Polypeptides with peroxidase activity

Abstract

The present invention provides a polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, wherein the amino acid sequence comprises at least one amino acid substitution compared to SEQ ID NO:1, the at least one amino acid substitution being a substitution of amino acid P146 or amino acid N275 of SEQ ID NO:1. The present invention further relates to a nucleic acid molecule comprising a sequence encoding the polypeptide, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector. Also provided are methods for producing the polypeptide, and compositions and kits comprising the polypeptide.

Description

[Technical Field]

[0001] The present invention relates to novel polypeptides having peroxidase activity. [Background technology]

[0002] Horseradish peroxidase (HRP) is an industrially important enzyme with a wide range of applications, including immunoassays, diagnostic kits, probe-based assay techniques such as ELISA, EMSA, Western blotting and Southern blotting, waste treatment, as a reagent in organic synthesis, and potential therapeutic applications.

[0003] To date, HRP has been primarily produced by extraction from hairy root cultures of horseradish (Armoracia rusticana). This is, however, an inefficient and time-consuming process. Disadvantages include low yields, long cultivation times, seasonal availability, heterogeneous isoenzyme composition (different preparations have different biochemical properties), dependence on conditions during root growth, and a plant glycosylation pattern that may be immunogenic to humans, making it unsuitable for medical use.

[0004] Recombinant production of HRP is a desirable alternative to hairy root culture because it is a more promising production method that ensures a stable supply of defined HRP preparations in high quality. In particular, recombinant production in E. coli may enable obtaining defined preparations of one isoform with stable biochemical properties while lacking glycosylation and, therefore, immunogenicity.

[0005] However, the original plant-based HRP contains glycosylation at eight asparagine residues, which makes recombinant production difficult, and wild-type HRP produced in E. coli exhibits significantly lower stability compared to the plant-derived enzyme.

[0006] Numerous attempts have been made to recombinantly produce HRP in various hosts, including mammalian cells, insect cells, yeast (Pichia pastoris and Saccharomyces cerevisiae), other plants (Nicotiana tabacum, Nicotiana benthamiana, and Armorasia lafatifolia), and Escherichia coli. However, these production strategies have resulted in very low yields and / or reduced enzyme activity and stability. In the case of production in E. coli, this occurs due to intracellular production of the protein, which leads to inclusion body formation due to the reducing conditions in the E. coli cytoplasm. Therefore, downstream processing is more elaborate than in, for example, yeast. The enzyme can be translocated to the periplasm by adding a signal sequence, but this results in even lower yields, and enzyme activity can be reduced by adding a translocation tag. Furthermore, E. coli cannot perform post-translational modifications, and therefore the enzyme is unglycosylated, leading to reduced protein stability.

[0007] Several studies have been conducted in the past with the goal of improving the properties of recombinantly produced HRP. An overview of such efforts is given by Humer and Spadiut ("Improving the performance of horseradish peroxidase by site-directed mutagenesis," International Journal of Molecular Sciences 20.4 (2019): 916), who disclosed the effects of numerous mutations on stability and / or enzymatic activity. Summary of the Invention [Problem to be solved by the invention]

[0008] However, despite all of these efforts, new and improved variants of HRP remain lacking. In particular, there is a need for HRP variants that can be recombinantly produced in a suitable host, especially E. coli, and that have improved stability and / or enzymatic activity. It is an object of the present invention to provide such HRP variants. [Means for solving the problem]

[0009] Accordingly, the present invention provides a polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said amino acid sequence comprises at least one amino acid substitution compared to SEQ ID NO: 1, and said at least one amino acid substitution is a substitution of amino acid P146 or amino acid N275 of SEQ ID NO: 1.

[0010] The present invention also provides a nucleic acid molecule comprising a sequence encoding a polypeptide according to the invention; an expression vector comprising a nucleic acid molecule according to the invention; and a host cell comprising an expression vector according to the invention.

[0011] The present invention further relates to a method for producing a polypeptide according to the invention, comprising the steps of culturing a host cell according to the invention and recovering said polypeptide.

[0012] In a further aspect, the present invention provides a composition comprising a polypeptide according to the invention. In a further aspect, the present invention provides a kit comprising said polypeptide or a kit according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the context of the present invention, it has surprisingly been found that the properties of HRP can be improved by introducing mutations at proline-146 (P146) and / or asparagine-275 (N275) of wild-type HRP. In particular, it has been found that such HRP variants can be recombinantly produced in E. coli with high yields and good purity, and exhibit surprisingly good thermostability (see Example 2) and surprisingly high enzymatic activity (see Example 3).

[0014] Although many mutations to HRP have been investigated (see Humer and Spadiut, "Improving the performance of horseradish peroxidase by site-directed mutagenesis," International Journal of Molecular Sciences 20.4 (2019): 916), substitutions of amino acids P146 and / or N275 have not been described in the prior art. A peroxidase from Rubera herbacea is disclosed in UniProt entry R0FP47. A predicted peroxidase-like protein from Camelina sativa is disclosed in NCBI protein entry XP_010503561.

[0015] The sequence of wild-type HRP is known in the art, for example, from Gajhede et al. ("Crystal structure of horseradish peroxidase C at 2.15 Å resolution," Nature Structural Biology 4.12 (1997): 1032-1038, Protein Data Bank PDB ID 1ATJ) or UniProt entry P00433. Unless otherwise noted, residue numbering used in this disclosure refers to the sequence of wild-type HRP as set forth in SEQ ID NO: 1 (positions P146 and N275 are shown in bold below).

[0016] Wild-type HRP (SEQ ID NO: 1): [ka]

[0017] Plant-derived enzymes undergo post-translational modifications that, among other things, result in a free N-terminus without an added methionine. However, when recombinantly produced in, for example, E. coli, they are typically produced with an N-terminal methionine residue derived from the start codon. Recombinantly produced wild-type HRP therefore typically has the following sequence:

[0018] Recombinantly produced wild-type HRP (SEQ ID NO: 2): [ka]

[0019] Preferably, the polypeptide of the present invention comprises an amino acid sequence containing two or more amino acid substitutions compared to SEQ ID NO: 1, and the two or more amino acid substitutions are substitutions of amino acids P146 and N275 of SEQ ID NO: 1. As can be seen from the experimental results shown in Example 2, the combination of amino acid substitutions at both P146 and N275 (in HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K) can unexpectedly confer higher thermostability than substitutions at only one position (HRP N13D / N57S / P146Q / N175S / N255D / N268D or HRP N13D / N57S / N175S / N255D / N268D / N275K).

[0020] In the context of the present invention, it is particularly preferred that the amino acid sequence comprises at least one amino acid substitution compared to SEQ ID NO: 1, wherein the at least one amino acid substitution is selected from the group consisting of P146Q, P146A, P146R, P146V, P146E, N275K, N275R, N275D, N275S, N275Q, N275A and N275E, with P146Q and N275K being particularly preferred.

[0021] In the context of all embodiments of the present invention, it is preferred that the amino acid sequence of the polypeptide according to the invention comprises an amino acid substitution selected from the group consisting of P146Q, P146A, P146R, P146V and P146E, in particular P146Q, compared to SEQ ID NO: 1. It is further preferred that said amino acid sequence comprises an amino acid substitution selected from the group consisting of N275K, N275R, N275D, N275S, N275Q, N275A and N275E, preferably N275K, compared to SEQ ID NO: 1. Most preferred is a combination of one of the above preferred amino acid substitutions for P146 (in particular P146Q) and one of the above preferred amino acid substitutions for N275 (in particular N275K). As can be seen from the experimental results shown in Examples 2 and 3, such combinations can lead to both increased thermostability (for HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275 compared to HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275 compared to HRP N13D / N57S / N175S / N255D / N268D / N275K) and increased enzymatic activity (for HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275 compared to HRP N13D / N57S / N175S / N255D / N268D).

[0022] In the context of the present invention, particularly good results have been observed when the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. Said sequence comprises the amino acid substitutions P146Q and N275K, as well as the amino acid substitutions N13D, N57S, N175S, N255D, and N268D (all marked in bold below) relative to SEQ ID NO: 1.

[0023] HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3): [ka]

[0024] As described above for wild-type HRP, when produced recombinantly, for example in E. coli, mutant HRPs are typically produced with an N-terminal methionine residue derived from the start codon. Thus, the HRP variants are typically produced recombinantly as proteins having the following sequence:

[0025] Recombinantly produced HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4): [ka]

[0026] As mentioned above, typically, variants according to the present invention contain an N-terminal methionine. However, in some embodiments of the present invention, variants do not contain this N-terminal methionine. In all cases, the amino acid numbering used according to the present invention is applied according to the wild-type sequence (SEQ ID NO: 1) lacking an N-terminal methionine; that is, this numbering also applies mutatis mutandis to variants containing an N-terminal methionine, which means, for example, that in SEQ ID NO: 4, the mutation N13D is at (absolute) position 14, etc. This also applies to further variant embodiments having deletions or insertions.

