Enzyme having lactate oxidizing activity and its use

Abstract

The present invention relates to an enzyme having lactate oxidizing activity, the use of such an enzyme for the determination of lactate in a sample, an electrode comprising such an enzyme, and methods for producing such an enzyme.

Description

[0001] ENZYME HAVING LACTATE OXIDIZING ACTIVITY AND ITS USE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of enzymes and their use for the detection and quantification of analytes, in particular to enzymes and enzyme-based methods for the detection and / or quantification of lactate. The invention further relates to specific enzymes having lactate oxidizing activity, their application for the detection of lactate, and to the recombinant production of such enzymes.

[0004] BACKGROUND OF THE INVENTION

[0005] In the food and beverage industries, as well as clinical diagnostics, there is a need for highly selective, sensitive, rapid, and reliable methods of determining the presence of key ingredients or metabolites which determine the quality of the product, serve as markers for diseases or as markers for the physiological state of humans. L-lactate is an example of such an ingredient or metabolite.

[0006] For example, analysis of lactate levels in blood is important for the clinical diagnosis of hypoxia, lactic acidosis, for drug toxicity tests, hyperlactemia in diabetic and liver diseases, sepsis, and thiamine deficit. Lactate is also measured when monitoring the performance of athletes and developing optimum training regimens.

[0007] In pharmaceutical industry, the presence of the metabolite lactate has to be frequently monitored. For example, during the production of recombinant proteins in Chinese hamster ovary (CHO) cell cultures, lactate is one of the critical parameters because of its toxic and growth inhibiting effect on the cells.

[0008] Thereby, the relevant concentration ranges for L-lactate vary among the specific sample types and aims of the measurement. For example, the physiological relevant range of L-lactate in blood is usually between 1 and 25 mM (Goodwin et al., 2007) but the physiological relevant range of L-lactate can also be up to >100 mM in sweat after exhaustive exercise (Mitsubayashi et al., 1994).

[0009] Currently, the determination of lactate is based on an enzymatic test system measuring the oxidation of L-lactate to pyruvate. Traditionally, these enzymatic methods are based on NAD+-dependent lactate dehydrogenases (LDH) isolated from animal muscles or heart, or on bacterial lactate oxidase (LOx). Thereby, the lactate content is determined by the spectrophotometric detection of NADH or a colorimetric assay of H2O2. Many other methods have been proposed as well; for example, spectrophotometry, fluorimetry, and pH potentiometric measurements. Also, amperometric biosensors have been proposed based on O2 consumption and H2O2 formation which is detected on electrodes.

[0010] Rathee et al. (2016) describe basic principles of lactate biosensors. In general, a biosensor consists of a biological recognition element e.g., an enzyme, which is able to specifically interact with a target molecule and a transducer able to convert this interaction into a measurable signal. In biosensors, enzymes are connected to electrodes and electric currents are measured that are proportional to the enzymes’ substrate concentration in a sample. Biosensors are easy to operate, highly specific and do not need expensive and heavy equipment like it is the case for HPLC or NMR.

[0011] The currently preferred enzyme used for the construction of L-lactate biosensors is L-lactate oxidase (LOx), especially LOx from Aerococcus viridans (AvLOX) (see e.g., Taurino, I. et al. (2013)). LOx belongs to the family of FMN-dependent a-hydroxy acid oxidoreductases (HAOx; EC: 1.1.3.15; Maeda-Yorita et al., 1995) and as such oxidizes a-hydroxy acids to their corresponding a-keto acids via a ping-pong reaction mechanism (Maeda-Yorita et al., 1995). L-lactate, the preferred substrate of LOx, is oxidized to pyruvate in the first (reductive) half-reaction while FMN is reduced. In the second (oxidative) half-reaction, oxygen is used as an electron acceptor to re-oxidize FMN and is itself reduced to hydrogen peroxide (H2O2). Therefore, a biosensor based on an oxidase such as LOx is dependent on oxygen and is a first-generation biosensor. In second-generation biosensors, redox mediators other than the O2 / H2O2 redox mediator are applied to transfer electrons from the enzyme to the electrode.

[0012] US2013 / 071868A1 discloses a mutant lactate oxidase and uses thereof in determining lactate.

[0013] WO2022 / 072677A1 discloses an engineered lactate oxidoreductase having dehydrogenase activity fused to a cytochrome domain.

[0014] However, currently known enzymes having lactate oxidizing activity are not stable enough, are available only with added stabilizers, and are not easy to produce in high yields and quality.

[0015] Thus, there is an urgent need in the art for improved enzymes, their application, and their recombinant production. SUMMARY OF THE INVENTION

[0016] It is the objective of the present invention to provide improved enzymes, their application, and their recombinant production.

[0017] The objective is solved by the subject matter of the present invention.

[0018] According to the invention there is provided an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1.

[0019] Specifically, the enzyme described herein comprises a sequence having at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1.

[0020] Specifically, the enzyme described herein has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0021] Specifically, the enzyme described herein is a recombinant enzyme.

[0022] Specifically, it further has a purification tag at its N-terminus.

[0023] Specifically, the enzyme further has a Met at its N-terminus.

[0024] Specifically, the sequence is selected from any one of SEQ ID NOs:2, 3, 4, 5 or 6.

[0025] According to the invention there is further provided the use of the enzyme described herein for the determination of lactate in a sample.

[0026] According to the invention there is further provided an electrode comprising the enzyme described herein.

[0027] According to the invention there is further provided a biosensor comprising the electrode described herein.

[0028] According to the invention there is further provided a nucleotide sequence encoding the enzyme described herein.

[0029] According to the invention there is further provided a host cell expressing the enzyme described herein.

[0030] According to the invention there is further provided a method of producing the enzyme described herein in a host cell, said method comprising the steps of: a. transfecting a host cell with one or more expression cassettes comprising the nucleotide sequence described herein, b. cultivating the host-cell under conditions wherein the enzyme is expressed in said host cell, and c. isolating the enzyme from the host cell.

[0031] FIGURES

[0032] Figure 1. Kinetic properties determination. Reaction rates of SEQ ID NO:1 (Seq.1 ), SEQ ID NO:2 (Seq.2), and SEQ ID NO:3 (Seq.3) dependent of substrate (L- lactate) concentration, Michaelis-Menten fit.

[0033] Figure 2: Temperature stability test of SEQ ID NO:1 (Seq.1 ), SEQ ID NO:2 (Seq.2), and SEQ ID NO:3 (Seq.3) in comparison to AvLOx.

[0034] Figure 3: Response of an electrochemical sensor of SEQ ID NO:1 (Seq.1 ), SEQ ID NO:2 (Seq.2), and SEQ ID NO:3 (Seq.3) versus the commercial reference AvLOX.

[0035] Figure 4: Results of electrochemical sensor using a mediator.

[0036] DETAILED DESCRIPTION

[0037] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017); and Berg et al, “Stryer Biochemie” Springer Verlag, (2018).

[0038] The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wildtype) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods, and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, modified proteins, and enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.

[0039] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0040] The term “about” as used herein refers to the same value or a value differing by + / -5 % of the given value. As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0041] As used herein, amino acids refer to twenty naturally occurring amino acids encoded by sixty-one triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:

[0042] The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: Alanine (Ala, A; nonpolar, neutral), Asparagine (Asn, N; polar, neutral), Cysteine (Cys, C; nonpolar, neutral), Glutamine (Gin, Q; polar, neutral), Glycine (Gly, G; nonpolar, neutral), Isoleucine (lie, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, M; nonpolar, neutral), Phenylalanine (Phe, F; nonpolar, neutral), Proline (Pro, P; nonpolar, neutral), Serine (Ser, S; polar, neutral), Threonine (Thr, T; polar, neutral), Tryptophan (Trp, W; nonpolar, neutral), Tyrosine (Tyr, Y; polar, neutral), Valine (Vai, V; nonpolar, neutral), and Histidine (His, H; polar, positive (10%) neutral (90%)).

[0043] The “positively” charged amino acids are: Arginine (Arg, R; polar, positive), and Lysine (Lys, K; polar, positive).

[0044] The “negatively” charged amino acids are: Aspartic acid (Asp, D; polar, negative), and Glutamic acid (Glu, E; polar, negative).

[0045] The term "enzyme" as used herein refers to any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reaction(s).

[0046] According to the invention, the enzyme described herein is an enzyme having lactate oxidizing activity.

[0047] The amino acid sequences of the enzymes described herein are the following:

[0048] SEQ ID NO: 1 - lactate oxidizing enzyme

[0049] SKDYQTSTAEGPIDFINVYDLEEAAKKVIPKGGFGYISSGAGDLFTLRENIRSFN HKLIVPHVLKDVENPDTSTTFDGDKLTSPIIMAPVAAHGLANVQGEVASAKGVAEFGTI YTISSYASKTLEEIREAGNGAPQWFQFYMSKDDGINRNILDMAKRNGAKAIVLTADAT VGGNRETDKRNGFTFPLGMPIVQAYQSGVGQTMDAVYKSAKQKLSPKDVEFIASYS GLPVYVKGVQSAEDVERALDAGANGIWVSNHGGRQLDGGPAAFDSLQEVAEAVNK RVPIVFDSGVRRGQHVFKALASGADLVAIGRPVIYGLALGGSTGVKQVFDYFKNELEM VMQLAGTQTIEDIKNTKLRDNPYM SEQ ID NO: 2 - lactate oxidizing enzyme

[0050] SKTYQTSTAEGPIDFINMYDLEEAAKKVIPKGGFGYISSGAGDLFTYRENIRAFN HKLIVPHVLKDVENPDTSTTFDGDKLTAPIIMAPVAAHGLANVQGEKASAKGVAEFGTI YTASSYASRTLEEIREAGGPDAPQWFQFYMSKDDGINLNILDMAKRNGAKAIVLTADA TVGGNRETDRRNGFTFPLPMPIVQAYQSGVGQTMDAVYKSAKQKLSPKDVEFIASHS DLPVYVKGVQSAEDVERALDAGANGIWVSNHGGRQLDGGPAAFDSLQYVAEAVDG RVPIVFDSGVRRGQHVFKALASGADLVAIGRPAIYGLALGGSTGVKQVFDFFKNELEM VMQLAGTQTIEDIKNTTLRDNRYM

