Bioelectrochemical determination of lactate

Abstract

The present invention relates to methods and means for detecting and / or quantifying lactate. In particular, the present invention relates to a method for detecting and / or quantifying lactate in a sample, comprising the steps of: a) detecting and / or quantifying lactate in a sample by combining flavin mononucleotide (FMN) with the following sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 in SEQ ID NO: 2, F is or corresponds to F152 in SEQ ID NO: 2, and X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45, n3 is an integer from 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V; a) providing an electrode containing lactate dehydrogenase (LDH) comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, wherein X1 is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45, n3 is an integer from 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V; b) contacting a sample with the electrode; and c) detecting oxidation of lactate by LDH, wherein the oxidation of lactate is carried out in the presence of a redox mediator.

Description

[Technical field]

[0001] The present invention relates to the field of analyte detection and quantification, and in particular to methods and means for the detection and / or quantification of lactate by providing an electrode containing lactate dehydrogenase. [Background technology]

[0002] The food and beverage industry, as well as clinical diagnostics, require highly selective, sensitive, rapid and reliable methods to determine the quality of products and the presence of important components or metabolites that act as markers for disease or for human physiological conditions. L-lactate is one of these metabolites. Analysis of blood lactate levels is also important for the clinical diagnosis of hypoxia, lactic acidosis, hyperlactemia in diabetes and liver disease, sepsis and thiamine deficiency, as well as for drug toxicity testing. Lactate is also measured in monitoring athletes' performance and developing optimal training plans.

[0003] In the pharmaceutical industry, the presence of the metabolite lactate needs 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 due to its toxic and growth inhibitory effects on cells.

[0004] Thereby, the relevant concentration range for L-lactate varies depending on the particular sample type and the purpose of the measurement: for example, the physiologically relevant range of L-lactate in blood is usually 1-25 mM (Goodwin et al., 2007), but the physiologically relevant range of L-lactate can also be up to more than 100 mM in sweat after exhaustive exercise (Mitsubayashi et al., 1994).

[0005] Currently, lactate determination is based on enzymatic test systems that measure the oxidation of L-lactate to pyruvate. Traditionally, these enzymatic methods are based on NAD+-dependent lactate dehydrogenase (LDH) isolated from animal muscle or heart, or on bacterial lactate oxidase (LOx). Thereby, the lactate content is determined by spectrophotometric detection of NADH or colorimetric assay of H2O2. Many other methods have been proposed as well; for example, spectrophotometry, fluorimetry, and pH potentiometric measurements. Also, amperometric biosensors based on O2 consumption and H2O2 formation detected on an electrode have been proposed.

[0006] Biosensors are the preferred method to monitor L-lactate levels. Rathee et al. (2016) describe the basic principles of lactate biosensors. Broadly, a biosensor consists of a biological recognition element, e.g., an enzyme, capable of specifically interacting with a target molecule, and a transducer capable of converting this interaction into a measurable signal. In a biosensor, the enzyme is connected to an electrode and a current is measured that is proportional to the concentration of the enzyme's substrate in the sample. Biosensors are easy to operate, highly specific, and do not require expensive and heavy equipment, as is the case for HPLC or NMR.

[0007] The currently preferred enzyme used to construct L-lactate biosensors is L-lactate oxidase (LOx), specifically LOx from Aerococcus viridans (AvLOx). LOx belongs to the family of FMN-dependent α-hydroxyacid oxidoreductases (HAOx; EC: 1.1.3.15; Maeda-Yorita et al., 1995) and thus oxidizes α-hydroxyacids to their corresponding α-ketoacids 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 the electron acceptor to reoxidize FMN, which itself is reduced to hydrogen peroxide (H2O2). Thus, biosensors based on oxidases such as LOx are oxygen-dependent and are first-generation biosensors.

[0008] In second generation biosensors, redox mediators other than O2 / H2O2 redox mediators are applied to transfer electrons from the enzyme to the electrode. Such a scheme allows the use of lower potentials, avoids oxygen dependency, and also avoids the effects of interfering molecules. However, the use of oxidases as recognition elements in such second generation biosensors leads to oxygen interference in the presence of oxygen, since oxygen is usually still present and competes with the alternative electron acceptor at the active site of the oxidase, i.e., LOx. Thereby, oxygen "steals" electrons from the detectable electron flow, leading to a reduction in the sensor signal.