[0027] In a preferred embodiment, the polypeptide comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 3, preferably at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, particularly at least 98% sequence identity, and most preferably at least 99% sequence identity to SEQ ID NO: 3. In order of increasing preference, the amino acid sequence has at least 70% sequence identity to SEQ ID NO: 3, preferably at least 71% sequence identity, more preferably at least 72% sequence identity, more preferably at least 73% sequence identity, more preferably at least 74% sequence identity, more preferably at least 75% sequence identity, more preferably at least 76% sequence identity, more preferably at least 77% sequence identity, more preferably at least 78% sequence identity, more preferably at least 79% sequence identity, more preferably at least 80% sequence identity, more preferably at least 81% sequence identity, more preferably at least 82% sequence identity, more preferably at least 83% sequence identity, more preferably at least 84% sequence identity, more preferably at least 85% sequence identity, more preferably at least 86% sequence identity, more preferably at least 87% sequence identity, more preferably at least 88% sequence identity, more preferably at least 89% sequence identity, more preferably at least 90% sequence identity, more preferably at least 91% sequence identity, more preferably at least 92% sequence identity, more preferably at least 93% sequence identity, more preferably at least 94% sequence identity, more preferably at least 95% sequence identity, more preferably at least 96% sequence identity, more preferably at least 97% sequence identity, more preferably at least 98% sequence identity, more preferably at least 99% sequence identity, more preferably at least 100% sequence identity to SEQ ID NO: 3. It is particularly preferred that the amino acid sequence is the sequence set forth in SEQ ID NO: 3. It is particularly preferred that the polypeptide of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 3. It is even more preferred that the polypeptide of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 4, especially when the polypeptide is produced recombinantly, for example in E. coli.

[0028] It is further preferred that the amino acid sequence further comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four, and especially five amino acid substitutions selected from the group consisting of N13D, N57S, N175S, N255D, and N268D compared to SEQ ID NO: 1.

[0029] Mutations N13D, N57S, N255D, and N268D were disclosed by Capone et al. ("Glyco-variant library of the versatile enzyme horseradish peroxidase," Glycobiology 24.9 (2014): 852-863) and Humer and Spadiut ("Improving the performance of horseradish peroxidase by site-directed mutagenesis," International Journal of Molecular Sciences 20.4 (2019): 916). Independently, and in the context of expression in yeast only, mutation N175S was disclosed by Morawski et al. ("Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae," Biotechnology and Bioengineering 76.2 (2001): 99-107).

[0030] All of the above mutations N13D, N57S, P146Q, N175S, N255D, N268D, and N275K relative to SEQ ID NO: 1 are preferred in the context of the present invention: this applies both individually and in combination with one another. It was surprisingly found that the combination of the five mutations N13D, N57S, N175S, N255D, and N268D leads to particularly high thermostability (see Example 2). Furthermore, a further combination with the hitherto completely unknown mutations P146Q and N275K led to a significant improvement in enzyme activity (see Example 3; SEQ ID NO: 4).

[0031] Several additional mutations to the original HRP are known in the art and are also preferred in the context of the present invention. For example, Ryan et al. ("Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide," Biochimie 89.8 (2007): 1029-1032) describe the beneficial effects of the mutations K232N and T110V. Therefore, in a preferred embodiment, the amino acid sequence further comprises the amino acid substitution T110V or K232N compared to SEQ ID NO: 1.

[0032] In the context of the present invention, "peroxidase activity" preferably means activity towards at least one of the substrates 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide (HO). Preferably, when measured in 50 mM phosphate-citrate buffer at pH 5 and 30°C containing 1 mM HO, the polypeptides of the present invention have a peroxidase activity of 0.01 mM. -1 s -1 or more, preferably 0.1 mM -1 s -1 or more, more preferably 1 mM -1 s -1 More preferably, 10 mM -1 s -1 More preferably, 20 mM -1 s -1 More preferably, 100 mM or more -1 s -1 More preferably, 1000 mM or more -1 s -1 More preferably, 10,000 mM or more -1 s -1 Above 20,000mM -1 s -1In another preferred embodiment, the polypeptide has a peroxidase activity corresponding to a kcat / Km value for the substrate TMB of 0.1 mM or more when measured in 50 mM phosphate-citrate buffer containing 1 mM H2O2 at pH 5 and 30°C. -1 s -1 or more, preferably 1 mM -1 s -1 or more, more preferably 10 mM -1 s -1 More preferably, 100 mM -1 s -1 or more, most preferably 250 mM -1 s -1 In yet another preferred embodiment, the polypeptide has a peroxidase activity corresponding to a kcat / Km value for the substrate ABTS of 1 mM or more when measured in a 50 mM phosphate-citrate buffer at pH 5 and 30°C containing 10 mM ABTS. -1 s -1 or more, preferably 10 mM -1 s -1 or more, more preferably 100 mM -1 s -1 More preferably, 1000 mM or more -1 s -1 or more, most preferably 2500 mM -1 s -1 The peroxidase activity corresponds to a kcat / Km value for the substrate HO of 0.5 or more. Preferably, the peroxidase activity is measured as described in Example 1. A 50 mM phosphate-citrate buffer can be obtained by adding 25.7 ml of 0.2 M dibasic sodium phosphate and 24.3 ml of 0.1 M citric acid to 50 mL of deionized water.

[0033] The peroxidase activity of the polypeptide according to the present invention is preferably 10% or more, preferably 20% or more, more preferably 50% or more of the peroxidase activity of wild-type HRP. The wild-type HRP herein is particularly a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably consisting of the amino acid sequence set forth in SEQ ID NO: 2, and particularly both polypeptides are produced and measured under the same conditions.

[0034] In a preferred embodiment, the polypeptide of the present invention has increased thermostability compared to wild-type HRP. For a polypeptide of the present invention comprising an amino acid sequence having a certain percent sequence identity (%) with SEQ ID NO: 3, the polypeptide comprising said amino acid sequence preferably has increased thermostability compared to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. A methionine residue may be added to the N-terminus of the amino acid sequence having a certain percent sequence identity (%) with SEQ ID NO: 3 to enable recombinant production in E. coli. That is, a polypeptide comprising an N-terminal methionine followed by the above-described amino acid sequence preferably has increased thermostability compared to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. To compare the thermostabilities of two such polypeptides, these polypeptides can be produced and measured under the same conditions. "Increased thermostability" preferably means a longer half-life when incubated at 60°C in a buffer consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol, and 500 mM NaCl. The half-life of the polypeptide consisting of the above-mentioned amino acid sequence (and / or the polypeptide of the present invention comprising said amino acid sequence) is preferably at least 1.5 times, preferably at least 3 times, more preferably at least 6 times, and even more preferably at least 12 times longer than that of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2. Preferably, the half-life is determined at various time points by measuring the residual peroxidase activity using 7 mM ABTS in 50 mM phosphate-citrate buffer containing 1 mM HO at pH 5 and 30°C. Preferably, the concentration of the polypeptide is 2.86 μM for half-life measurements. Preferably, the half-life is determined as described in Example 1.

[0035] In a preferred embodiment, the polypeptide of the present invention consisting of the amino acid sequence and / or comprising the amino acid sequence has a half-life at 60°C in a buffer solution consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol, and 500 mM NaCl of 0.5 hours or more, preferably 1 hour or more, more preferably 2 hours or more, even more preferably 4 hours or more, and most preferably 6 hours or more, as measured by residual peroxidase activity using 7 mM ABTS in a 50 mM phosphate-citrate buffer solution containing 1 mM HO at pH 5 and 30°C.

[0036] In many applications, HRP is used as a conjugate with other molecules. Such conjugates are useful in techniques such as Western blot, ELISA, and immunohistochemistry. One type of widely used conjugate is a conjugate with an antibody or another binding protein. In this case, the addition of a substrate for HRP can generate a detectable signal at the site where the binding protein binds to its target. Other frequently used HRP conjugates include HRP-streptavidin conjugates (e.g., for use in sandwich ELISA applications to detect biotinylated antibodies) or HRP-protein A, G, or L conjugates (proteins A, G, and L bind to immunoglobulins and are therefore useful for detecting primary antibodies in, for example, ELISA, ELISPOT, IHC, or Western blotting).

[0037] In a preferred embodiment, the polypeptide according to the invention therefore further comprises the amino acid sequence of streptavidin. In yet another preferred embodiment, the polypeptide further comprises the amino acid sequence of Protein A, Protein G or Protein L.