[0051] SEQ ID NO: 3 - lactate oxidizing enzyme

[0052] SEIEKDMQATHSLDNVQEESASANTADYQASTAEKPIDIINVYDLEEEAKKVIPK GGFGYISSGAGDLWTLKQNIKSFNHKLIVPRVLKDIENPDTSTTIFGDKLSTPIIMAPVA AHGLANVQGEVATAKGVAESGTIMTISSYANKTFEEISEAGNGAPQWFQFYMSKDDG INRNILDEAKANGVKAIVLTADATVGGNREADKRNGFTFPLGMPIVQAYQSGVGQTM DAVYGSSKQKLSPKDVEFIASYSGLPVFVKGVQTAEDALIAIEAGAGGIWVSNHGGRQ LDGGPAAFDSLQEVAEAVNKRVPIVFDSGVRRGQHVFKALASGADLVAIGRPVIYGLA LGGSQGVKSVFDYFKNELEMVMQLAGTQTIEDIKNTKLLDNPYS

[0053] SEQ ID NO: 4 lactate oxidizing enzyme

[0054] MSKDYQTSTAEGPIDFINVYDLEEAAKKVIPKGGFGYISSGAGDLFTLRENIRSF NHKLIVPHVLKDVENPDTSTTFDGDKLTSPIIMAPVAAHGLANVQGEVASAKGVAEFG TIYTISSYASKTLEEIREAGNGAPQWFQFYMSKDDGINRNILDMAKRNGAKAIVLTADA TVGGNRETDKRNGFTFPLGMPIVQAYQSGVGQTMDAVYKSAKQKLSPKDVEFIASYS GLPVYVKGVQSAEDVERALDAGANGIWVSNHGGRQLDGGPAAFDSLQEVAEAVNK RVPIVFDSGVRRGQHVFKALASGADLVAIGRPVIYGLALGGSTGVKQVFDYFKNELEM VMQLAGTQTIEDIKNTKLRDNPYM

[0055] SEQ ID NO: 5 lactate oxidizing enzyme

[0056] MSKTYQTSTAEGPIDFINMYDLEEAAKKVIPKGGFGYISSGAGDLFTYRENIRA F N H KL I VP H VL KD VE N P DTSTTF D G D KLTAP 11 M AP VAAH G LAN VQ G E KAS AKGVAE F GTIYTASSYASRTLEEIREAGGPDAPQWFQFYMSKDDGINLNILDMAKRNGAKAIVLT ADATVGGNRETDRRNGFTFPLPMPIVQAYQSGVGQTMDAVYKSAKQKLSPKDVEFIA SHSDLPVYVKGVQSAEDVERALDAGANGIWVSNHGGRQLDGGPAAFDSLQYVAEAV DGRVPIVFDSGVRRGQHVFKALASGADLVAIGRPAIYGLALGGSTGVKQVFDFFKNEL

[0057] E M VM Q LAGTQTI E D I KNTTL R D N RYM

[0058] SEQ ID NO: 6 lactate oxidizing enzyme

[0059] MSEIEKDMQATHSLDNVQEESASANTADYQASTAEKPIDIINVYDLEEEAKKVIP KGGFGYISSGAGDLWTLKQNIKSFNHKLIVPRVLKDIENPDTSTTIFGDKLSTPIIMAPV AAHGLANVQGEVATAKGVAESGTIMTISSYANKTFEEISEAGNGAPQWFQFYMSKDD GINRNILDEAKANGVKAIVLTADATVGGNREADKRNGFTFPLGMPIVQAYQSGVGQT MDAVYGSSKQKLSPKDVEFIASYSGLPVFVKGVQTAEDALIAIEAGAGGIWVSNHGG RQLDGGPAAFDSLQEVAEAVNKRVPIVFDSGVRRGQHVFKALASGADLVAIGRPVIY GLALGGSQGVKSVFDYFKNELEMVMQLAGTQTIEDIKNTKLLDNPYS

[0060] The term “lactate” refers to lactic acid or the salt thereof.

[0061] According to a specific embodiment, the enzyme described herein is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L-lactate.

[0062] The term "activity” as used herein e.g., in the context of an enzyme activity, shall refer to the catalyzed reaction of the enzyme. Thereby, an enzyme having an activity is a functionally active molecule such as a functional enzyme. A functional enzyme is specifically characterized by a catalytic centre recognizing the enzyme substrate and catalysing the conversion of the substrate to a conversion product. Enzyme variants are considered functional or functionally active upon determining their enzymatic activity in a standard test system, e.g., wherein the enzymatic activity is at least 30% of the activity of the parent (not modified or wild-type enzyme), or at least any of 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even more than 100%.

[0063] Enzyme activity is generally given in units. Thereby, one unit of enzymatic activity is defined as the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per min under the respective conditions of the determination method. For example, one unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of substrate such as e.g., lactate, per min under the respective conditions of the determination method. The specific activity is given in “ll / mg”, “U mg’1” or “U per mg”. Volumetric activity is given in units per volume such as in “U / mL”, “ll / ml”, “U per mL”, “U per ml”, “U mL’1”, or “U ml’1”. The term “oxidizing” in the context of an enzyme having lactate oxidizing activity, refers to an enzyme that oxidizes a lactate molecule and gains or “accepts” one or more electrons from said lactate molecule. Generally, this is also referred to as “substance oxidizing activity”. Therefore, an enzyme having lactate oxidizing activity catalyzes the oxidation of lactate. Herein, the term “capable of oxidizing” may be alternatively used for the term “oxidizing” in the context of an enzyme having oxidizing activity.

[0064] In general, an enzyme having substance oxidizing activity gains or accepts one or more electrons from the substance. Thereby, the enzyme itself or a cofactor of the enzyme, gets reduced. In the reduced state, an enzyme cannot catalyze another oxidation reaction of a substance. Therefore, the enzyme or the cofactor of the enzyme needs to be re-oxidized by transferring the gained electrons to an electron acceptor before another oxidation reaction of a substance can be catalyzed.

[0065] According to one embodiment, in the enzyme described herein, the enzyme or the cofactor may be re-oxidized by transferring the gained electrons to oxygen, to a molecule with electrochemical activity such as a redox mediator, or to an electrode.

[0066] In general, the preference or degree of electron transfer of an enzyme to an electron acceptor differs depending on the specific enzyme and on the specific electron acceptor used.

[0067] In general, an enzyme having substance oxidizing activity is known as “oxidase” if the enzyme uses dioxygen as preferred electron acceptor for the reoxidation of the enzyme. If an enzyme having substance oxidizing activity uses an electron acceptor different than dioxygen as preferred electron acceptor for the reoxidation of the enzyme, then such an enzyme is generally known as a “dehydrogenase”.

[0068] The term “lactate oxidase activity” refers to the activity of an enzyme catalyzing the oxidation of lactate with dioxygen as electron acceptor forming pyruvate and hydrogen peroxide as products. Thereby, two electrons are transferred from lactate to the cofactor of the enzyme e.g., to FAD, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide. An example of such an enzyme is a lactate oxidase (LOX).

[0069] The term “lactate dehydrogenase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of lactate with an electron acceptor different from dioxygen forming an oxidized lactate molecule e.g., pyruvate, and the respective reduced electron acceptor as products. An example of such an enzyme is a lactate dehydrogenase (LDH). Such electron acceptors different from dioxygen are also referred to as redox mediators.

[0070] In a specific embodiment, the enzyme described herein has lactate oxidase activity.

[0071] In a specific embodiment, the enzyme described herein is a functionally active variant of an enzyme peptide sequence. In a specific embodiment, the enzyme described herein is a functionally active variant of an enzyme peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the enzyme sequence, compared to the respective parent enzyme sequence. Specifically, it comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, point mutations, specifically resulting in one or more amino acid substitutions, additions, or deletions, or the like. Specifically, the functional variant of the enzyme peptide sequence is a full-length enzyme peptide sequence comprising point mutations, or it is a fragment of the full-length enzyme peptide sequence with retained enzymatic activity. Specifically, a variant of an enzyme sequence is functionally active if it is capable of converting L- lactate to pyruvate. Specifically, a functionally active variant of an enzyme sequence has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or even more % of the enzymatic activity of the corresponding parent enzyme sequence with lactate. Specifically, a functionally active variant of an enzyme sequence has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or even more % of the enzymatic activity of the corresponding parent enzyme sequence, wherein said enzymatic activity is determined with the following assays.

[0072] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0073] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 86 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0074] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 87 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0075] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 88 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0076] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 89 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0077] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 90 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0078] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 91 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0079] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 92 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0080] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 93 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0081] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 94 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0082] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 95 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0083] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 96 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0084] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 97 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0085] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 98 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0086] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 99 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0087] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 30 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1. According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 40%, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0088] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 50 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0089] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 60%, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0090] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 70 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0091] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 80 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0092] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 90 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0093] According to one embodiment, described herein is an enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1 , and wherein said sequence has at least 95 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0094] The lactate oxidizing activity of an enzyme can be determined using oxygen as electron acceptor. Thereby, the lactate oxidase activity of an enzyme can be determined for example by the Amplex Red assay. Thereby, oxidase activity is measured using a peroxidase-coupled reaction containing 7.1 U / mL horseradish peroxidase (181 ll / rng; Sigma) and 0.05 mM Amplex Red (resorufin: £seo nm = 54.0 mM-1cm-1). Oxygen is present at ambient concentrations of ~250 pM (Kadowaki, M. A. S. et al. (2020)).