[0009] Oxygen interference is usually avoided by applying dehydrogenases instead of oxidases for second generation biosensors. In contrast to oxidases, dehydrogenases do not use oxygen as the main preferred electron acceptor. The application of lactate dehydrogenase for the bioelectrochemical detection and / or quantification of lactate can prevent oxygen interference even in the presence of oxygen.

[0010] Unfortunately, the only L-lactate dehydrogenases (LDHs) known to date are either membrane-bound or NAD+-dependent, and the production, purification, and application of membrane-bound proteins pose various challenges and are not NAD + Both of them are unsuitable for application, for example, in biosensors for continuous lactate measurement in vivo, since it is continuously lost due to diffusion or degradation and needs to be resupplied during the measurement ( Cardosi and Liu, 2012 ; Kucherenko et al., 2019 ).

[0011] WO 2022 / 054044 A1 discloses an L-lactate biosensor using L-LDH (ScLDH) from baker's yeast. ScLDH is a fungal flavocytochrome b2 enzyme located in the mitochondrial intermembrane space. This membrane-bound enzyme shows impractical yields when recombinantly expressed.

[0012] There is therefore a pressing need in the art for methods and means that enable oxygen-independent second generation biosensor-based lactate detection and / or quantification for industrial applications. Summary of the Invention [Problem to be solved by the invention]

[0013] It is an object of the present invention to provide methods and means for bioelectrochemical detection and / or quantification of lactate in a sample that is oxygen independent. That object is solved by the subject matter of the present invention. [Means for solving the problem]

[0014] The inventors of the present invention have surprisingly found a unique class of flavin mononucleotide (FMN)-dependent lactate dehydrogenases capable of oxidizing lactate and transferring the acquired electrons to a redox mediator that has limited ability to transfer electrons to oxygen. Specifically, the inventors have found specific sequences with characteristic conserved amino acid residues that show this suitable dehydrogenase activity pattern of lactate oxidation for application on electrodes for second generation biosensors. These electrodes have no or very low oxygen interference, allowing the application of lactate biosensors in industry for applications such as continuous lactate measurement. The advantage of the ability of the enzymes described herein to transfer the acquired electrons from lactate oxidation to a redox mediator is that this transfer to the redox mediator occurs even when oxygen is present during lactate oxidation. The presence of oxygen does not interfere with the methods and means described herein, since the enzymes described herein have no or very low activity with oxygen as the electron acceptor. Another advantage of the ability to transfer the electrons gained from lactate oxidation to a redox mediator instead of oxygen is that several different redox mediators can be used in the methods and means described herein. If an enzyme capable of oxidizing a molecule, for example lactate, does not accept oxygen as an electron acceptor, i.e. is not lactate oxidase, and if this enzyme accepts a redox mediator as an electron acceptor, i.e. the enzyme is lactate dehydrogenase, it is generally known that such an enzyme will accept several different redox mediators commonly known to those skilled in the art, for example as described by Rathee et al. (2016).

[0015] According to the present invention there is provided a method for detecting and / or quantifying lactate in a sample, comprising the steps of: a) providing an electrode comprising lactate dehydrogenase (LDH), the LDH being an enzyme having the sequence: T-(X x )n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 of SEQ ID NO:2; F is or corresponds to F152 in SEQ ID NO:2; X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45; n3 is an integer from 5 to 20, X1 is N or F; X2 is L or F; X3 is G, T, or S; X4 is I or V) 2, or an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2; b) contacting the sample with electrodes; c) detecting the oxidation of lactate by LDH; A method is provided, comprising:

[0016] Specifically, the oxidation of lactate is carried out in the presence of a redox mediator. Specifically, the sample is selected from the group consisting of any one of food; beverages; fermented foods; fermented beverages; chemicals; water; soil; and samples provided by humans or animals, specifically any one of body fluids, interstitial fluid, blood, plasma, dermal fluid, urine, tears, sweat, saliva, skin, meat, tissue, eyeball, cornea, and gastric juice.