[0038] It is further preferred that the polypeptide of the present invention comprises the amino acid sequence of a binding protein. The polypeptide of the present invention may therefore be or comprise a conjugate of a mutant HRP and a binding protein. The binding protein may be any protein that can act as an agent for binding to a molecule of interest, for example, as an agent for detecting a specific protein or another molecule. Preferably, the binding protein is an antibody. In a further preferred embodiment, the binding protein is an antibody fragment, preferably a single-chain variable fragment (scFv) or an antigen-binding fragment (Fab). In a further preferred embodiment, the binding protein is an antibody mimetic, preferably selected from the group consisting of adnectins, affibodies, anticalins, DARPins, engineered Kunitz inhibitors, and monobodies. Many such suitable binding proteins are known in the art, for example, from Gebauer and Skerra ("Engineered protein scaffolds as next-generation antibody therapeutics," Current opinion in chemical biology 13.3 (2009): 245-255).

[0039] Conjugates between HRP and a binding protein can be obtained by linking HRP to the binding protein by known methods, for example, by creating a genetic fusion (linking a nucleic acid sequence encoding the HRP to a nucleic acid sequence encoding the binding protein, preferably including a linker sequence between the two proteins) and expressing the resulting fusion protein in a suitable host, for example, in E. coli. Alternatively, the conjugate can be obtained by chemical cross-linking. For this purpose, HRP (preferably purified) and the binding protein can be linked to each other by a chemical cross-linking agent to form a stable bond, preferably a covalent bond, between the two molecules. Such methods, often referred to as bioconjugation, are known in the art. Many suitable methods can be found, for example, in the book Hermanson, Greg T. Bioconjugate Techniques. Academic Press, 2013. Suitable cross-linking agents are also described later in this disclosure in connection with the kits of the present invention.

[0040] Conjugates between HRP and other proteins such as streptavidin or protein A, G, or L can be obtained by methods similar to those described above for conjugates with binding proteins. Preferably, the polypeptides of the present invention can be recombinantly produced by culturing host cells containing a suitable expression vector. Preferably, the host cells are yeast (Saccharomyces, Pichia) cells or prokaryotic cells. It is particularly preferred that the host cells are bacteria, most preferably Escherichia coli.

[0041] Preferably, the polypeptide is an isolated polypeptide, which may be isolated from a cell culture and may be purified and / or enriched as appropriate for a particular use.

[0042] Standard methods known in the art may be used to produce the polypeptides of the present invention. Suitable methods are described, for example, by Humer and Spadiut ("Improving the performance of horseradish peroxidase by site-directed mutagenesis," International Journal of Molecular Sciences 20.4 (2019): 916). Further suitable methods for producing and purifying the polypeptides are described by Smith et al. ("Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme," Journal of Biological Chemistry 265.22 (1990): 13335-13343) or Gundinger and Spadiut ("A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli," Journal of Biotechnology 248 (2017): 15-24). Preferably, the polypeptides of the present invention are produced as described in Example 1.

[0043] The composition of the present invention preferably comprises one or more formulation additives. Such formulation additives can, for example, further increase the stability of the polypeptide of the present invention during storage, ensuring a longer shelf life and a stable level of biological activity. Preferred formulation additives include pH buffers, stabilizers, bulking agents, tonicity adjusters, etc. Formulation additives suitable for use in lyophilization situations are particularly preferred.

[0044] Suitable formulation additives are known to those skilled in the art. For example, the composition preferably contains a pH buffer, preferably selected from the group consisting of glycine, histidine, glutamate, succinate, phosphate, acetate, and aspartate. The composition more preferably contains a bulking agent, preferably selected from the group consisting of mannitol, glycine, sucrose, dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. The composition preferably comprises a stabilizer selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCl, and polyhydroxy compounds including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid, and sodium chloride. The composition may further comprise a surfactant, such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, Preferably, the surfactant is selected from the group consisting of CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbates 20, 40, 60, 65, and 80, soybean lecithin, DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose.

[0045] Advantageously, the composition comprises at least 0.01 mg, preferably at least 0.1 mg, more preferably at least 1 mg, even more preferably at least 5 mg, and in particular at least 10 mg of the polypeptide of the invention. It is further preferred that the composition contains the polypeptide of the invention in a concentration of at least 0.0001% (weight / weight, w / w), preferably at least 0.001% w / w, more preferably at least 0.01% w / w, even more preferably at least 0.1% w / w, even more preferably at least 1% w / w, and in particular at least 10% w / w.

[0046] Advantageously, said composition is a solid composition (at 25°C and atmospheric pressure), preferably a freeze-dried composition. In another preferred embodiment, said composition is a liquid composition (at 25°C and atmospheric pressure). Advantageously, said composition contains the polypeptide according to the invention at a concentration of at least 0.001 mg / mL, preferably at least 0.01 mg / mL, more preferably at least 0.1 mg / mL, even more preferably at least 0.5 mg / mL, most preferably at least 2 mg / mL.

[0047] It is further preferred that the polypeptide of the present invention contained in the composition of the present invention is isolated from a host, for example, a bacterium such as E. coli. In particular, the composition of the present invention is substantially free of DNA, particularly dsDNA. Preferably, the composition contains less than 1 μg / g of DNA, preferably less than 100 ng / g, more preferably less than 10 ng / g, and most preferably less than 1 ng / g. DNA concentration can be determined by flowmetry using the dye SYBR Green I (N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine). Those skilled in the art are familiar with measuring DNA concentration using flowmetry. Measurements can be performed as described in Rengarajan, Kalpana, et al. (Technical Brief Quantifying DNA concentrations using fluorometry: A comparison of fluorophores, Molecular Vision 8 (2002): 416-421).

[0048] The polypeptides of the present invention can find use in a variety of therapeutic applications, for example, targeted cancer therapy. In a preferred embodiment, the composition is therefore a pharmaceutical composition, which preferably contains one or more formulation additives pharmaceutically acceptable for administration to an individual, particularly a mammal, especially a human. Suitable formulation additives are known to those skilled in the art, such as water (particularly water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank's solution, vesicle-forming compounds (e.g., lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances that improve isotonicity and chemical stability, buffers, and preservatives. Other suitable formulation additives include any compound that, when administered to a patient, does not induce the production of antibodies harmful to the patient. Examples include well-tolerated proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition is preferably suitable for parenteral administration, especially intravenous administration. The pharmaceutical composition may be formulated together with the above-defined pharmaceutically acceptable formulation additives and provided in an injectable unit dosage form, for example, as a solution, suspension, or emulsion. All preferred embodiments described above for the compositions of the invention in general (especially those relating to the concentration and amount of polypeptide) are also preferred when the composition is a pharmaceutical composition.

[0049] In the context of therapeutic applications, the polypeptides of the present invention may be part of an enzyme-prodrug system. Enzyme-prodrug systems containing HRP are known in the art, particularly for cancer treatment (see, for example, Tupper et al. "In vivo characterization of horseradish peroxidase with indole-3-acetic acid and 5-bromoindole-3-acetic acid for gene therapy of cancer," Cancer Gene Therapy 17.6 (2010): 420-428). For example, targeted cancer therapy may involve an enzyme-prodrug system containing HRP together with indoleacetic acid (IAA), in which HRP oxidizes IAA, thereby reducing the viability of cancer cells. Neither the prodrug IAA nor HRP is cytotoxic alone, indicating that combining these two substances in a pure, human-compatible, and non-immunogenic form is necessary to achieve the desired cytotoxic effect. Another study showed that HRP could convert paracetamol into a potent cytotoxin (Tupper et al. "Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol." British Journal of Cancer 90.9 (2004): 1858-1862). However, the authors found that commercially available HRP preparations from plants were not very effective in the conversion reaction, and therefore did not pursue this study further.The polypeptides of the present invention may find particular use in antibody-directed enzyme prodrug therapy (ADEPT) (see, e.g., Bagshawe, "Antibody-directed enzyme prodrug therapy (ADEPT) for cancer," Expert Review of Anticancer Therapy 6.10 (2006): 1421-1431). The principle of ADEPT is to localize an enzyme (e.g., HRP) to the tumor site, typically using an antibody directed against a tumor-associated antigen. A prodrug can be given to the patient, after which it is converted site-specifically to its active species.

[0050] The nucleic acid molecules of the present invention may also find use in therapy, particularly gene therapy such as gene-directed enzyme prodrug therapy (GDEPT) or ADEPT.