[0095] The lactate oxidizing activity of an enzyme can be determined with an electron acceptor different than oxygen. Thereby, the lactate oxidizing activity of an enzyme can be determined by a DCIP assay assessing the enzymatic activity from the colorimetric reduction of 2,6-dichlorophenol-indophenol sodium salt hydrate (DCIP) at 30 °C and 520 nm or alternatively at 600 nm (molar extinction coefficient £520nm= 6.8 mM-1 cm-1 ; molar extinction coefficient £6oonm= 8.98 mM-1 cm-1 ), e.g. as previously described (W.J. Bao, S.N. et al. (1993), Krondorfer, I., et al. (2014), Harreither, W. et al. (2011 )). The assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCI, 3 mM KCI and contains 10 mM lactate and 120 pM DCIP, which acts as an electron acceptor. One unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of lactate per min under the assay conditions. The reaction stoichiometry of lactate: DCIP is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single DCIP molecule. For the detection of activity with other substrates, lactate can be exchanged for other compounds.

[0096] Alternatively, the lactate oxidizing activity of an enzyme can also be determined by assessing the colorimetric reduction of 500 pM 1 ,4-benzoquinone (1 -4-BQ) (molar extinction coefficient £290 nm — 2.24 mM-1cm-1) or 160 pM ferrocenium hexafluorophosphate (FcPFe) (molar extinction coefficient £300 nm = 4.3 mM-1cm-1). The assay mixture is formulated as is described for the DCIP assay but contains 500 pM 1 ,4- benzoquinone or 160 pM ferrocenium hexafluorophosphate instead of DCIP. The reaction stoichiometry of lactate: 1 ,4-benzoquinone is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single molecule of 1 ,4-benzoquinone. The reaction stoichiometry of lactate : ferrocenium hexafluorophosphate is 1 : 2, since two electrons are gained per lactate molecule and transferred individually to two molecules ferrocenium hexafluorophosphate (Brugger, D, et al. (2014), Sygmund, C. et al. (2011 )). The terms "increase in activity", "increased activity", or the like used herein may refer to a detectable increase in activity of an enzyme. The terms "increase in activity", or "increased activity" used herein may mean that a modified enzyme (variant) shows higher activity than a comparable enzyme of the same type, like an enzyme that does not have the particular modification. As another example, the modified enzyme may comprise sequence alterations in the polypeptide or the nucleotide sequence encoding the enzyme. For example, activity of a modified or engineered enzyme may be higher than activity of a non-engineered enzyme of the same type, for example, a wild-type enzyme by about 5 % or more, about 10 % or more, about 15 % or more, about 20 % or more, about 30 % or more, about 50 % or more, about 60 % or more, about 70 % or more, or about 100 % or more. The activity of a particular protein or enzyme in a recombinant or engineered cell may be higher than the activity of a protein or enzyme of the same type in a parent cell, for example, a non-engineered cell by about 5 % or more, about 10 % or more, about 15 % or more, about 20 % or more, about 30 % or more, about 50 % or more, about 60 % or more, about 70 % or more, or about 100 % or more. Increased activity of an enzyme or protein in a cell may be verified by any methods known in the art. Similarly, the term “decrease in activity”, “decreased activity”, or the like used herein may refer to a detectable decrease in activity of an enzyme.

[0097] The term “functional variant” or “functionally active variant” also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelic variant, or also referred to as homologue, is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may comprise a substitution, deletion and / or addition of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, or a combination thereof. Specifically, substitutions, deletions and / or additions may be conservative modifications. Specifically, substitutions, deletions and / or additions do not decrease the enzyme’s specific activity. Specifically, a functionally active variant of the enzyme as described herein comprises specific enzymatic activity towards lactate of at least 1 ll / rng, as determined by the respective assay as described herein.

[0098] Specifically, a functional variant as described herein comprises no more than or up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity. Specifically, a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5, amino acid substitutions, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity.

[0099] Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence e.g., by one or more point mutations, wherein the sequence alterations retain or improve a feature of the enzyme, such as its stability or activity for example. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.

[0100] A point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the nonengineered amino acid sequence in the substitution, or exchange, deletion, or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.

[0101] According to a specific embodiment, the enzymes described herein may comprise one or more tag sequences, specifically N-terminal tag sequences. Specifically, such tag sequence is C-terminal of the N-terminal methionine of the enzymes described herein. Such tag sequence may comprise any number of amino acids of more than 2, 4, 5, 6 or 10 amino acids and up to 20 or 50 or more amino acids. Specifically, tag sequences used herein may be any tag sequence known to the person skilled in the art. Specifically, tag sequences used herein are selected from purification tags, solubility enhancement tags, or monitoring tags.

[0102] Purification tags are also known as affinity tags and are amino acid sequences that can be used for example for the purification of proteins where they are attached to. These affinity tags have high affinity to appropriate ligands of a solid support, like chromatography resins or directly to the resins. By selectively binding of the protein having the affinity tag to the particular resin, the protein can be purified highly effective by only one chromatography step. According to a specific embodiment, affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, poly-arginine tag, FLAG tag, Strep tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP) tag, S-tag, HA tag, c-Myc tag, and SUMO tag, or any other tag known to be useful for the efficient purification of a protein it is fused to. Preferably, the tag is a His tag comprising one or more H, specifically a hexa-histidine tag. Specifically, proteins comprising a poly-, or hexa-histidine tag (His-tag) can be captured and purified using chromatography, e.g. by Immobilized Metal Affinity Chromatography (IMAC).

[0103] Solubility enhancement tags can be fused N-terminal to the enzymes described herein. Solubility enhancement tags can increase the titer of the soluble protein when expressed in a host cell, e.g. in the cytosol of P. pastoris, compared to expression of the proteins without the tag. According to a further specific embodiment, solubility enhancement tag sequences used herein are selected from calmodulin-binding peptide (CBP), poly Arg, poly Lys, protein D tag (dTAG), Z domain of Staphylococcal protein A, and thioredoxin or any other tag known to improve the solubility of the protein it is fused to e.g. during expression in a host cell. Specifically, the solubility enhancement tag is a T7 tag, preferably selected from the group consisting of T7A, T7A1 , T7A2, T7A3, T7A4, T7A5, T7B, T7B1 , T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11 , T7B12, T7B13, and T7C.

[0104] According to a further specific embodiment, the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin or any other tag useful for detection or quantification of the recombinant enzyme during production steps including fermentation, isolation, and purification by simple in-situ, inline, online, or atline detectors, like UV, IR, Raman, fluorescence and the like.

[0105] The term “sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%. Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.

[0106] Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry. Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.

[0107] To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.

[0108] “Percent (%) identity” with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared. In case of percentages determined for sequence identities, it is possible that arithmetical decimal places may result which are not possible with regard to full nucleotides or amino acids. In this case, the percentages shall be rounded up to whole nucleotides or amino acids.

[0109] For purposes described herein, the sequence identity between two amino acid sequences may be determined using standard methods, e.g. using the NCBI BLAST program version 2.2.29 (Jan-06-2014) or online using the multiple sequence alignment tool EMBL-EBI Clustal Omega (Sievers, F. et al. (2011 )).

[0110] "Percent (%) identity" with respect to a nucleotide sequence e.g. , of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared.

[0111] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, MAFFT based algorithms: multiple alignment using fast fourier transform, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (Short Oligonucleotide Analysis Package, available at biotools:soap), and Maq (Mapping and Assembling with Qualities, available at maq.sourceforge.net).

[0112] According to a specific embodiment, the enzymes described herein may be active at acidic, neutral, or alkaline pH ranges. Specifically, the enzymes of the invention may be used at a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or at any pH of a body fluid e.g., of sweat or blood. Usually, blood has a pH between 7.35 and 7.45.

[0113] According to the invention, the enzyme described herein may be used for determining lactate in a sample.

[0114] The term “determining” or “determination” as used herein refers to detecting and / or quantifying an analyte such as lactate. The term “detecting” refers to the general determination if analyte is present. Detection does not require the exact quantification of analyte but rather provides the user of the method with the information if e.g., the analyte is present with a concentration above a certain threshold. These thresholds are to be adapted to the respective application and sample. The term “quantifying” refers to the determination of the concentration or amount of an analyte. Quantification may refer to the determination of an exact amount of an analyte or may alternatively refer to a semi- quantitative determination of an analyte e.g., if the amount of the analyte in a sample is in a certain range. Such a range may be a concentration range suitable for the respective purpose of the determination of the analyte.

[0115] According to one embodiment, the enzyme described herein may be used for the quantification or detection of lactate. Thereby, the enzyme described herein may be used for determining the amount of lactate or for determining if lactate is present in a certain concentration range. Further, the enzyme described herein may be used for determining if lactate is present above or below a certain threshold. According to one embodiment, the sample may be any material for which determining lactate is relevant or of interest. In particular, the sample is a human or animal sample, specifically any one of body fluid, interstitial fluid, blood, blood plasma, blood serum, dermal fluid, urine, tears, sweat, saliva, skin, flesh, tissue, eyeballs, cornea, and gastric fluid.

[0116] According to a specific embodiment, human blood is used as a sample in the methods and means described herein.

[0117] According to one embodiment of the invention, the sample contains or is suspected to contain lactate.

[0118] According to one embodiment of the invention, the electron acceptor used together with the enzyme described herein may be dioxygen or a molecule different from dioxygen.

[0119] According to an alternative embodiment of the invention, the enzyme described herein may be used with a molecule different from dioxygen as electron acceptor for the re-oxidation of the enzyme. In specific embodiments, a system for regeneration of the used electron acceptor may be implemented in the method and use described herein.

[0120] According to a specific embodiment, a system for regeneration of an electron acceptor may comprise an enzymatic, chemical, electrochemical, homogeneous catalytic, photocatalytic, or heterogeneous catalytic regeneration system.