[0017] According to the present invention, there is also provided an electrode comprising LDH, the LDH being a flavin mononucleotide (FMN) having the following sequence: T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x)n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 of SEQ ID NO:2; F is or corresponds to F152 in SEQ ID NO:2; X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45; n3 is an integer from 5 to 20, X1 is N or F; X2 is L or F; X3 is G, T, or S; X4 is I or V) and an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, comprising the amino acid sequence of SEQ ID NO:2.

[0018] Specifically, the electrode further comprises a redox mediator. In particular, according to said method or said electrode, the LDH is immobilized on the electrode, preferably by adsorption, preferably by complexation via a further complexing linker, a covalent or ionic bond, and / or preferably the LDH is cross-linked, in particular by a bifunctional agent.

[0019] Specifically, the LDH is immobilized on an electrode. Specifically, the LDH is immobilized by adsorption or complexation. Specifically, the complexation is via a complexing linker, a covalent bond or an ionic bond.

[0020] Specifically, the LDH is cross-linked. Specifically, the LDH is cross-linked by a bifunctional agent. The present invention also provides a biosensor comprising an electrode according to the present invention.

[0021] The present invention also provides a device comprising an electrode according to the present invention, or a biosensor according to the present invention. According to the present invention, there is also provided an enzyme composition comprising LDH and a redox mediator, wherein the lactate dehydrogenase is selected from the group consisting of flavin mononucleotide (FMN) and the following sequence: T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 of SEQ ID NO:2; F is or corresponds to F152 in SEQ ID NO:2; X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45; n3 is an integer from 5 to 20, X1 is N or F; X2 is L or F; X3 is G, T, or S; X4 is I or V) and an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, comprising the amino acid sequence of SEQ ID NO:2.

[0022] Specifically, according to the method of the present invention, the electrode of the present invention, or the enzyme composition of the present invention, the 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.

[0023] The present invention also provides a recombinant LDH containing flavin mononucleotide (FMN), the amino acid sequence of which, from the N-terminus to the C-terminus, is: i. dipeptide GP; ii. Array T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 of SEQ ID NO:2; F is or corresponds to F152 in SEQ ID NO:2; X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45; n3 is an integer from 5 to 20, X1 is N or F; X2 is L or F; X3 is G, T, or S; X4 is I or V) SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity to amino acids 2 to 373 of SEQ ID NO:2. The present invention provides a recombinant LDH comprising:

[0024] Specifically, according to the recombinant LDH comprising flavin mononucleotide (FMN) described herein, the dipeptide "GP" forms the N-terminus of the sequence, whereby "G" forms the N-terminus of the recombinant LDH, and the following sequence is linked to the C-terminus of the dipeptide "GP": the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 of SEQ ID NO:2; F is or corresponds to F152 in SEQ ID NO:2; X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45; n3 is an integer from 5 to 20, X1 is N or F; X2 is L or F; X3 is G, T, or S; X4 is I or V), or an amino acid sequence having at least 50% sequence identity to amino acids 2 to 373 of SEQ ID NO:2.

[0025] Specifically, the technical effect of the dipeptide "GP" in the recombinant LDH containing flavin mononucleotide (FMN) described herein is increased activity. Specifically, according to the method of the present invention, the electrode of the present invention, the enzyme composition of the present invention, or the recombinant LDH of the present invention, the amino acid sequence of the LDH comprises a sequence having at least 70%, 80%, 90%, or 95% sequence identity to amino acids G35 to K360 of SEQ ID NO:2, wherein the LDH comprises SEQ ID NO:1.

[0026] Specifically, according to the method of the present invention, the electrode of the present invention, the enzyme composition of the present invention, or the recombinant LDH of the present invention, the LDH is selected from SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:59, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:155, or a sequence having at least 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:59, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:155, wherein the sequence comprises SEQ ID NO:1.

[0027] Specifically, according to the method of the present invention, the electrode of the present invention, the enzyme composition of the present invention, or the recombinant LDH of the present invention, the LDH is selected from SEQ ID NO: 2 to SEQ ID NO: 249, or a sequence having at least 90%, or 95%, sequence identity to SEQ ID NO: 2 to SEQ ID NO: 249, wherein the LDH includes SEQ ID NO: 1.