[0051] For many applications, particularly industrial applications such as wastewater treatment (e.g., removal of chlorophenols), it is advantageous to immobilize the polypeptide of the present invention on a solid support. Therefore, it is preferred that the polypeptide included in the composition of the present invention be immobilized on a solid support. Enzyme immobilization offers, among other advantages, particularly high storage stability, improved reusability, and reduced operational process costs. Solid support-immobilized HRP and its industrial applications are known in the art. See, for example, Tatsumi, et al. ("Removal of chlorophenols from wastewater by immobilized horseradish peroxidase," Biotechnology and Bioengineering 51.1 (1996): 126-130) and Sarno and Iuliano ("Immobilization of Horseradish Peroxidase on Fe3O4 / Au_GO Nanoparticles to Remove 4-chlorophenols from Wastewater," Chemical Engineering Transactions 73 (2019): 217-222). Those skilled in the art are familiar with methods for immobilizing enzymes on solid supports, as described, for example, in Andreescu, et al. ("Chapter 7 - Nanostructured materials for enzyme immobilization and biosensors." The New Frontiers of Organic and Composite Nanotechnology. Elsevier, 2008, 355-394).

[0052] Nanomaterials are particularly suitable as solid supports due to their specific surface area and effective enzyme loading capacity. In the context of this embodiment, it is therefore preferred that the solid support is a nanoparticle. In the present disclosure, a "nanoparticle" preferably has a surface area of ​​less than 1×10 in all three dimensions. -12 ~1×10 -6m, more preferably in the range of 1×10 -9 ~1×10 -7 The nanoparticles may be, for example, magnetite (Fe3O4) nanoparticles or gold nanoparticles. Magnetic nanoparticles are particularly preferred, as they offer the added advantage of being easily separated by applying a magnetic field. Further preferred examples of solid supports are nanofibers, carbon / polyvinyl materials, carbon nanotubes, nanowires, nanorods, nanocrystals, mesoporous silica, and composite materials.

[0053] In a further preferred embodiment, the solid support is a membrane, particularly a synthetic membrane such as a polymeric membrane. Membranes containing immobilized HRP, methods for producing such membranes, and uses of such membranes (e.g., in wastewater treatment) are known in the art, for example, from Vasileva et al. ("Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing phenol from water," International Journal of Biological Macromolecules 44.2 (2009): 190-194).

[0054] Preferably, for the kit according to the invention, said polypeptide is provided in lyophilized form. In an alternative preferred embodiment, said polypeptide is provided in solution.

[0055] The kits according to the present invention preferably include components selected from buffers, reagents, and instruction manuals.

[0056] In a preferred embodiment, the kit of the present invention further comprises a cross-linking agent suitable for conjugating the polypeptide to another molecule. Preferably, the cross-linking agent is suitable for conjugating the polypeptide to another protein, preferably a binding protein. Kits for conjugating HRP to a protein of interest are known in the art and are available from many commercial suppliers, such as the HRP Conjugation Kit / HRP Labeling Kit ab102890 from Abcam, the LYNX Rapid HRP Antibody Conjugation Kit (product code LNK001P) from Bio-Rad, or the EZ-Link Plus Activated Peroxidase Kit (catalog number 31489) from Thermo Fisher Scientific. The purpose of such kits is typically to allow users to conjugate HRP to any protein of interest, such as an antibody. Such kits may contain an activated form of HRP (for direct reaction with another protein) or may contain HRP and an appropriate cross-linking agent for conjugation by the user.

[0057] Suitable techniques for conjugation and suitable crosslinkers can be found, for example, in the book Hermanson, Greg T. Bioconjugate Techniques. Academic Press, 2013. Preferably, a crosslinker suitable for conjugating the polypeptide to another molecule is capable of forming a covalent bond with the polypeptide. Preferably, the crosslinker contains one, particularly two, reactive groups selected from the group consisting of NHS ester, succinimidyl ester, imidoester, difluoro, haloacetyl, maleimide, pyridyldithiol, and hydrazide. For example, the crosslinker may be a bifunctional PEG linker having a maleimide group and an active ester group, such as maleimide-PEG8-succinimidyl ester (CAS No. 756525-93-6, commercially available, for example, from Sigma-Aldrich under catalog number 746207).

[0058] In a further preferred embodiment, the polypeptide of the present invention is activated for conjugation. This embodiment is particularly preferred in the context of a kit comprising the polypeptide of the present invention. Preferably, "activated for conjugation" means that the polypeptide comprises a reactive group capable of forming a covalent bond with another protein, preferably with a binding protein, in particular with an antibody. It is particularly preferred that the polypeptide is covalently linked to the above-mentioned chemical cross-linking agent, in particular, where at least one reactive group of the cross-linking agent is available for reaction with another molecule, preferably with another protein. This can be achieved, for example, by reacting an additional reactive group of the cross-linking agent with the polypeptide of the present invention to form the covalent bond.

[0059] Polypeptides of the present invention may further find use as reactants for coupled enzyme assays, in which an enzyme of interest (e.g., glucose oxidase) produces HO, which can then be utilized by HRP (see, e.g., Aumiller et al. "Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG / citrate aqueous two-phase system," The Journal of Physical Chemistry B 118.9 (2014): 2506-2517).

[0060] The polypeptides of the present invention may also be used as reactants for polymer cross-linking. For example, HRP has been successfully used in the art for enzymatic cross-linking of dextran-tyramine conjugates for the preparation of hydrogels, such as 3D scaffolds for cartilage tissue engineering applications (Jin et al. "Enzymatically cross-linked dextran-tyramine hydrogels as injectable scaffolds for cartilage tissue engineering." Tissue Engineering Part A 16.8 (2010): 2429-2440).

[0061] To facilitate understanding of the present invention, several terms are defined below. Terms defined in this disclosure have meanings commonly understood by one of ordinary skill in the art in the area to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to a single entity but include general classes, specific examples of which may be used for illustration. While terms in this disclosure are used to describe specific embodiments of the present invention, the use of such terms does not delimit the invention except as outlined in the claims.

[0062] "Percent (%) amino acid sequence identity," "X% sequence identity," or "X% identical" (e.g., "70% sequence identity" or "70% identical") to a reference polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, where any conservative substitutions are not considered part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR), or the "needle" pairwise alignment application in the EMBOSS software package. Those of skill in the art can determine appropriate parameters for aligning sequences, including the algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes of this disclosure, percent amino acid sequence identity values ​​are calculated using sequence alignments in the computer program "needle" from the EMBOSS software package (publicly available from the European Molecular Biology Laboratory; Rice et al., EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet. 2000 Jun;16(6):276-7, PMID: 10827456).

[0063] The needle program can be accessed at the website http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / or downloaded for local installation as part of the EMBOSS package from http: / / emboss.sourceforge.net / It runs on many popular UNIX operating systems, such as Linux.

[0064] To align two protein sequences, the needle program is preferably run with the following parameters: Command line: needle -auto -stdout -asequence SEQUENCE_FILE_A-bsequence SEQUENCE_FILE_B -datafile EBLOSUM62 -gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 -aformat3 pair -sprotein1 -sprotein2(Align_format: pair Report_file:stdout)

[0065] The percent amino acid sequence identity of a given amino acid sequence A "to," "with," or "against" a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity "to," "with," or "against" a given amino acid sequence B) is calculated as follows:

[0066] Ratio (X / Y) x 100 where X is the number of amino acid residues scored as perfect matches by the sequence alignment program "needle" in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. In the case where "the sequence of A is more than N % identical to the entire sequence of B," Y is the entire sequence of B (i.e., the total number of amino acid residues in B). Unless specifically stated otherwise, all % amino acid sequence identity values ​​used in this disclosure are obtained as described in the immediately preceding paragraph using the needle computer program.

[0067] Unless otherwise stated, all parameters used in this disclosure correspond to IUPAC SATP conditions (standard ambient temperature and pressure), specifically a temperature of 25° C. and a pressure of 101,300 Pa.

[0068] Percentages (%) used in this disclosure correspond to weight / volume (w / v) unless specified as weight / weight (w / w) or otherwise specified.

[0069] The present invention relates to the following preferred embodiments: Embodiment 1. A polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, wherein said amino acid sequence comprises at least one amino acid substitution compared to SEQ ID NO:1, and wherein said at least one amino acid substitution is a substitution of amino acid P146 or amino acid N275 of SEQ ID NO:1.

[0070] Embodiment 2. A polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, wherein said amino acid sequence comprises at least two amino acid substitutions compared to SEQ ID NO:1, said at least two amino acid substitutions being substitutions of amino acids P146 and N275 of SEQ ID NO:1.

[0071] Embodiment 3. A polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, wherein said amino acid sequence comprises at least one amino acid substitution compared to SEQ ID NO:1, wherein said at least one amino acid substitution is selected from the group consisting of P146Q, P146A, P146R, P146V, P146E, N275K, N275R, N275D, N275S, N275Q, N275A, and N275E; preferably P146Q or N275K.