[0121] According to one embodiment of the invention, the enzyme described herein may be used for determining lactate in a sample. Thereby, determining lactate may be performed by using a suitable electron acceptor. Such an electron acceptor may be any suitable molecule which can be used for the colorimetric, photometric, fluorimetric, or electrochemical detection of the electron transfer reaction. Thereby, the signal obtained from detecting the electron transfer reaction is used for the determination of the lactate in the sample.

[0122] According to one embodiment of the invention, the enzyme described herein may be any enzyme capable of oxidizing lactate and transferring the so gained electrons to a suitable electron acceptor such as dioxygen. In this case, hydrogen peroxide is produced through the electron transfer from lactate to dioxygen. Hydrogen peroxide can also be detected by suitable methods such as e.g., by colorimetric, photometric, fluorimetric, bioelectrochemical, electrochemical methods, possibly using enzyme based assays. Thereby, the presence or amount of lactate can be calculated from the detection of hydrogen peroxide. According to one embodiment of the invention, determining of lactate is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.

[0123] The term “colorimetrically” as used herein refers to the application of a colorimetric analysis for the determination of the presence or the amount of lactate in the sample. In a colorimetric analysis the concentration of a chemical element or chemical compound in a solution is determined with the aid of a color reagent. The color reagent may be visually detected or detected with suitable equipment e.g., a colorimeter. In enzymatic analysis as described herein, the color reaction is preceded by a reaction catalyzed by an enzyme such as the enzyme capable of oxidizing a lactate. A general example of such a colorimetric determination is the detection of a colored complex formed by a peroxidase from hydrogen peroxide and ABTS.

[0124] The term “photometrically” as used herein refers to the usage of a photometer or a spectrophotometer for enzymatically determining a substance in a sample, or alternatively also for determining the enzyme activity, by following the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution. A colorimetric detection may be coupled or performed by a photometer.

[0125] The term “fluorimetrically” as used herein refers to the detection of fluorescence by determining the difference in the fluorescence spectra of substrate from product to measure the enzyme reaction or determine the substrate or product concentration. The Amplex Red assay which is elsewhere described herein is an example of an assay based on a fluorimetric detection method.

[0126] The term “electrochemically” as used herein refers to the usage of an electrochemical biosensor based on the measurement of biological binding, oxidation or reduction event-dependent changes in conductance, resistance, or capacitance of the biosensor surface. In such an electrochemical biosensor, one of the electrodes is immobilized with a biological recognition molecule. The binding of the analyte to the biological recognition molecule triggers a change in the electrical properties due to oxidation and reduction reactions taking place as a result of biological interaction activity, thus providing the sensor signal. Electrochemical biosensors rely mostly on enzyme- catalyzed reactions to produce current / potential difference which is then detected.

[0127] According to one embodiment of the invention, the electron acceptor used in the oxidation reaction of the lactate may also be an electrode which is equipped with an enzyme described herein. Such an electrode may be part of a biosensor. According to one embodiment of the invention, detecting the oxidation of lactate by the enzyme described herein may be carried out by a sensor, specifically a bio- electrochemical sensor, configured to detect and / or quantify lactate in a sample via (bio)electrochemical redox reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of the analyte such as lactate.

[0128] Electrochemical biosensors can be impedimetric, potentiometric, or amperometric. In an amperometric biosensor, a biochemical signal is transduced into a quantifiable amperometric signal.

[0129] As described in Rocchitta G. et al. (2016) amperometric biosensors are commonly divided into three main generations depending on the electron transfer method used for the measurement of the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediators, and cofactors). First-generation biosensors measure the concentration of analytes and / or products of enzymatic reactions that diffuse to the transducer surface and generate an electrical response. Commonly, oxidases are used in first-generation biosensors. Oxidases need molecular oxygen as a second substrate so the oxidase-based biosensors are oxygen dependent. Second-generation biosensors require an electron mediator for the transfer of electrons obtained from enzymatic reactions to the transducer surface and thereby generate an electrical response. In third-generation biosensors, direct electron transfer is enabled between the redox-active biomolecule i.e. , the enzyme, and the electrode surface.

[0130] According to one embodiment of the invention, for the determination of lactate by electrochemical means, an electrode comprising an enzyme described herein is contacted with the sample. This contact between electrode and sample can be performed by any approach which brings the electrode and the sample in contact in order that the enzyme is allowed to react with the lactate or with the sample suspected to contain lactate.

[0131] The term “electrode” refers to any suitable surface for accepting electrons from the enzyme via analytes and / or products of enzymatic reactions such as hydrogen peroxide, mediated, or direct electron transfer. Thereby the electrode is of a material capable of accepting electrons. Furthermore, the electrode may be of any material suitable or modified with any material to adsorb or immobilize the enzyme described herein. Non-limiting examples of such a material are platinum, gold, boron doped diamond and carbons such as graphite, pyrolytic graphite and glassy carbon where all of them can additionally be modified with carbon nanotubes (single or multi-walled), carbon fibers, nanoparticles e.g. gold nanoparticles or promoters as e.g., thiols. The electrode may be also of any material to increase the specific surface are of the electrode.

[0132] According to one embodiment of the invention, the electrode may be used as single electrode or as a stack of electrodes of e.g., 2, 3, 4, 5, or more electrodes.

[0133] According to one embodiment of the invention, the electrode provided herein is a working electrode.

[0134] According to one embodiment, the electrode comprising the enzyme described herein enables the detection and / or quantification of lactate based on electron transfer via analytes and / or products of enzymatic reactions such as hydrogen peroxide, mediated electron transfer, or direct electron transfer.

[0135] Electron transfer via analytes and / or products of enzymatic reactions typically employs the transfer of electrons from hydrogen peroxide, produced through the enzymatic reaction, to the electrode.

[0136] Mediated electron transfer in biosensors typically employs a two-step procedure in which the enzyme takes part in a first redox reaction with the substrate and is in turn re-oxidized by a redox mediator. Finally, the redox mediator is oxidized by the electrode.

[0137] In the context of enzyme electrodes and biosensors, redox mediators are artificial electron transferring agents that can readily participate in the redox reaction with the biological component and thus help in rapid electron transfer to the electrode. A "redox mediator" is an electron-transfer agent for carrying electrons between an analyte, an analyte-reduced or analyte-oxidized enzyme, and an electrode, either directly, or via one or more additional electron-transfer agents. A redox mediator that includes a polymeric backbone may also be referred to as a redox polymer.

[0138] Specifically, said redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.

[0139] According to one embodiment of the invention, the oxidation of lactate is performed in the presence of a redox mediator. The method of the invention can also be performed in the presence of more than one redox mediator e.g., in the presence of two or more different redox mediators. Thereby, the redox mediator may be present on the electrode, in an enzyme composition comprising the enzyme and the redox mediator, or may be present in the sample. According to one embodiment, the redox mediator may be any molecule or material able to accept electrons from the enzyme capable of oxidizing lactate. Such a redox mediator may be also referred to as electron acceptor different from dioxygen and may transfer the gained electrons to an electrode or may be directly used for determining lactate in a sample, e.g., by colorimetric, photometric, or fluorimetric determination methods. Specifically, said redox mediator is selected from the group consisting of any one of organic redox mediators, soluble redox mediators, insoluble redox mediators, redox polymers, transition metal complexes, polymeric transition metal complexes, wired redox mediators, sandwich compounds, and derivatives of these redox mediators.

[0140] Polymeric transition metal complexes comprise a polymeric backbone, spacers, and transition metal complexes.

[0141] Specifically, redox polymers are polymers comprising redox species. Non-limiting examples of such redox species used in redox polymers are osmium (Os), ruthenium (Ru), iron (Fe), cobalt (Co), or any transition metal. Non-limiting examples of polymers used for redox polymers are poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). An example of a redox polymer is Os-containing poly(vinylpyridine).

[0142] According to a specific embodiment, in the case the redox mediator comprises osmium, the redox mediator may be an osmium transition metal complex with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2'- bipyridine, 1 ,10-phenanthroline, 1 -methyl, 2-pyridyl biimidazole, or derivatives thereof. The redox mediator may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. One example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2' -bipyridine, 1 ,10- phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. Some derivatives of 2,2' -bipyridine for complexation with the osmium cation include but are not limited to 4,4'-dimethyl-2,2'- bipyridine and mono-, di-, and polyalkoxy-2, 2'-bipyri dines, including 4,4'-dimethoxy-2,2'- bipyridine. Derivatives of 1 , 10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl- 1 , 10- phenanthroline and mono, di-, and polyalkoxy-1 , 10- phenanthrolines, such as 4,7- dimethoxy- 1 ,10-phenanthroline. Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1 -vinyl imidazole) and poly(4-vinyl pyridine). Suitable copolymer substituents of poly(1 -vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1 -vinyl imidazole). An example of a redox polymer is also derived from poly(l -vinylimidazole) or a copolymer of (1 -vinyl imidazole) bound to a metal ion selected from the group consisting of Os.sup.3+ / 2+, Ru.sup.3+ / 2+, and Fe.sup.3+ / 2+.

[0143] The term “transition metal” refers to an element whose atom has a partially filled d sub-shell, or which can give rise to cations with an incomplete d sub-shell. Thereby, transition metals are elements in the d-block of the periodic table and also lanthanides and actinides.

[0144] Non-limiting examples of transition metal complexes include complexes comprising titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, indium, and platinum. Specific examples of transition metal complexes are ferricyanide, ruthenium hexamine, metalloporphyrins such as heme b or heme c. In these complexes, the transition metal is coordinatively bound to one or more ligands, which are typically mono-, di-, tri-, or tetradentate.

[0145] Non-limiting examples of transition metal complexes include complexes comprising lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0146] Non-limiting examples of transition metal complexes include complexes comprising actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, and lawrencium.

