucose source and / or a halide or pseudohalide, for use as a combined preparation for simultaneous, separate or sequential use as a medicament, preferably for treating a microbial infection.

22. A peptide linker as defined in claim 5 or 6.

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