[0072] Embodiment 4. The polypeptide of any one of embodiments 1 to 3, wherein the amino acid sequence comprises an amino acid substitution selected from the group consisting of P146Q, P146A, P146R, P146V, and P146E, preferably P146Q, compared to SEQ ID NO: 1.

[0073] Embodiment 5. The polypeptide of any one of embodiments 1 to 4, wherein the amino acid sequence comprises an amino acid substitution selected from the group consisting of N275K, N275R, N275D, N275S, N275Q, N275A, and N275E, preferably N275K, compared to SEQ ID NO: 1.

[0074] Embodiment 6. The polypeptide of any one of embodiments 1 to 5, wherein the amino acid sequence comprises the amino acid substitutions P146Q and N275K compared to SEQ ID NO: 1.

[0075] Embodiment 7. A polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, wherein the amino acid sequence is at least one amino acid substitution selected from the group consisting of P146Q, P146A, P146R, P146V, and P146E, preferably P146Q, compared to SEQ ID NO: 1; and An additional amino acid substitution selected from the group consisting of N275K, N275R, N275D, N275S, N275Q, N275A, and N275E, preferably N275K, compared to SEQ ID NO: 1. The polypeptide comprising:

[0076] Embodiment 8. A polypeptide described in any one of embodiments 1 to 7, wherein the amino acid sequence has a sequence identity of 75% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly 98% or more, and even more preferably 99% or more to SEQ ID NO: 3, and most preferably the amino acid sequence is the sequence described in SEQ ID NO: 3.

[0077] Embodiment 9. The polypeptide of any one of embodiments 1 to 8, wherein the amino acid sequence is the sequence set forth in SEQ ID NO:4.

[0078] Embodiment 10. A polypeptide described in any one of embodiments 1 to 9, wherein the amino acid sequence further comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four, and particularly five amino acid substitutions selected from the group consisting of N13D, N57S, N175S, N255D, and N268D compared to SEQ ID NO: 1.

[0079] Embodiment 11. A polypeptide according to any one of embodiments 1 to 10, wherein the amino acid sequence further comprises the amino acid substitution N13D compared to SEQ ID NO:1.

[0080] Embodiment 12. A polypeptide according to any one of embodiments 1 to 11, wherein the amino acid sequence further comprises the amino acid substitution N57S compared to SEQ ID NO: 1.

[0081] Embodiment 13. A polypeptide according to any one of embodiments 1 to 12, wherein the amino acid sequence further comprises the amino acid substitution N175S compared to SEQ ID NO: 1.

[0082] Embodiment 14. A polypeptide according to any one of embodiments 1 to 13, wherein the amino acid sequence further comprises the amino acid substitution N255D compared to SEQ ID NO: 1.

[0083] Embodiment 15. A polypeptide according to any one of embodiments 1 to 14, wherein the amino acid sequence further comprises the amino acid substitution N268S compared to SEQ ID NO: 1.

[0084] Embodiment 16. A polypeptide according to any one of embodiments 1 to 15, wherein the amino acid sequence further comprises the amino acid substitution T110V or K232N compared to SEQ ID NO: 1.

[0085] Embodiment 17. The peroxidase activity is greater than or equal to 0.01 mM when measured in 50 mM phosphate-citrate buffer containing 1 mM H2O2 at pH 5 and 30°C. -1 s -1 or more, preferably 0.1 mM -1 s -1 or more, more preferably 1 mM -1 s -1 More preferably, 10 mM -1 s -1 More preferably, 20 mM -1 s -1 More preferably, 100 mM -1 s -1 More preferably, 1000 mM or more -1 s -1 More preferably, 10,000 mM or more -1 s -1Above 20,000mM -1 s -1 The polypeptide according to any one of embodiments 1 to 16, which has a kcat / Km value for the substrate 3,3',5,5'-tetramethylbenzidine (TMB) of at least 1.

[0086] Embodiment 18. The peroxidase activity is greater than or equal to 0.1 mM when measured in 50 mM phosphate-citrate buffer containing 10 mM H2O2 at pH 5 and 30°C. -1 s -1 or more, preferably 1 mM -1 s -1 or more, more preferably 10 mM -1 s -1 More preferably, 100 mM -1 s -1 or more, most preferably 250 mM -1 s -1 The polypeptide according to any one of embodiments 1 to 17, which has a kcat / Km value for the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) that is equal to or greater than this value.

[0087] Embodiment 19. The peroxidase activity is greater than or equal to 1 mM ABTS when measured in 50 mM phosphate-citrate buffer at pH 5 and 30°C. -1 s -1 or more, preferably 10 mM -1 s -1 or more, more preferably 100 mM -1 s -1 More preferably, 1000 mM or more -1 s -1 or more, most preferably 2500 mM -1 s -1 The polypeptide according to any one of embodiments 1 to 18, which has a kcat / Km value for the substrate hydrogen peroxide (H2O2) of equal to or greater than this value.

[0088] Embodiment 20. A polypeptide described in any one of embodiments 1 to 19, wherein the polypeptide consisting of the amino acid sequence has increased thermal stability compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2.

[0089] Embodiment 21: The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide consisting of the amino acid sequence has a half-life at 60°C in a buffer solution consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol, and 500 mM NaCl of at least 0.5 hours, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 4 hours, and most preferably at least 6 hours, as measured by residual peroxidase activity in a 50 mM phosphate-citrate buffer solution containing 1 mM HO at pH 5 and 30°C using 7 mM ABTS.

[0090] Embodiment 22: The polypeptide of any one of embodiments 1 to 21, wherein the polypeptide consisting of the amino acid sequence has a longer half-life at 60°C in a buffer solution consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol, and 500 mM NaCl than the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2, when measured by residual peroxidase activity using 7 mM ABTS in a 50 mM phosphate-citrate buffer solution containing 1 mM HO at pH 5 and 30°C.

[0091] Embodiment 23. A polypeptide described in embodiment 22, having a half-life at 60°C that is at least 1.5 times, preferably at least 3 times, more preferably at least 6 times, and even more preferably at least 12 times longer than a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2.

[0092] Embodiment 24. The polypeptide of any one of embodiments 1 to 23, further comprising the amino acid sequence of a binding protein, preferably the amino acid sequence of an antibody.

[0093] Embodiment 25. A polypeptide according to any one of embodiments 1 to 24, further comprising the amino acid sequence of streptavidin.

[0094] Embodiment 26. A polypeptide according to any one of embodiments 1 to 25, further comprising the amino acid sequence of Protein A, Protein G, or Protein L.

[0095] Embodiment 27. A nucleic acid molecule comprising a sequence encoding the polypeptide according to any one of embodiments 1 to 26.

[0096] Embodiment 28. An expression vector comprising the nucleic acid molecule of embodiment 27.

[0097] Embodiment 29. A host cell comprising the expression vector of embodiment 28.

[0098] Embodiment 30. The host cell of embodiment 29, which is a bacterium, preferably E. coli.

[0099] Embodiment 31. A method for producing a polypeptide described in any one of embodiments 1 to 26, comprising culturing a host cell described in embodiment 30 and recovering the polypeptide.

[0100] Embodiment 32. A composition comprising a polypeptide according to any one of embodiments 1 to 26.

[0101] Embodiment 33. The composition of embodiment 32, further comprising one or more formulation additives.

[0102] Embodiment 34. The composition of embodiment 32 or embodiment 33, comprising at least 0.01 mg, preferably at least 0.1 mg, more preferably at least 1 mg, even more preferably at least 5 mg, especially at least 10 mg of said polypeptide.

[0103] Embodiment 35. A composition described in any one of embodiments 32 to 34, containing the polypeptide at a concentration of 0.0001% w / w or more, preferably 0.001% w / w or more, more preferably 0.01% w / w or more, even more preferably 0.1% w / w or more, even more preferably 1% w / w or more, particularly 10% w / w or more.

[0104] Embodiment 36. The composition of any one of Embodiments 32 to 35, which is a pharmaceutical composition, wherein the pharmaceutical composition preferably comprises a pharmaceutically acceptable formulation excipient.

[0105] Embodiment 37. A composition according to any one of embodiments 32 to 36, wherein the polypeptide is immobilized on a solid support, preferably a nanoparticle.

[0106] Embodiment 38. A kit comprising a polypeptide according to any one of embodiments 1 to 26 or a composition according to any one of embodiments 32 to 37.

[0107] Embodiment 39. The kit of embodiment 38, wherein the polypeptide is provided in lyophilized form.

[0108] Embodiment 40. The kit of embodiment 38 or embodiment 39, wherein the polypeptide is provided in solution.

[0109] Embodiment 41. A kit according to any one of embodiments 38 to 40, further comprising a cross-linking agent suitable for conjugating the polypeptide to another protein.