[0028] The present invention also provides a kit for detecting and / or quantifying lactate or a salt thereof, the kit comprising the electrode of the present invention, the biosensor of the present invention, or the device of the present invention, and further comprising an instruction manual. [Brief description of the drawings]

[0029] [Figure 1] Maximum likelihood phylogenetic tree of the LOx cluster, showing the location of some enzyme sequences and their relative activities with L-lactate. The tree shows a clear separation of the oxidases and dehydrogenases into two distinct clades. [Figure 2-1] UV-Vis absorption spectra showing the oxidized (solid line) and reduced (dashed line) forms of purified flavoprotein. The maximum of the oxidized FMN cofactor is indicated in each spectrum. nd - not determinable. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Diagram 3] Effect of overnight digestion of the purification tags of the enzymes with protease HRV 3C on their specific activity. Activity was measured with L-lactate and DCIP. [Figure 4-1] pH activity curves of LDH and AvLOx measured with 10 mM L-lactate and oxygen (dashed line) or DCIP (solid line) as electron acceptor. Activities are given in % relative to the highest activity of each. Measurements were performed in quadruplicate in 40 mM Britton-Robinson universal buffer (BRB) pH 4.5-10. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Diagram 5] Current response (left) and calibration function (right) of the biosensor prototype at increasing concentrations of lactate using EaLDH. [Figure 6]Current response (left) and calibration function (right) of the biosensor prototype at increasing concentrations of lactate using LjLDH. [Figure 7] Current response (left) and calibration function (right) of the biosensor prototype at increasing concentrations of lactate using SsLDH. [Figure 8] Current response (left) and calibration function (right) of the biosensor prototype at increasing concentrations of lactate using EaLDH. [Figure 9] Current response (left) and calibration function (right) of the biosensor prototype at increasing concentrations of lactate using AvLOX. [Figure 10] Comparison of catalytic currents in the biosensor prototype in the presence and absence of oxygen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Unless otherwise specified or defined, all terms used herein have their usual meaning in the art, which are clear to those skilled in the art.For example, refer to standard handbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th edition), vol. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017); Berg et al., "Stryer Biochemie", Springer Verlag, 2018; and Murphy & Weaver, "Janeway's Immunobiology" (9th edition or more recent edition), Taylor & Francis Inc, 2017.

[0031] The subject matter of the claims specifically refers to man-made products or methods of using or producing such man-made products, which may be variants of natural (wild-type) products. Although there may be a certain degree of sequence identity to natural structures, it is well understood that the materials, methods, and uses of the invention, which specifically refer to, for example, isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, and modified proteins and enzymes, are "artificial" or synthetic and therefore are not considered the result of the "laws of nature."

[0032] The terms "comprise", "contain", "have" and "comprising" as used herein can be used synonymously and should be understood as open definitions, allowing for additional members or moieties or elements. "Consisting of" is considered the most closed definition without additional elements characteristic of the definition of "consisting of". Thus, "comprising" is broader and contains the definition of "consisting of".

[0033] The term "about" as used herein refers to the same value or a value that differs by + / - 5% from a given value. As used in this specification and the claims, the singular forms, e.g., "a," "an," and "the," include the plural forms unless the context clearly dictates otherwise.

[0034] As used herein, amino acid refers to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be divided into neutrally charged, positively charged, and negatively charged amino acids.

[0035] The "neutral" amino acids are shown below with their respective three letter and one letter codes and polarities: alanine (Ala, A; nonpolar, neutral), asparagine (Asn, N; polar, neutral), cysteine ​​(Cys, C; nonpolar, neutral), glutamine (Gln, Q; polar, neutral), glycine (Gly, G; nonpolar, neutral), isoleucine (Ile, I; nonpolar, neutral), leucine (Leu, L; nonpolar, neutral), methionine (Me, F; nonpolar, neutral), riboflavin (Rri ... The amino acids in the ribozyme are: methacridine (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 (Val, V; nonpolar, neutral), and histidine (His, H; polar, positive (10%) and neutral (90%).