[0147] A sandwich compound is a chemical compound featuring a metal bound by haptic covalent bonds to two arene ligands. The arenes have the formula CnHn, substituted derivatives (for example Cn(CH3)n) and heterocyclic derivatives (for example BCnHn+i). A special class of sandwich complexes are metallocenes. A metallocene contains a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure, i.e. , the two cyclopentadienyl anions are on parallel planes with equal bond lengths and strengths. Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1 ,1'-dimethyl ferrocene [DMF], and ferrocene monocarboxylic acid. Organic redox compounds are organic molecules capable to act as a redox mediator. Non-limiting examples of organic redox mediators are organic molecules such as quinones, compounds having a quinoid structure such as benzoquinones or phenanthroline quinones, phenazine such as 1 -methoxyphenazine methosulfate, tetracyanoquinodimethane (TCNQ), N,N,N', N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivative of these molecules.

[0148] According to one embodiment of the invention, the enzyme described herein is immobilized on the electrode by adsorption, physical entrapment in a polymer, complex formation, covalent binding, and / or cross-linking. Cross-linking agents are e.g., dialdehydes such as glutaraldehyde.

[0149] According to one embodiment of the invention, the electrode of the invention is part of a biosensor. Thereby, a specific use of the electrodes of the invention is in the provision of a biosensor, more specifically a first, second, or third-generation lactate biosensor. The biosensor may be suitable for use at acidic, neutral, or alkaline pH. The biosensor may be suitable for use at room temperature or at body temperature. Specifically, the biosensor may be suitable for the detection and / or quantification at 4°C, 10°C, 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, or higher.

[0150] According to another embodiment, the biosensor may have one or more electrodes comprising the enzyme described herein. In further embodiments, the lactate biosensor includes: a working electrode comprising a conductive material, wherein the enzyme capable of oxidizing the lactate is in proximity to the conductive material. One or more other electrodes may be included such as one or more counter electrodes, one or more reference electrodes and / or one or more counter / reference electrodes.

[0151] The particular configuration of the biosensor may depend on the use for which the biosensor is intended and the conditions under which it will operate.

[0152] In a specific embodiment of the present invention, the biosensor may be a single use biosensor for the determination of lactate. Thereby, the biosensor may be a biosensor strip.

[0153] According to one embodiment of the present invention, the enzymes described herein may be recombinantly expressed by methods commonly known in the art. For example, the enzymes described herein may be expressed using standard methods for cloning, transformation, and recombinant production in suitable host organisms e.g., in Escherichia coli or in Pichia pastoris.

[0154] According to one embodiment, the enzyme described herein is produced in Escherichia coli or in Pichia pastoris.

[0155] Also described herein is a “host cell”, “host cell line” or “production cell line”, which is commonly understood to be a host cell or cell line ready-to-use for cultivation / culturing in a bioreactor to obtain the product of a production process, such as the enzymes described herein. The host cell or cell line as described herein is particularly understood as a recombinant bacterial or yeast organism, which may be cultivated / cultured to produce the desired protein.

[0156] Genetic modifications of such host cells include modifications that introduce a polynucleotide encoding a polypeptide into a cell; a modification that substitutes, adds (i.e. , inserts), or deletes one or more nucleotides of the genetic material of a parent cell, including a chemical modification (exposure to a chemical) resulting in a change to the genetic material of a parent cell. Genetic modification includes a heterologous or homologous modification of referenced species. Genetic modification further includes a modification of a coding region for polypeptides. Genetic modification also includes a modification of non-coding regulatory regions that changes expression of a gene or function of an operon. Non-coding regions include 5'-non-coding sequence (5' of a coding sequence) and 3'-non-coding sequence (3' of a coding sequence).

[0157] The "gene" or “nucleotide sequence encoding” used herein refers to a nucleic acid fragment that encodes a particular protein such as the enzyme described herein, which may optionally include at least one regulatory sequence, such as a 5'-non-coding sequence and a 3'-non-coding sequence (3' and 5' in reference to the position relative to the coding sequence).

[0158] In some embodiments, the gene encoding the enzyme described herein may be introduced into a bacterial or yeast cell and may be e.g., inserted into an endogenous genetic material (for example, chromosome) of a yeast cell. This gene may be inserted into one or more locations of a particular endogenous gene of the yeast cell to disrupt the endogenous gene.

[0159] The gene or nucleotide sequence that encodes the enzyme described herein may be introduced in the bacterial or yeast cell, but not inserted into the endogenous genetic material of the cell. For example, the gene may be included in an expression vector, such as a plasmid, and remain separated from the endogenous genetic material of the cell.

[0160] The gene or nucleotide sequence that encodes the enzyme described herein may be introduced in an expressible form into a bacterial or yeast cell, and expressed to generate a gene product thereof in the cell. The expressible form may be a structure in which the gene is operably linked to an expression regulatory sequence. For example, the gene may be operably linked to at least one sequence selected from an exogenous enhancer, an operator, a promoter, and a transcription terminator to be expressible in a cell, or may be linked to an endogenous regulatory sequence of the cell to be expressible in the cell.

[0161] The gene or nucleotide sequence that encodes the enzyme described herein may be introduced into the host cell using any known method of introducing a genetic material into a cell (see e.g. R. Gietz et al., Biotechniques 30:816-831 , April 2001 ). The introducing may include a spheroplast method, intact cell transformation, electroporation, or a combination thereof. For example, intact cell transformation may use a particular monovalent alkali cation (Na+, K+, Rb+, Cs+, and Li+) in combination with PEG to promote uptake of DNA, such as a plasmid, by a cell. For example, intact cell transformation may include applying a heat shock to an aqueous solution of PEG, LiAc, carrier ssDNA, plasmid DNA, and a cell. For example, electroporation may include applying an electric pulse to a mixed medium including a cell and DNA, such as plasmid DNA.

[0162] In the introduction into the yeast, the gene may be included in a vector, with a homologous sequence with respect to an endogenous genetic material of a parent cell of a yeast cell. The homologous sequence is complementary to a target sequence present in an endogenous genetic material of a parent yeast cell, and accordingly may be substituted with the target sequence by homologous recombination. The vector may include two sequences which are respectively homologous to the 5' end and 3' end of the target sequence. In this regard, the introducing may include culturing the yeast cell under a selection pressure during or after the contacting. The selection pressure may indicate a material or state that enables to select only cells where homologous recombination has occurred. The selection pressure may include culturing in the presence of antibiotics. In this regard, the vector may include a gene that provides antibiotic resistance to the yeast cell, for example, a gene that encodes an enzyme that decomposes antibiotics. The term “cell culture” or “cultivation” (“culturing” is herein synonymously used), also termed “fermentation”, with respect to a host cell line is meant to be the maintenance of cells in an artificial, e.g., an in vitro environment, under conditions favoring growth, differentiation or continued viability, in an active or quiescent state, of the cells, specifically in a controlled bioreactor according to methods known in the industry. When cultivating, a cell culture is brought into contact with the cell culture media in a culture vessel or with substrate under conditions suitable to support cultivation of the cell culture. In certain embodiments, a culture medium as described herein is used to culture cells according to standard cell culture techniques that are well-known in the art for cultivating or growing bacterial or yeast cells.

[0163] Cultivation of the host cells may be in one, two or multiple phases.

[0164] According to a specific embodiment, cell growth and production of the recombinant protein is in a single phase. In this case, the medium used in the cultivation process comprises the respective components required for the production of the protein from the beginning of the cultivation process.

[0165] Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolality, pH, and nutrient formulations. Feeding of nutrients may be done in a continuous or discontinuous mode according to methods known in the art.

[0166] Cell culture may be a batch process or a fed-batch process. A batch process is a cultivation mode in which all the nutrients necessary for cultivation of the cells, and optionally including the substrates necessary for production of the enzyme as described herein, are contained in the initial culture medium, without additional supply of further nutrients during fermentation. In a fed-batch process, a feeding phase takes place after the batch phase. In the feeding phase one or more nutrients, such as an inducer, e.g. methanol, are supplied to the culture by feeding. In certain embodiments, the method described herein is a fed-batch process. Specifically, a host cell transformed with a nucleic acid construct encoding the enzyme as described herein, is cultured in a growth phase medium and transitioned to an induction phase medium in order to produce the recombinant proteins described herein.

[0167] In another embodiment, host cells described herein are cultivated in continuous mode, e.g. a chemostat. A continuous fermentation process is characterized by a defined, constant and continuous rate of feeding of fresh culture medium into the bioreactor, whereby culture broth is at the same time removed from the bioreactor at the same defined, constant and continuous removal rate. By keeping culture medium, feeding rate and removal rate at the same constant level, the cultivation parameters and conditions in the bioreactor remain constant.

[0168] Expression products such as polypeptides, proteins, or enzymes as described herein may be introduced into a host cell either by introducing the respective coding polynucleotide or nucleotide sequence for expressing the expression products within the host cell, or by introducing the respective expression products which are within an expression system or isolated. Any of the known procedures for introducing expression cassettes, vectors or otherwise introducing (e.g., coding) nucleotide sequences into host cells may be used (see, e.g., Sambrook et al.).

[0169] The invention specifically allows for the production process to be performed on a pilot or industrial scale.

[0170] According to one embodiment, a growth medium allowing the accumulation of biomass as described herein, specifically a basal growth medium, typically comprises a carbon source, a nitrogen source, a source for sulphur and a source for phosphate. Typically, such a medium comprises furthermore trace elements and vitamins, and may further comprise amino acids, peptone, or yeast extract.

[0171] According to one embodiment, the fermentation is carried out at a pH ranging from 3 to 7.5.

[0172] Typical fermentation times are about 24 to 120 hours with temperatures in the range of 20°C to 35°C, preferably 22 to 30°C.

[0173] Specifically, the cells are cultivated under conditions suitable to produce the recombinant enzymes described herein, which can be purified from the cells or culture medium.