[0110] Embodiment 42. A kit according to any one of embodiments 38 to 41, further comprising components selected from a buffer, a reagent, and an instruction manual.

[0111] The present invention is further illustrated, but not limited, by the following figures and examples.

[0112] Figure 1: Residual activity of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (mHRP, SEQ ID NO: 4) compared with recombinant wild-type HRP (rHRP, SEQ ID NO: 2) and plant-derived HRP (pHRP). 150 μl of pHRP (triangles), mHRP (squares), and rHRP (circles) at a concentration of 2.86 μM were incubated at 60°C for up to 10 hours in 50 mM BisTris / HCl (pH 7), 0.5 M NaCl, and 7% glycerol. Initial and residual enzyme activities were measured by ABTS as described in Example 1 and plotted as percentages versus incubation time. mHRP exhibited a greater than 13-fold improved thermostability compared with rHRP and a 1.7-fold improved thermostability compared with plant-derived (i.e., glycosylated) pHRP.

[0113] Example 1. Materials and Methods -Expression and purification of HRP mutants- Plant HRP type VI-A (catalog number: P6782) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All HRP variants produced in E. coli consisted of the sequence set forth in SEQ ID NO: 2 with the indicated mutations unless otherwise noted.

[0114] Expression hosts and plasmids Standard molecular screening techniques were performed as previously described (Humer and Spadiut, "Improving the performance of horseradish peroxidase by site-directed mutagenesis," International Journal of Molecular Sciences 20.4 (2019): 916). The hrp gene encoding HRP variant C1A (wild-type HRP; SEQ ID NO: 2) was codon-optimized for E. coli and obtained from GenScript USA Inc. (Piscataway, NJ, USA). HRP was produced in E. coli strain BL21(DE3) (Lucigen, Middleton, WI, USA) from pSF-T7-LacO-NH2-dsbA(OG4591) (Oxford Genetics Ltd., Oxford, UK) or pET21d+ (Novagen, San Diego, CA, USA). Plasmid pSFT7 encodes a Dsb tag for periplasmic export, which is cleaved after export. Plasmid pET21d+ was used for HRP inclusion body production in the cytoplasm. A stop codon was introduced so that the protein was produced without any tag.

[0115] Protein expression and purification from inclusion bodies (IBs) For the cultivation of BL21(DE3) cells harboring the vector pET21d+ without any N- or C-terminal tag but carrying the hrp gene (or its variant), SB medium (32 g L -1 Tryptone; 20gL -1 Yeast extract; 5gL -1 NaCl; 5 mM NaOH) was used. Ampicillin was 100 mg L -1 The preculture was performed in 50 mL of SB Amp The cells were grown overnight at 37°C with shaking (250 rpm) in SB medium and transferred to 2.5 L Ultra Yield Flasks (UYF). Amp In a final volume of 500 mL of medium, the optical density (OD) of 0.3 was reached. 600OD 600 Cells were grown at 37°C with shaking (250 rpm) until the ΔΨ was 0.5, after which hrp expression was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 20 h of growth at 25°C and 250 rpm, cells were harvested by centrifugation (5000 g, 20 min, 4°C).

[0116] Biomass was resuspended in Buffer A (50 mM TRIS / HCl; pH 8; 500 mM NaCl; 1.5 mM EDTA) using an IKA T10 Basic Ultra-Turrax homogenizer at 3–5 mL per gram of wet biomass and homogenized (using a GEA Niro Soavi Panda PLUS) at >1300 bar, 3 passages, with cooling. The homogenized suspension was centrifuged at 15,650 g for 20 min at 4°C, the supernatant was discarded, and the cell debris was resuspended in Buffer B (50 mM TRIS / HCl; pH 8; 500 mM NaCl; 2 M urea) at 10 mL per gram of wet cell debris, and centrifuged again at 15,650 g for 20 min at 4°C. The washing step with Buffer B was repeated once. The IBs / cell debris were then resuspended in water (5 mL water per 1 g wet cell debris), the suspension was aliquoted into pre-weighed 50 mL reaction tubes, centrifuged (15650 g; 20 min, 4°C), and the pellets were stored at -20°C until further use.

[0117] For solubilization, an aliquot of frozen IBs was thawed, weighed to calculate the wet inclusion body (wIB) weight, and resuspended in the appropriate solubilization buffer (50 mM TRIS / HCl; pH 8.5; 6 M urea) to a wIB concentration of 100 g / L. After resuspension, DTT was added (using a 1 M DTT stock solution) to a final concentration of 7.11 mM DTT in the solubilization mix, and the solubilization mix was incubated (4°C; 0.5 h; gentle agitation) and subsequently centrifuged (20379 g; 20 min; 4°C). The supernatant was used immediately for refolding, and the pellet was discarded.

[0118] The solubilized material was diluted 1:40 in an appropriate refolding buffer (e.g., 20 mM TRIS / HCl (pH 8.5), 2 M urea, 7% glycerol, 2 mM CaCl, 1.27 mM GSSG), to which hemin was added to a final concentration of 20 μM, and refolding was carried out at 10°C for 19 hours.

[0119] The protein was further purified by hydrophobic interaction chromatography (HIC). An 80 ml column packed with Butyl Sepharose 4 Fast Flow (GE Healthcare) was used. The column was equilibrated with Buffer A (Buffer A: 20 mM Bis-Tris, pH 7; 4 M NaCl) at a flow rate of 113 cm / h until all signals remained constant. A 1250-1300 ml load was then applied at a flow rate of 90 cm / h. After loading, a wash step with 20% Buffer B (Buffer B: 20 ​​mM Bis-Tris, pH 7) was performed for two column volumes (CV) at a flow rate of 90 cm / h. Stepwise elution was then performed using 75% Buffer B (flow rate 79 cm / h) and 100% Buffer B (flow rate 90 cm / h). Active HRP eluted at 75% Buffer B.

[0120] -Kinetic parameters- Enzyme kinetic parameters were determined for the substrates ABTS, TMB, and hydrogen peroxide in a 96-well plate assay using a Tecan Infinite M200 PRO instrument (Tecan, Manndorf, Switzerland).

[0121] For assays using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate, the reaction mixture in each well of a 96-well plate contained a saturating hydrogen peroxide concentration of 1 mM and varying TMB concentrations (20-550 μM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein sample (5 μL) was mixed with 175 μL of the TMB-buffer mixture, and the reaction was initiated with 20 μL of 10 mM hydrogen peroxide solution. The increase in absorbance was followed at 652 nm for 60 seconds at 30°C in a Tecan Infinite M200PRO instrument. Reaction kinetic parameters were calculated using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and the extinction coefficient ε 652 =39mM -1 cm -1 (See Josephy, et al. "The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates." Journal of Biological Chemistry 257.7 (1982): 3669-3675.)

[0122] For measurements using ABTS as the substrate, the reaction mixture in each well of a 96-well plate contained a saturating hydrogen peroxide concentration of 1 mM and varying ABTS concentrations (0.1–7 mM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein sample (5 μL) was mixed with 175 μL of the ABTS-buffer mixture, and the reaction was initiated with 20 μL of 10 mM hydrogen peroxide solution. The increase in absorbance was followed at 420 nm for 120 s at 30°C in a Tecan Infinite M200PRO instrument. Reaction kinetic parameters were calculated using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and the extinction coefficient ε420 =36mM -1 cm -1 (See Childs and Bardsley, "The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen," Biochemical Journal 145.1 (1975): 93-103.)

[0123] For measurements using hydrogen peroxide as the substrate, the reaction mixture in each well of a 96-well plate contained a saturating ABTS concentration of 10 mM and varying hydrogen peroxide concentrations (0.001–1 mM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein sample (5 μL) was mixed with 145 μL of the hydrogen peroxide-buffer mixture, and the reaction was initiated with 50 μL of ABTS solution (40 mM). The increase in absorbance was followed at 420 nm for 120 seconds at 30°C in a Tecan Infinite M200PRO instrument. Kinetic parameters were calculated using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and the extinction coefficient ε 420 =36mM -1 cm -1 (See Childs and Bardsley, "The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen," Biochemical Journal 145.1 (1975): 93-103.)