[0036] The "positively" charged amino acids are arginine (Arg, R; polar, positive) and lysine (Lys, K; polar, positive). The "negatively" charged amino acids are aspartic acid (Asp, D; polar, negative) and glutamic acid (Glu, E; polar, negative).

[0037] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and alpha-methylserine) may be substituted for amino acid residues in wild-type polypeptides. A limited number of non-conservative amino acids, which are amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" are modified after protein synthesis and / or have a chemical structure in their side chain(s) that differs from the chemical structure of the standard amino acids. Unnatural amino acids can be chemically synthesized and are preferably commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0038] The term "enzyme", as used herein, refers more or less specifically to any substance consisting exclusively or primarily of a protein or polypeptide that catalyzes or facilitates one or more chemical or biochemical reactions.

[0039] The term "lactate" as used herein refers to lactic acid or its salts. The preferred enantiomer of lactate is L-lactate. According to the present invention, a method for detecting and / or quantifying lactate is provided. Thereby, the term "detecting" lactate refers to a general determination if lactate is present. Detection does not require a precise quantification of lactate, but provides information to the user of the method, for example, if lactate is present at a concentration above a certain threshold value. These threshold values ​​should be adapted to each application and sample. The term "quantification" refers to the determination of the concentration or amount of lactate.

[0040] According to the invention, the detection of the oxidation of lactate by LDH is carried out by a sensor, in particular a bioelectrochemical sensor, configured to detect and / or quantify lactate in a sample via (bio)electrochemical redox reactions, which can usually be converted into an electrical signal that can be correlated to the amount or concentration of the analyte lactate.

[0041] Electrochemical biosensors can be impedimetric, potentiometric, or amperometric. In amperometric biosensors, a biochemical signal is converted into a quantifiable amperometric signal.

[0042] As described in Rocchitta G. et al. (2016), amperometric biosensors are usually divided into three main generations according to the electron transfer method used to measure the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediator and cofactor). 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. First generation biosensors are also called mediator-less amperometric biosensors. Usually, oxidases are used in first generation biosensors. Oxidases require oxygen molecules as a second substrate, and therefore, oxidase-based biosensors are oxygen-dependent. Therefore, first generation biosensors that use oxygen as an electron acceptor suffer from errors resulting from changes or low concentrations of dissolved oxygen, affecting the sensor response and reducing linearity. This oxygen dependency limits the applicability of first generation amperometric biosensors in biological systems, e.g., first generation amperometric biosensors are not suitable for use under ischemic conditions. Ischemia is a condition in which blood flow (and therefore oxygen) is restricted or reduced in a part of the body. Cardiac ischemia is a condition in which blood flow and oxygen to the heart muscle is reduced. It has been described that lactate concentrations at the incision site indicate an ischemic-like condition and may contribute to postoperative pain (Kim, TJ et al., 2007).

[0043] According to one embodiment, the sample may be any material in which lactate concentration is relevant or of interest. In particular, the sample is selected from the group consisting of any one of: food; beverages; fermented foods; fermented beverages; chemicals; water; soil; and samples provided by humans or animals, in particular any one of body fluids, interstitial fluid, blood, plasma, skin fluid, urine, tears, sweat, saliva, skin, meat, tissue, eyeball, cornea, and gastric juice.

[0044] For detection and / or quantification by the methods provided herein, the electrodes are contacted with the sample. This contact between the electrodes and the sample can be performed by any approach that contacts the electrodes and the sample in a manner that allows the enzyme to react with lactate or with a sample suspected of containing lactate.

[0045] The term "lactate dehydrogenase", abbreviated herein as LDH, refers to an enzyme that catalyzes the oxidation of lactate to pyruvate, whereby two electrons are transferred from lactate to the LDH cofactor FMN, and subsequent transport of the electrons so acquired is toward a suitable electron acceptor, such as DCIP, while generally being insensitive or nearly insensitive to accepting dioxygen as the electron acceptor.

[0046] According to certain embodiments, the LDH is characterized by its enantiomeric selectivity and specificity for the natural lactate substrate L-lactate. According to certain embodiments, the LDH of the present invention can be active in the acidic, neutral, or alkaline pH range. Specifically, the LDH of the present invention can be used at a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or at any pH of body fluids, such as sweat or blood. Usually, blood has a pH of 7.35 to 7.45.