[0174] The term "exogenous" as used herein refers to a referenced molecule (e.g., nucleic acid) that has been introduced into a host cell. A nucleic acid may be exogenously introduced into a host in any suitable manner. For example, a nucleic acid can be introduced into a host cell and inserted into a host chromosome, or the nucleic acid can be introduced into the host as non-chromosomal genetic material, such as an expression vector (e.g., a plasmid) that does not integrate into the host chromosome. A nucleic acid encoding a protein should be introduced in an expressible form (i.e., so that the nucleic acid can be transcribed and translated). The term "endogenous" refers to a referenced molecule (e.g., nucleic acid) already present in the host cell prior to a particular genetic modification (e.g., a genetic composition, trait, or biosynthetic activity of a "wild-type" cell or a parent cell).

[0175] The term "heterologous" refers to a referenced molecule (e.g., nucleic acid) derived from a source other than referenced species; and the term "homologous" refers to a molecule (e.g., nucleic acid) or activity derived from a host parent cell. Accordingly, an exogenous molecule (e.g., expression of an exogenous coding nucleic acid) may be heterologous (e.g., a coding nucleic acid from a different species) or homologous (e.g., an additional copy of a coding nucleic acid from the same species).

[0176] The term “expression” as used herein regarding expressing a polynucleotide or nucleotide sequence, is meant to encompass at least one step selected from the group consisting of DNA transcription into mRNA, mRNA processing, non-coding mRNA maturation, mRNA export, translation, protein folding and / or protein transport. Nucleic acid molecules containing a desired nucleotide sequence may be used for producing an expression product encoded by such nucleotide sequence e.g., proteins or transcription products such as RNA molecules. To express a desired nucleotide sequence, an expression system is conveniently used, which can be an in vitro or in vivo expression system, as necessary to express a certain nucleotide sequence by a host cell or host cell line. Typically, host cells are transfected or transformed with an expression system comprising an expression cassette that comprises the desired nucleotide sequence and a promoter operably linked thereto optionally together with further expression control sequences or other regulatory sequences. Specific expression systems employ expression constructs such as vectors comprising one or more expression cassettes.

[0177] The term “expression construct” as used herein, means the vehicle, e.g. vectors or plasmids, by which a DNA sequence is introduced into a host cell so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. “Expression construct” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences.

[0178] The terms "vector”, “DNA vector” and "expression vector” mean the vehicle by which a DNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vector” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences, such as artificial chromosomes. Plasmids are preferred vectors of the invention. In specific embodiments, an expression vector may contain more than one expression cassettes, each comprising at least one coding sequence and a promoter in operable linkage.

[0179] A "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. An “expression cassette” as used herein refers to nucleic acid molecules containing a desired coding sequence and control sequences in operable linkage, so that an expression system can use such expression cassette to produce the respective expression products, including e.g., encoded proteins or other expression products. Certain expression systems employ host cells or host cell lines which are transformed or transfected with an expression cassette, which host cells are then capable of producing expression products in vivo. In order to effect transformation of host cells, an expression cassette may be conveniently included in a vector, which is introduced into a host cell; however, the relevant DNA may also be integrated into a host chromosome. A coding sequence is typically a coding DNA or coding DNA sequence which encodes a particular amino acid sequence of a particular polypeptide or protein, or which encodes any other expression product.

[0180] The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Vectors typically comprise DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism. A coding DNA sequence or segment of DNA molecule coding for an expression product can be conveniently inserted into a vector at defined restriction sites. To produce a vector, heterologous foreign DNA can be inserted at one or more restriction sites of a vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. It is preferred that a vector comprises an expression system, e.g. one or more expression cassettes. Expression cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.

[0181] To obtain expression, a sequence encoding a desired expression product, such as e.g. any of the polypeptides or proteins described herein, is typically cloned into an expression vector that contains a promoter to direct transcription. Appropriate expression vectors typically comprise regulatory sequences suitable for expressing coding DNA. Examples of regulatory sequences include promoter, operators, enhancers, ribosomal binding sites, and sequences that control transcription and translation initiation and termination. The regulatory sequences are typically operably linked to the DNA sequence to be expressed.

[0182] A promoter is herein understood as a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, one or more nuclear localization signals (NLS) and one or more expression cassettes.

[0183] According to one embodiment, the following items are described herein:

[0184] ITEMS

[0185] 1. An enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO: 1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1.

[0186] 2. The enzyme of item 1 , wherein the sequence has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1.

[0187] 3. The enzyme of item 1 or 2, wherein the sequence having at least 85 % sequence identity with SEQ ID NO:1 is selected from SEQ ID NO:2 and SEQ ID NO:3.

[0188] 4. The enzyme of any one of items 1 to 3, wherein said enzyme comprising a sequence having at least 85 % sequence identity with SEQ ID NO:1 has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1 .

[0189] 5. The enzyme of any one of items 1 to 4, wherein said enzyme is a recombinant enzyme.

[0190] 6. The enzyme of any one of items 1 to 5, further having a purification tag at its N-terminus.

[0191] 7. The enzyme of any one of items 1 to 6, further having a Met at its N-terminus.

[0192] 8. The enzyme of any one of items 1 to 7, wherein the sequence is selected from any one of SEQ ID NOs:2 to 6. 9. An enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO: 1 or a sequence having at least 85 % sequence identity with SEQ ID NO:1.

[0193] 10. The enzyme of item 9, wherein the sequence has at least 86, 87, 88, 89, 90,

[0194] 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1.

[0195] 11. The enzyme of item 9 or 10, wherein said enzyme comprising a sequence having at least 85 % sequence identity with SEQ ID NO:1 has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:1.

[0196] 12. The enzyme of any one of items 9 to 11 , wherein said enzyme is a recombinant enzyme.

[0197] 13. The enzyme of any one of items 9 to 12, further having a purification tag at its N-terminus.

[0198] 14. The enzyme of any one of items 9 to 13, further having a Met at its N-terminus.

[0199] 15. An enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:2 or a sequence having at least 85 % sequence identity with SEQ ID NO:2.

[0200] 16. The enzyme of item 15, wherein the sequence has at least 86, 87, 88, 89, 90,

[0201] 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:2.

[0202] 17. The enzyme of item 15 or 16, wherein said enzyme comprising a sequence having at least 85 % sequence identity with SEQ ID NO:2 has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:2.

[0203] 18. The enzyme of any one of items 15 to 17, wherein said enzyme is a recombinant enzyme.

[0204] 19. The enzyme of any one of items 15 to 18, further having a purification tag at its N-terminus.

[0205] 20. The enzyme of any one of items 15 to 19, further having a Met at its N- terminus.

[0206] 21. An enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO:3 or a sequence having at least 85 % sequence identity with SEQ ID NO:3.

[0207] 22. The enzyme of item 21 , wherein the sequence has at least 86, 87, 88, 89, 90,

[0208] 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:3. 23. The enzyme of item 21 or 22, wherein said enzyme comprising a sequence having at least 85 % sequence identity with SEQ ID NO:3 has at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 100 %, or more of the lactate oxidizing activity of an enzyme comprising SEQ ID NO:3.

[0209] 24. The enzyme of any one of items 21 to 23, wherein said enzyme is a recombinant enzyme.

[0210] 25. The enzyme of any one of items 21 to 24, further having a purification tag at its N-terminus.

[0211] 26. The enzyme of any one of items 21 to 25, further having a Met at its N- terminus.

[0212] 27. Use of the enzyme of any one of items 1 to 26 for the determination of lactate in a sample.

[0213] 28. An electrode comprising the enzyme of any one of items 1 to 26.

[0214] 29. A biosensor comprising the electrode of item 28.

[0215] 30. A nucleotide sequence encoding the enzyme of any one of items 1 to 26.

[0216] 31 .A host-cell expressing the enzyme of any one of items 1 to 26.

[0217] 32. A method of producing the enzyme of any one of items 1 to 26 in a host-cell, said method comprising the steps of: a. transfecting a host-cell with one or more expression cassettes comprising the nucleotide sequence of item 30, b. cultivating the host-cell under conditions wherein the enzyme is expressed in said host-cell, and c. isolating the enzyme from the host-cell.

[0218] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention.

[0219] EXAMPLES

[0220] Example 1: Phylogenetic analysis

[0221] Methods

[0222] Literature was searched for soluble and FMN-dependent L-lactate oxidizing enzyme sequences for which activity was shown experimentally. The sequence identity matrix was created using the Clustal Omega online server (Clustal Omega < Multiple Sequence Alignment < EMBL-EBI).

[0223] The enzyme sequences used in the example are the following:

[0224] SEQ ID NO:7 (A. viridans Q44467)

[0225] MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRA

[0226] NDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAV

[0227] SEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIIL

[0228] TADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEE

[0229] IAGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGSFDTLPAIAERV

[0230] NKRVPIVFDSGVRRGEHVAKALASGADWALGRPVLFGLALGGWQGAYSVLDYFQK

[0231] DLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY

[0232] SEQ ID NO:8 (S. iniae A9QH69)

[0233] MENKSEMINATTIEFKTSSAEGSVDFVNVFDLEKMAQKVIPKGAFGYIASGAGD

[0234] TFTLHENIRSFNHKLIVPHGLKGVENPSTEITFDGDKLASPIILAPVAAHKLANEQGEIAS

[0235] AKGVKEFGTIYTTSSYSTTDLPEISQTLGDSPHWFQFYYSKDDGINRHIMDRLKAEGV

[0236] KSIVLTVDATVGGNREVDKRNGFVFPVGMPIVQEYLPNGAGKTMDYVYKATKQALSP

[0237] KDVEYIAQYSGLPVYVKGPQCAEDAFRALEAGASGIWVTNHGGRQLDGGPAAFDSL

[0238] QEVAEAVDRRVPIVFDSGVRRGQHVFKALASGADLVALGRPVIYGLAMGGSVGTRQ

[0239] VFEKINDELKMVMQLAGTQTIDDVKHFKLRHNPYDSSIPFSQNALKLD

[0240] SEQ ID NO: 9 (Enterococcus A0A4Y3JPV3)