[0124] -Thermal stability- The thermal stability of the enzyme variants was evaluated in 50 mM BisTris / HCl (pH 7), 7% glycerol, 500 mM NaCl at 60° C. Enzyme activity for ABTS was measured for HRP wild-type (SEQ ID NO: 2) and HRP N13D / N57S / N255D / N268D after 0, 30, 60, 90, and 120 minutes; and for variants HRP N13D / N57S / N175S / N255D / N268D, HRP N13D / N57S / P146Q / N175S / N255D / N268D, HRP N13D / N57S / N175S / N255D / N268D, and HRP N13D / N57S / P146Q / N175S / N255D / N268D after 0, 90, 180, 300, 420, and 588 minutes. The enzyme activity was measured for N13D / N57S / P146Q / N175S / N255D / N268D / N275K; and for plant HRP after 0, 90, 180, 300, and 420 minutes. The enzyme concentration of all variants, including plant HRP, was 2.86 μM upon heat treatment. The samples were then cooled on ice for 5 minutes and then centrifuged at 16162 g for 15 minutes at 4°C. Residual activity was then measured with 7 mM ABTS using a Tecan Infinite M200PRO instrument. The reaction mixture contained 5 μL of protein, a saturating hydrogen peroxide concentration of 1 mM, and 7 mM ABTS in a total volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). The increase in absorbance was followed at 420 nm for 120 seconds at 30°C. Residual enzyme activity was plotted against incubation time and the half-life at 60°C was calculated using the inactivation rate in the following formula:

[0125] t 1 / 2 =ln(2) / k in In the formula, t 1 / 2 is the half-life, and k in is the slope of the log residual activity.

[0126] Example 2. Thermal stability of HRP variants Several mutants of HRP were expressed and purified, and their thermostabilities were measured as described in Example 1. Many mutations were found to increase thermostability. The most preferred mutant, HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4), exhibited greater than 13-fold improved thermostability compared to wild-type HRP (SEQ ID NO: 2) and a 1.7-fold improvement in thermostability compared to the plant-derived (i.e., glycosylated) enzyme. Importantly, the combination of mutations P146Q and N275K (HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K) exhibited significantly higher thermal stability than either mutations P146Q alone (HRP N13D / N57S / P146Q / N175S / N255D / N268D) or mutation N275K alone (HRP N13D / N57S / N175S / N255D / N268D / N275K).

[0127] Table 1. Thermal stability of plant HRPs and recombinantly produced HRP variants [Table 1]

[0128] Example 3. Kinetic parameters of HRP variants Several mutants of HRP were expressed and purified, and kinetic parameters were determined for the substrates ABTS, TMB, and hydrogen peroxide as described in Example 1. HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) was found to be significantly more active than HRP N13D / N57S / N175S / N255D / N268D with the substrates TMB and hydrogen peroxide. Mutations P146Q and N275K were found to have a strong beneficial effect on enzyme activity.

[0129] Table 2. Kinetic parameters for the substrate TMB. [Table 2]

[0130] Table 3. Kinetic parameters for the substrate H2O2 [Table 3]

[0131] Table 4. Kinetic parameters for the substrate ABTS [Table 4]

[0132] Example 4. Comparison between plant-derived HRP and wild-type HRP The kinetic parameters and thermostability of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) were compared with those of wild-type HRP (SEQ ID NO: 2) and plant-derived HRP (pHRP). The kinetic parameters and thermostability were determined as described in Example 1.

[0133] Thermal stability measurements are shown in Figure 1. As previously described in Example 2 of the present disclosure, the most preferred mutant HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (mHRP, SEQ ID NO: 4) exhibited greater than 13-fold improved thermal stability compared to wild-type HRP (rHRP, SEQ ID NO: 2), and even 1.7-fold improved thermal stability compared to plant-derived (i.e., glycosylated) pHRP (see Table 1). It was also found that, despite this strongly improved thermal stability, catalytic efficiencies similar to those of both wild-type HRP (rHRP, SEQ ID NO: 2) and plant-derived HRP were observed (see Tables 5 and 6 below).

[0134] Table 5. Kinetic parameters for the substrate ABTS [Table 5]

[0135] Table 6. Kinetic parameters for the substrate TMB. [Table 6]

[0136] Example 5. Site-saturation mutagenesis at positions 146 and 275 To test the effects of all possible amino acid substitutions at positions P146 and N275, site-saturation mutagenesis was performed at these positions. The preferred mutant HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3) was selected as the starting point for the site-saturation mutagenesis. The effects of amino acid substitutions at positions 146 and 275 were examined individually.

[0137] -Library creation- The following plasmids were constructed using standard molecular cloning techniques. To introduce mutations into the hrp gene at positions 146 and 275 by site-saturation mutagenesis, whole-plasmid PCR of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3) in pSF-T7 was used. A 6.3 kb fragment was amplified with each oligonucleotide to generate a site-saturation library (Table 7). All oligonucleotides were purchased from Microsynth (Balgach, Switzerland). The oligonucleotides were phosphorylated using the following protocol: 300 pmol primer DNA, 1x T4PNK buffer (NEB), 1 mM ATP, 5% PEG, 10 units T4 polynucleotide kinase (PNK, NEB). The reaction was incubated at 37°C for 45 minutes and then heat-inactivated at 65°C for 20 minutes. Each PCR reaction contained 1x Q5 reaction buffer, 200 μM dNTP Mix, 200 nM of both forward and reverse phosphorylated primers, 100 ng of template vector DNA, and 1 U of Q5 High-Fidelity DNA polymerase. PCR products were purified using the Monarch PCR & DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA), and template plasmid DNA was removed by FastDigest DpnI digestion (Thermo Scientific™, Waltham, MA, USA). Two FastDigest units (FDU) of DpnI were added to the cleaned PCR products and incubated at 37°C for 4 hours. After heat inactivation at 80°C for 20 minutes, the plasmids were blunt-end ligated: 50 ng of plasmid DNA, 1x T4 DNA ligase buffer (NEB), 400 sticky end units of T4 DNA ligase (NEB), overnight at 16°C. After heat inactivation at 65°C for 20 minutes, the plasmid was transformed into BL21(DE3).

[0138] Table 7. Primers used for whole-plasmid PCR [Table 7]

[0139] -screening- Positively transformed cells were harvested from the selection plate and cultured in 200 μl of SB medium (32 g L ) in a 96-well plate in a plastic box to prevent drying. -1 Tryptone; 20gL -1 Yeast extract; 5gL -1 NaCl; 5 mM NaOH; 50 mg L -1 The cells were grown for 16 hours at 37°C and 250 rpm in 100% ethanol (100% kanamycin). Afterwards, 90 μl of 75% glycerol was added to the master plates, which were then stored at -80°C. 10 μl of the master plate was inoculated into a slave plate containing 190 μl of SB medium, which contained 2 mM CaCl2; 6 μM hemin; and 0.1 mM IPTG in a final volume of 200 μl. The cells were grown in a plastic box at 25°C and 250 rpm for 16 hours, and cell density was determined by measuring absorbance at 600 nm in a Tecan Infinite M200PRO (Tecan, Manndorf, Switzerland) plate reader. The plates were then centrifuged at 5000 g for 6 minutes at 4°C in a Thermo-Fisher Lynx Sorvall centrifuge, and the cells were inoculated with 5 μl of SB medium. -1The cells were thoroughly resuspended in 200 μl / well of B-PER Bacterial Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) with 100 mM DNase I and 1 / 2 protease inhibitor cocktail tablets (Complete Tablets, EDTA-free; Roche Diagnostics GmbH, Mannheim, Germany) and 200 mM MgCl2. Cell lysis was carried out at room temperature for 15 min, followed by centrifugation at 5000 g for 20 min at 4°C. Total protein content was then measured by the Bradford method. 90 μl from each well was transferred to a new plate and incubated at 80°C for 20 min (position 146) or 80°C for 15 min (position 275). Both the heated and control plates were then centrifuged again at 5000 g for 20 min at 4°C. Enzyme activity was then measured with 395 μM TMB (position 146) or 406 μM TMB (position 275), 1 mM HO, and 10 μl of cell lysate in a total volume of 200 μl of 50 mM phosphate-citrate buffer (pH 5). Measurements were performed at 30°C and measured as the increase in absorbance at 652 nm (ε = 3.9 × 10 for the blue TMB radical). 4 M -1 cm -1 ) was monitored for 120 seconds using a Tecan Infinite M200 PRO plate reader. Initial and residual activities were normalized using total protein concentration, and thermostability was expressed as the ratio of residual activity to initial activity. 180 colonies were screened for each position, corresponding to a predicted library completeness of >99%. Each plate contained six colonies of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (corresponding to SEQ ID NO: 3) as a positive control.

[0140] -result- The results for selected mutants are shown below in Table 8 (position P146) and Table 9 (position N275). Because this example uses a 96-well plate-based screening assay, the variability (standard deviation) is higher than with other assays reported in this disclosure. Therefore, results for each mutant should only be compared within each plate measured.

[0141] As can be seen from Tables 8 and 9, several different amino acid substitutions at positions 146 and 275 were found to lead to excellent thermostability. Several amino acid substitutions at each position gave advantageous results. For position 146, 146A, 146R, 146V, 146E, and especially 146Q were found to be particularly advantageous (all within the standard deviation of the most preferred variant 146Q). For position 275, the best results were observed for 275R, 275D, 275S, 275Q, 275A, 275E, and especially 275K (all within the standard deviation of the most preferred variant 275K).