[0047] The LDH according to the present invention has the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 in SEQ ID NO: 2, F is or corresponds to F152 in SEQ ID NO: 2, and X xis any amino acid, n1 is an integer of 25 to 35, n2 is an integer of 40 to 45, n3 is an integer of 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V), or an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 2.

[0048] According to a particular embodiment of the invention, the LDH of the invention comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or even 95% sequence identity to SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 90%, or even 95% sequence identity to amino acids Gly35 to Lys360 of SEQ ID NO: 2, wherein the amino acid sequence of the LDH comprises the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 in SEQ ID NO: 2, F is or corresponds to F152 in SEQ ID NO: 2, and X x is any amino acid, n1 is an integer from 25 to 35, n2 is an integer from 40 to 45, n3 is an integer from 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V).

[0049] The LDH sequences according to the invention comprise SEQ ID NO:1 as defined herein, wherein said LDH sequences comprising SEQ ID NO:1 are characterized in that these LDHs catalyse the lactate dehydrogenase reaction as defined herein.

[0050] SEQ ID NO: 2 is the amino acid sequence of LDH (PaLDH) from Pediococcus acidilactici. In the present invention, it was surprisingly found that the enzyme "PaLCTO" from Pediococcus acidilactici described in Ashok Y. et al. (2020) is in fact capable of oxidizing lactate when a redox mediator other than molecular oxygen is used as the electron acceptor. According to Ashok Y. et al. (2020), PaLCTO does not use lactate as a substrate. Even more surprisingly, in the present invention, it was found that the enzyme named PaLCTO in Ashok Y. et al. (2020) is a lactate dehydrogenase that allows the detection and / or quantification of lactate by an electrode using mediated electron transfer.

[0051] According to one embodiment, the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x ) n3 In -X3-X4 (SEQ ID NO: 1), the amino acids and variables are the following amino acids and integers in SEQ ID NO: 2: T is T122, F is F152, n1 is 29, n2 is 43, n3 is 11, X1 is N196, X2 is L197, X3 is G209, and X4 is I210. In SEQ ID NO: 1 of the LDH of the present invention, the amino acids and integers correspond to these amino acids and integers.

[0052] The term "corresponding to" as used herein refers to respective corresponding amino acids or integers determined by sequence alignment and / or by structural alignment of two or more sequences. For example, for determining corresponding amino acids in LDH, the LDH amino acid sequence is aligned in a sequence and / or structural alignment and / or by superposition with, for example, SEQ ID NO:2.

[0053] According to certain embodiments, n1 is an integer of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. According to certain embodiments, n2 is an integer of 40, 41, 42, 43, 44, or 45.

[0054] According to certain embodiments, n3 is an integer of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. According to a particular embodiment of the invention, the electrode comprises a nucleotide sequence comprising flavin mononucleotide (FMN) and T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3-X3-X4 (SEQ ID NO: 1) (wherein T is or corresponds to T122 in SEQ ID NO: 2, F is or corresponds to F152 in SEQ ID NO: 2, and X x is any amino acid, n1 is an integer of 25 to 35, n2 is an integer of 40 to 45, n3 is an integer of 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V.

[0055] In Table 1 below, the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:249 are given.

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058]

Table 1-3

[0059]

Table 1-4

[0060]

Table 1-5

[0061]

Table 1-6

[0062]

Table 1-7

[0063]

Table 1-8

[0064]

Table 1-9

[0065]

Table 1-10

[0066]

Table 1-11

[0067]

Table 1-12

[0068]

Table 1-13

[0069]

Table 1-14

[0070]

Table 1-15

[0071]

Table 1-16

[0072]

Table 1-17

[0073]

Table 1-18

[0074]

Table 1-19

[0075]

Table 1-20

[0076]

Table 1-21

[0077]

Table 1-22

[0078]

Table 1-23

[0079]

Table 1-24

[0080]

Table 1-25

[0081]

Table 1-26

[0082]

Table 1-27

[0083]

Table 1-28

[0084]

Table 1-29

[0085]