[0241] MEKTYQAGTNEGIVDFINMEDLEIAASQVIPAGGYGYISSGAGDLFTYQENERA

[0242] FNHQLIIPHVLRDVELPDTTTHFDEETLTAPIIMAPVAAHGLAHVKAEKASAKGVADFG

[0243] TIYTASSYASCTLEEIREAGGEKAPQWFQFYMSKDNGINLDILEVAKRNGAKAIVLTAD

[0244] ATVGGNRETDRRNGFTFPLPMPIVQAYQSGVGQTMDAVYKSSKQKLSPKDVEFIAAH

[0245] SDLPVYVKGVQSEEDVYRSLESGAGGIWVSNHGGRQLDGGPAAFDSLQYVADAVD

[0246] KRVPIVFDSGVRRGQHVFKAIASGADLVAIGRPVIYGLSLGGSTGVRQVFDFFKTELE

[0247] MVMQLAGTQTVEDIKKIKLRENRFI

[0248] SEQ ID NO: 10 (P. acidilactici E0NE46)

[0249] MTMINGYEQSDREEKIDILNLESLEKQAEEIIPAGGFGYIAGGSEDEWTLKQNR

[0250] MAFHHRQIAPKALSGIEKPELNTEIFGIPLNTPVMMAPAAAQGLAHSQGEKDTARGLA AVGGLMAQSTYSSVSIAETAAAGGDAPQFFQLYMSKDWNFNESLLDEAKKANVKAIIL TVDATVDGYREADIKNKFTFPLPMANLIKFSEGNGQGKGIEEIYASAAQNIRPEDVKRI ADYTNLPVIVKGIQTPEDAIRAIDAGAAGIYVSNHGGRQLNGGPASFDVLEDIATAVNK QVPIIFDSGVRRGSDVFKALASGADLVALGRPVIYGLALGGAKGVQSVFEHLNHELEIV MQLAGTKTIEDVKNNSLLNIKY

[0251] SEQ ID NO:11 (S. pneumoniae WP000120722)

[0252] MSYKTSNAEGHVDFINTYDLEPMAQQVIPKAAFGYIASGAEDTFTLRENIRAFN HKLIVPHTLCNVENPSTEIEFAGEKLSSPIIMAPVAAHKLANEQGEVATARGVHEFGSL YTTSSYSTVDLPEISEALQGTPHWFQFYFSKDDGINRHIMDRVKAEGYKAIVLTADAT VGGNREVDKRNGFVFPVGMPIVEEYLPEGAGKSMDFVYKSAKQRLSPRDVEFIAEYS GLPVYVKGPQCREDVERSLAAGASGIWVTNHGGRQIDGGPAAFDSLQEVAEAVDRR VPIVFDSGVRRGQHVFKALASGADLVAIGRPVIYGLALGGSVGVRQVFEHLNAELKTV MQLSGTQTIEDVKHFKLRHNPYNPTFPVDPRDLKLY

[0253] SEQ ID NO: 12 (L lactis WP038603448)

[0254] MTYKTSTENKALEIVNVKSLEGKVKQSMEAAGNKGAFGYIRGGSEDEWTLNE NTSAFNKKQIMPRVLRGVDSADLSTSLFGIKLKTPIIQAPVAAQGLAHEEGEVATAKAM AEVGSIFSISTYGSTSVEDAAKAAPDAPQFFQLYMSKDDRFNEFLLKKAVSAGVKAIIL TADSTLGGYREEDIVNHFQFPLPMPNLAAFSESDGTGKGISEIYAAAKQGLVLEDIQKI KKITNLPVIVKGVQSPIDADDAINAGADGIWVSNHGGRQLDGGPASIDVLPLIAKSVNH RVPIVFDSGVRRGEHVFKALAQGADWAVGRPVLYGLNLGGAKGVQSVFEHLNKELS ITMQLAGTKNIEEIKHTSLID

[0255] SEQ ID NO: 13 (E .hirae I6SYK8)

[0256] MEKVYQAGTHEGMIDFINMEDLELAATQVIPSGGYGYISSGAGDLFTYRENQK AFNHQLVIPHVLKDVELPDTTTYFSDETLAAPIIMAPVAAHGLAHEQAEKASAKGVSEF GTIYTASSYASCTLEEIRAAGGPEAPQWFQFYMSKDDGINLDILEMAKRNGAKAWLT ADATVGGNRETDRRNGFTFPLPMPIVQAYQSGVGQTMDAVYKSSKQKLSPKDIEFIT THSELPVYVKGVQSEDDVYRSLDAGAQGIWVSNHGGRQLDGGPASFDSLRYVAEAV DKRVPIVFDSGVRRGQHIFKAIASGADLVAIGRPAIYGLSLGGSTGIKQVFDFFKTELE MVMQLAGTQTVE D IKNAKLRE N RFMC SEQ ID NO: 14 (L Johnsonii A0A137PML5)

[0257] MTTYYKGFPQSTREEKLHMVNLDELENEAKYVMPEAAYYYVASGAENEWTW RNNTQAFNHFQIVPRALTGMQNPELNTEFLGMKLKTPVMICPIACHGIANAEAEIDTAK GAKAAGALFAMSTYANKSVQEVQSAVGDSPRFMQLYLSKNWDFNKMVIEESVKAGF TGFFLTVDALVSGYREANLRTNFTYPVPLAFFNEWNGGKGEGQSVAQMYASSAQNI GPDDIRKIKEIADVPVIVKGVECAEDAMLAIGAGADGIWSNHGGREVDGAPATIDVLP EIAKAVRSCDHRVPIILDGGVRRGSHVFKALALGADLVGIGRPFLYGLALGGAQGVQS VIEQLNKELLIDMQLTGCKTIEDIKHAKIDHINYSADWGISSTSRSVMKPYPVTKENQLT GEAADAVSGASRH

[0258] SEQ ID NO: 15 (L helsingborgensis A0A0F4LW09)

[0259] MTSYYNGFPQSDRDEAIDMINLDELEERAKKVMPEGAYYYIASGSENEWTWR NNISAFNHFQIVPRALTNMDHPQLDTEFMGMKLKTPVMISPIACHGIAHKDAEIATQKG AAAAGALFSSSTYANKSVEDIAAAAPDAPRFFQLYLSKDWGFNKMVFDAVKKAGYKG IFLTVDALVSGFREANLRTKFAYPVPLDFFTRYQGAKGEGQTVAQMYASSAQKIGPE DIKRIKEMSGLPVFVKGVVCAEDAYLAMGAGADGIYVTNHGGREVDCGPATIDMLPEI AKAVNHRVPIVFDSGVRRGSHVFKALALGADMVGIGRPYLYGLALGGAKGVQSVIEQ LNQELLIDMQLTGCKTIEDVKHAKITHINYTADNLKSNTDPSRVKPYPVTAENQLKEND SDAVTGASQA

[0260] SEQ ID NO: 16 (Shigella SP.FC1655 A0A1 D3DHR6)

[0261] M KRQ I LKTTALAM ALSVGVAQAAEYKASTAE G P I KI VN LKAM EAQVQAN M E KG AFGYIRGGAEDENNLRSNTSAFDKKYIMPRSLQGIEFSDLDLTTEFLGIKLDTPIIQAPM AAQGLAHQQGEVATAKGMAKAGSIFSLSTYGNKTIKEVADAQPGYPFFFQLYMSKND AFNEYILSQAKQYGAKGIIMTIDSSVGGYREDDVKNNFQFPLGFANLEAFAKISDDKSK TGKGAGISEIYAQAKQAFTPADIQYVKKMSGLPVIVKGIESPEDADTAIKAGADAIVWS NHGGRQLDSAPATIDVLPAIAKWNKRVPIVFDSGVRRGSHVFKALASGADWAVGR PILYGLNLGGAEGVNSVIQHLNKELKINMMLGGAKTVKDIQATQLYTDASFNQ

[0262] SEQ ID NO: 17 (G. bombicola A0A1 C4CJ33)

[0263] MSYHTSNEEHAIEIVNIASLEALVKARMEAGAFGYIRGGAEDEWTMRENTLSF NRKKIIPRVLQGIDHADLSTKLWDIPLKTPIIQAPSAAQGLAHEQGEKDTAKGVAAAGSI FCISTYANTSIEDAANAAPNVPYFFQLYMSKDDDFNRFIIDKAVKAGTKAIILTVDSTLG GYREEDIVNKFQFPLPMKNLSAYSQSNGNGDGSGKGISEIYAAAKQGIVPSDIQKIKD MANLPVIVKGIQSPEDASIAISAGADGIWISNHGGRQLDGAPASFEVLPSIAATVAKRV

[0264] PIIFDSGVRRGEHVFKALASGADLVAIGRPILYGLNLGGAEGVKSVFDHLNKELSITMQ

[0265] LAGTKTIEDIKNTLLI

[0266] SEQ ID NO: 18 (E avium S0K8K6)

[0267] MTYQAS N E E H P I E I VN I AS LE G RVKE RM E KGAFGYI RGGS E D E WTM KE NTS S F NKKTIMPRVLRGIDHADLSTKLWDMDLATPIIQAPSAAQGLAHEKGEADTAKGVADAG SIFSISTYANTTIEDAAAAAPNAPQFFQLYMSKDDGFNAFILEKAVKAGAKAIILTADSTL GGYREEDIVNKFQFPLPMPNLAAYSAKSASGDGEGKGIAEIYAAAKQGLVPEDIKKIK DLTSLPVFVKGIQSPEDAEVAIAAGADGIWVSNHGGRQLDGGPASFDVLPLITGWNK RVPIVFDSGVRRGEHVFKALASGADLVAIGRPVIYGLNLGGAEGVKSVFDHLNKELSIT MQLAGTKTINEVKNTRLHDTK

[0268] Resu / ts

[0269] Table 1 describes the sequence identity of three variants of the novel lactate oxidase enzyme (SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3) compared to LOx sequences known from literature. Thereby, the matrix shows that the most closely related lactate oxidizing enzymes from literature to SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3 are Enterococcus _A0A4Y3JPV3 and E. / ? / rae_l6SYK8 which show 84.7 and 84.2 % sequence identity to SEQ ID NO:2 respectively. The commercially relevant A. viridans_Q44467 shows its highest sequence identity to SEQ ID NO: 1 with 59.9 %. The sequences SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3 are compared with multiple L-lactate oxidizing sequences known from literature. All sequences are given with their database sequence ID from NCBI or UniProt.