[0142] Table 8. Selected mutants generated by site-saturation mutagenesis at position 146. Two separate plates were measured (results should be compared within each plate). Some mutants contained the same amino acid substitution. For the control (SEQ ID NO: 3), the mean and standard deviation from six replicates are shown. [Table 8]

[0143] Table 9. Selected mutants generated by site-saturation mutagenesis at position 275. Two separate plates were measured (results should be compared within each plate). Some mutants contained the same amino acid substitution. For the control (SEQ ID NO: 3), the mean and standard deviation from six replicates are shown. [Table 9]

[0144] Example 6. Kinetic parameters and thermostability of additional HRP variants The beneficial effects of mutations at positions P146 and N275 were investigated for wild-type HRP (SEQ ID NO: 2), HRP N175S, and HRP N13D / N57S / N175S / N255D / N268D. Furthermore, the beneficial effects of single mutants and their combinations were investigated. In this context, the role of mutations at positions P146 and N275 on biochemical properties was determined by measuring specific enzyme activity and thermostability at 60°C.

[0145] -Materials and Methods- The following mutants of wild-type HRP (SEQ ID NO: 2) were created and verified by Sanger sequencing: HRP P146Q, HRP N175S, HRP N275K, HRP P146Q / N275K, HRP P146Q / N175S, HRP N175S / N275K, HRP P146Q / N175S / N275K. Purification was performed as described in Example 1. Kinetic parameters and thermostability were also determined as described in Example 1.

[0146] (Specific enzyme activity (ABTS)) The specific activity (units / mg protein) of all HRP variants was tested using the substrate ABTS in a Tecan plate reader (Table 10). HRP P146Q showed a 1.4-fold higher specific activity than HRP wild-type. Interestingly, HRP N175S showed a lower specific activity; however, HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) was able to attenuate this activity reduction, restoring it to the value of HRP wild-type.

[0147] Table 10. Specific activity of selected HRP variants compared to HRP wild type and HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K using ABTS as substrate. [Table 10]

[0148] -Specific enzyme activity (H2O2)- Furthermore, specific activity (units / mg protein) was determined using hydrogen peroxide substrate. Here, the same trend as observed in the data for ABTS was observed: variant P146Q led to increased specific activity, while introduction of N175S led to lower values. This was also the case for the double mutants P146Q / N175S and N175S / N275K, as well as the triple mutant P146Q / N175S / N275K. When N175S was absent or when the additional mutation of SEQ ID NO:4 was present, specific activity was similar to or even better than SEQ ID NO:2.

[0149] Table 11. Specific activity of selected HRP variants compared to HRP wild type and HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K using H2O2 as substrate. [Table 11]

[0150] -Specific enzyme activity (TMB)- For the substrate TMB, the differences in specific activity between the HRP variants were less pronounced, but P146Q again showed a significant increase in specific enzyme activity, while N175S showed the lowest U / mg compared to the wild type.

[0151] Table 12. Specific activity of selected HRP variants compared to HRP wild type and HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K using TMB as substrate. [Table 12]

[0152] -Thermal stability- When the enzyme stability at 60°C was examined for all HRP variants, surprisingly, N175S alone was not responsible for the increased stability at high temperatures. While HRP N13D / N57S / N255D / N268D increased the enzyme half-life by 1.5-fold, HRP N175S improved stability by 7.7-fold, and their combination increased stability by 13-fold compared to the HRP wild-type, suggesting a synergistic effect (Table 13). The fact that HRP N13D / N57S / N175S / N255D / N268D and HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) showed similar stability suggests that the quadruple mutant in combination with N175S is responsible for this improvement.

[0153] For P146Q and N275K, the stability of the single mutant and the double mutant P146Q / N275K was slightly reduced compared to the wild-type HRP. However, the triple mutant P146Q / N175S / N275K showed the same stability as N175S alone, while the double mutants P146Q / N175S and N175S / N275K were less stable. This indicates a negative effect on stability when only one of the mutants is combined with N175S, but this is alleviated in the combination, suggesting a synergistic effect between P146Q, N275K, and N175S. The same effect is seen for HRP N13D / N57S / P146Q / N175S / N255D / N268D and HRP N13D / N57S / N175S / N255D / N268D / N275K when compared to HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4).

[0154] Table 13. Thermal stability of several HRP variants, expressed as half-life at 60° C. Data in grey shaded area are taken from Example 2 for comparison. [Table 13]

[0155] -Conclusion- It was found that the substitution P146Q itself led to a strong increase in HRP enzyme activity with all substrates tested, for example with the substrate ABTS, which resulted in a 1.4-fold increase over the wild-type enzyme (SEQ ID NO: 2) and a 2-fold higher specific activity compared to HRP N175S.

[0156] The substitution N175S was found to strongly increase the thermal stability of HRP. This effect was observed with N175S alone and even more strongly in combination with the mutated N-glycosylation site amino acids N13D / N57S / N255D / N268D, where a synergistic effect was observed. The single mutation P146Q or the single mutation N275K combined with N175S led to a slight decrease in stability. However, this decrease was alleviated when both P146Q and N275K were combined with N175S, suggesting a synergistic effect between P146Q, N275K, and N175S.

[0157] N175S was found to reduce enzyme activity for some substrates. This effect, however, was attenuated by N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4). Therefore, this mutant offers a combination of high thermostability and optimal kinetic performance.

Claims

1. A polypeptide having peroxidase activity and comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, wherein the amino acid sequence in the polypeptide comprises at least one amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:1, wherein the at least one amino acid substitution is selected from the group consisting of P146Q, P146A, P146R, P146V, and P146E; the amino acid sequence of the polypeptide further comprises an N175S amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:1; The polypeptide has increased thermal stability compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:

2. The polypeptide.

2. The polypeptide of claim 1, wherein the at least one amino acid substitution is P146Q.

3. 3. The polypeptide of claim 1 or claim 2, wherein the amino acid sequence in the polypeptide has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

3.

4. The polypeptide according to any one of claims 1 to 3, wherein the amino acid sequence in the polypeptide has at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO:

3.

5. 5. The polypeptide of any one of claims 1 to 4, wherein the amino acid sequence in the polypeptide further comprises an amino acid substitution selected from the group consisting of N275K, N275R, N275D, N275S, N275Q, N275A, and N275E.

6. The polypeptide of any one of claims 1 to 5, wherein the amino acid sequence of the polypeptide comprises the amino acid substitutions P146Q and N275K compared to the amino acid sequence set forth in SEQ ID NO:

1.

7. The polypeptide according to any one of claims 1 to 6, wherein the amino acid sequence in the polypeptide further comprises at least one amino acid substitution selected from the group consisting of N13D, N57S, N255D, and N268D compared to the amino acid sequence set forth in SEQ ID NO:

1.

8. The polypeptide according to any one of claims 1 to 7, wherein the amino acid sequence in the polypeptide comprises amino acid substitutions of P146Q, N175S, and N275K compared to the amino acid sequence set forth in SEQ ID NO:

1.

9. The polypeptide of any one of claims 1 to 8, wherein the amino acid sequence in the polypeptide further comprises the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: N13D, N57S, N255D, and N268D.

10. The polypeptide of claim 1, wherein the amino acid sequence in the polypeptide is the amino acid sequence set forth in SEQ ID NO:

3.

11. The peroxidase activity was determined by 1 mM H ２ O ２ When measured in 50 mM phosphate-citrate buffer at pH 5 and 30°C containing －１ s －１ The polypeptide according to any one of claims 1 to 10, which has a kcat / Km value corresponding to the above for the substrate 3,3',5,5'-tetramethylbenzidine (TMB).

12. The polypeptide consisting of the amino acid sequence has a half-life of 1 mM H at 60° C. in a buffer solution consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol, and 500 mM NaCl. ２ O ２ The polypeptide according to any one of claims 1 to 11, wherein the residual peroxidase activity is measured using 7 mM ABTS in a 50 mM phosphate-citrate buffer containing the compound at pH 5 and 30°C, for 1 hour or more.

13. A nucleic acid molecule comprising a sequence encoding the polypeptide of any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the expression vector of claim 14.

16. A method for producing a polypeptide according to any one of claims 1 to 12, said method comprising culturing a host cell according to claim 15 and recovering said polypeptide.

17. A composition comprising the polypeptide of any one of claims 1 to 12, said composition comprising one or more formulation additives.

18. A kit comprising the polypeptide of any one of claims 1 to 12, further comprising components selected from buffers, reagents, and instruction manuals.

19. 20. The kit of claim 18, further comprising a cross-linking agent suitable for conjugating the polypeptide to another protein.

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