[0270] Tablel Example 2: Biochemical properties and stability

[0271] Activity of oxidase enzyme variants such as Vmax or KM value can be determined in biochemical assays leveraging the conversion of O2 to H2O2.

[0272] Assay conditions

[0273] One unit of enzyme activity is defined as the amount of enzyme that catalyses the transformation of 1 pmol of product (formation of H2O2) per minute at 30 °C. Formation of H2O2 fuels the oxidation of Amplex Red to pink resorufin in the presence of a suitable peroxidase (see Tsvik et al. 2022). When considering a molar extinction coefficient of Amplex Red of 54.0 mM’W (560 nm) the enzymatic activity can be translated from the change in absorbance per time and hence enzyme activity at given substrate concentrations.

[0274] Using nonlinear regression, the Michaelis Menten enzyme kinetic constants can be derived. The maximal reaction rate (vmax) is a good approximator of heterologous electron transfer rates to electrode materials, while the KM value predicts the linear range of the sensor response.

[0275] Resu / ts

[0276] The results of the determination of the kinetic properties are shown in Figure 1. The following Table 2 shows the determined kinetic data.

[0277] Table 2

[0278] The results show that the enzymes having SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3 can be used for the determination of lactate e.g., by using a colorimetric, photometric, or fluorimetric detection methods and determining lactate visually or from the measured absorbance.

[0279] The results of the thermostability determination are shown in Figure 2. Activities were measured at 30°C and 20 mM Lactate in PBS + 0.01 % Triton X-100 using the Amplex Red assay (described above) after storage of the enzyme with 0.5 mg / mL at 70°C for a given time in minutes. Example 3: Electrochemical Properties

[0280] In electrochemical setups the generated peroxide can be oxidized at a platinum surface when sufficient overpotential is applied. Resulting electrical currents correlate to the added substrate concentration.

[0281] Procedures and Materials

[0282] • DRP-C550 Platinum electrodes.

[0283] • Glutaraldehyde 25%. SIGMA G5882-50mL o Freshly diluted to 1 % using water

[0284] Electrode cleaning

[0285] • Perform cyclic voltammetry o -0.5 V to +0.9 V, 100 mV / s, 5 scans

[0286] ■ In drop of 100 pl 0.5 M NaOH, then rinse with water o -0.5 V to +1 .1 V, 100 mV / s, 5 scans

[0287] ■ In drop of 100 pl 0.5 M H2SO4, then rinse with water

[0288] Electrode preparation

[0289] • Mix 9 pl of each LOX with 1 pl of 0.2% Triton X-100. Yields 0.02% Triton X-100

[0290] • Modify electrodes with 1 pl LOX / Triton per working electrode and spread manually to entire surface

[0291] • Cure for 1 h in sealed boxed filled with silica at room temperature

[0292] • Add 5 pl of 1 % glutaraldehyde to working electrode. o Wait for 5 min. o Gently rinse with water. o Measure

[0293] Measurement sequence

[0294] • immerse electrodes horizontally in PBS buffer at 37°C

[0295] • chronoamperometric 3 electrodes setup with an applied voltage of 0.6 Volt vs Ag|AgCI (0.14 M NaCI)

[0296] • subsequent addition of Lactate stock solution to reach a 30 mM concentration Results

[0297] The results are shown in Figure 3. Sensor responds to the addition of Lactate with similar currents then the commercial standard AvLOX. The results show that the enzymes having SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3 can be used for the electrochemical determination of lactate using an electrode comprising such an enzyme. Example 4: Recombinant expression

[0298] Methods

[0299] Recombinant expression and purification were performed as previously described (Tsvik et al. 2022). In short, a E. coli PET 21 system induced by IPTG was used to produce biomass. After cell disruption, purification was performed following the common HisTrap protocols. Before characterization, the His-Tag was cleaved using a HRV3C protease.

[0300] Results

[0301] Table 3 shows the production yields of LOX enzymes.

[0302] Table 3

[0303] Example 5: Electrochemical sensor using a mediator

[0304] In electrochemical setups the electrons can be shuffled from the enzyme to an electrode by a mediator, which gets then oxidized at the electrode at appropriate overpotential. Resulting electrical currents correlate to the added substrate concentration.

[0305] Procedures and Materials

[0306] • DRP-C110 Carbon electrodes.

[0307] • Triton X-100, SIGMA T8787-50ML o Fresh diluted to 0.6% using water

[0308] • Potassium ferricyanide SIGMA 702587-50G o dissolved at 200 mM in 1 mM sodium phosphate buffer, pH 7.4

[0309] Electrode preparation

[0310] • Mix 20 pl of enzyme (SEQ ID NO:1 ) (10 mg / ml) o with 10 pl of 0.6% Triton o with 10 pl of 200 mM ferricyanide

[0311] • Add 2 pl of enzyme / triton / femcyanide mixture to working electrode. Cover working electrode entirely.

[0312] • dry in silica at room temperature for 2 h. Measurement sequence

[0313] • horizontal measurement at ambient temperature

[0314] • samples o buffer o 5 mM lactate in buffer o 50 mM lactate in buffer

[0315] • Add 100 pl of sample to sensor, wait 10 s

[0316] • Perform Chronoamperometry at 0.25 V vs. Ag|AgCI for 120 s Resu / ts The results are shown in Figure 4. Sensor responds to the addition of Lactate with increasing currents. The results show that the enzymes described herein can be used for the electrochemical determination of lactate using an electrode comprising such an enzyme.

[0317] REFERENCES

[0318] Bao W.J., et al (1993), Purification and Characterization of Cellobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-Rot Fungus Phanerochaete chrysosporium, Archives of Biochemistry and Biophysics, 300(2) 705- 713.

[0319] Brugger D, et al. (2014) Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity. PLOS ONE 9(10): e109242.

[0320] Harreither, W. et al. (2011 ) Catalytic Properties and Classification of Cellobiose Dehydrogenases from Ascomycetes, Applied and Environmental Microbiology, 77(5), 1804-1815.

[0321] Kadowaki, M. A. S. et al. (2020) 'Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77', Biochimica et Biophysica Acta, 1864(10), 0304-4165.

[0322] Krondorfer I,, et al. (2014) Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity. PLOS ONE 9(3): e91145.

[0323] Maeda-Yorita, K. et al. (1995) ‘L-lactate oxidase and L-lactate monooxygenase: Mechanistic variations on a common structural theme’, Biochimie, 77(7-8), pp. 631-642. doi: 10.1016 / 0300-9084(96)88178-8.

[0324] Mitsubayashi, K. et al. (1994) ‘Analysis of metabolites in sweat as a measure of physical condition. The increasing desire to naturally enhance the performance of sportsmen and athletes through’, Analytica Chimica Acta, 289, pp. 27-34.

[0325] Kavita Rathee, et al., Biosensors based on electrochemical lactate detection: A comprehensive review, Biochemistry and Biophysics Reports, Volume 5, 2016, pp. 35- 54, ISSN 2405-5808, https: / / doi.Org / 10.1016 / j.bbrep.2015.11.010.

[0326] Rocchitta, G., et al. (2016). Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids. Sensors (Basel, Switzerland), 16(6), 780. https: / / doi.org / 10.3390 / s16060780

[0327] Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011 )

[0328] Sygmund, C. et al. (2011 ) 'Reduction of Quinones and Phenoxy Radicals by Extracellular Glucose Dehydrogenase from Glomerella Cingulata Suggests a Role in Plant Pathogenicity' Microbiology, 157(11 ):3203-12.

[0329] Tsvik, Lidiia, Beate Steiner, Peter Herzog, Dietmar Haltrich, and Leander Sutzl. 2022. “Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors.” ACS Omega 7 (45): 41480-92. https: / / doi.org / 10.1021 / acsomega.2c05257

[0330] Taurino, I. et al. (2013) ‘Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications’, Electrochi mica Acta, 93, pp. 72-79. doi: 10.1016 / j.electacta.2013.01 .080.

Claims

CLAIMS1 . An enzyme having lactate oxidizing activity, wherein said enzyme comprises SEQ ID NO: 1 or a sequence having at least 85 % sequence identity with SEQ ID NO: 1 .

2. The enzyme of claim 1 , wherein the sequence has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1.

3. The enzyme of claim 1 or 2, wherein said enzyme is a recombinant enzyme.

4. The enzyme of any one of claims 1 to 3, further having a purification tag at its N- terminus.

5. The enzyme of any one of claims 1 to 4, further having a Met at its N-terminus.

6. The enzyme of any one of claims 1 to 5, wherein the sequence is selected from any one of SEQ ID NOs:2 to 6.

7. Use of the enzyme of any one of claims 1 to 6 for the determination of lactate in a sample.

8. An electrode comprising the enzyme of any one of claims 1 to 6.

9. A biosensor comprising the electrode of claim 8.

10. A nucleotide sequence encoding the enzyme of any one of claims 1 to 6.11 . A host cell expressing the enzyme of any one of claims 1 to 6.

12. A method of producing the enzyme of any one of claims 1 to 6 in a host cell, said method comprising the steps of: a. transfecting a host cell with one or more expression cassettes comprising the nucleotide sequence of claim 10, b. cultivating the host cell under conditions wherein the enzyme is expressed in said host-cell, and c. isolating the enzyme from the host cell.

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