Table 1-30

[0086]

Table 1-31

[0087]

Table 1-32

[0088]

Table 1-33

[0089]

Table 1-34

[0090]

Table 1-35

[0091]

Table 1-36

[0092]

Table 1-37

[0093]

Table 1-38

[0094]

Table 1-39

[0095]

Table 1-40

[0096]

Table 1-41

[0097]

Table 1-42

[0098] In the three-dimensional structure of LDH, SEQ ID NO:1 is located in or interacts with the lid of the active site (see Ashok Y. et al., 2020). Residues T, F, X1, X2, X3, and X4 of SEQ ID NO:1 form a structural motif in the three-dimensional structure of LDH.

[0099] According to one embodiment of the present invention, residues T, F, X1, X2, X3, and X4 of SEQ ID NO: 1 form a structural motif in the three-dimensional structure of LDH. The distance ranges of these residues in the structural motif are given in the α-carbon distance matrix below in Table 2. The distances are given in units of angstroms (Å). The distances given in Table 2 should be understood to include the decimal places of each number. For example, if a distance of 10 Å is given, this designation of the distance includes 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, and 10.9. Furthermore, when considering the distances between residues in the three-dimensional structure of an enzyme, it is necessary to consider the variability between different methods of determining protein structure and the uncertainty of a single determined structure, especially when a computer-simulated model of the structure is used to determine the distances between residues. The distances between residues X3 and X4, and between residues T, F, X1 and X2 of SEQ ID NO:1 may vary between the conformations of the different active site loops, and therefore the distances for X3 and X4 may vary in these different conformations of the three-dimensional structure.

[0100] [Table 2]

[0101] In the method of the present invention, an electrode with LDH is used. The term "electrode" refers to any suitable surface for receiving electrons from LDH via mediated electron transfer. The electrode thereby has a material capable of receiving electrons from the redox mediator used in the present invention. Furthermore, the electrode may have any material suitable for adsorbing or immobilizing LDH, or may be modified with any material to adsorb or immobilize LDH. Non-limiting examples of such materials are platinum, gold, boron-doped diamond, and carbon such as graphite, pyrolytic graphite, and glassy carbon, all of which can be further modified with carbon nanotubes (single-walled or multi-walled), carbon fibers, nanoparticles, such as gold nanoparticles, or promoters, such as thiols. The electrode may also have any material to increase the specific surface area of ​​the electrode.

[0102] According to one embodiment of the invention, the electrodes may be used as single electrodes or as a stack of, for example, 2, 3, 4, 5 or more electrodes. According to one embodiment of the present invention, the electrode provided herein is a working electrode.

[0103] The LDH-containing electrodes of the present invention allow the detection and / or quantification of lactate based on mediated electron transfer. Mediated electron transfer in biosensors usually uses a two-step procedure in which an enzyme first participates in a redox reaction with a substrate, which is then reoxidized by a redox mediator, which is finally oxidized by the electrode.

[0104] A redox mediator is an artificial electron transfer agent that can easily participate in a redox reaction with a biological component and thus facilitates rapid electron transfer to an electrode. A "redox mediator" is an electron transfer agent for transporting electrons between an analyte, an enzyme that is reduced by the analyte or oxidized by the analyte, and an electrode, either directly or via one or more additional electron transfer agents. A redox mediator that includes a polymer backbone may also be referred to as a redox polymer.

[0105] Generally, oxygen is not a redox mediator. According to one embodiment of the present invention, the oxidation of lactate is carried out in the presence of a redox mediator.The method of the present invention can also be carried out in the presence of more than one redox mediator, for example in the presence of two or more different redox mediators.Therefore, the redox mediator can be present on the electrode, in an enzyme composition comprising the enzyme and the redox mediator, or in the sample.

[0106] According to another embodiment of the present invention, the enzyme composition comprises an LDH of the present invention and a redox mediator as defined herein. In a particular embodiment, the enzyme composition comprises an LDH and a redox polymer. In particular, the redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.

[0107] According to one embodiment, the redox mediator can be any molecule or material capable of transporting electrons between the LDH and the electrode. Specifically, the redox mediator is selected from the group consisting 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.

[0108] The polymeric transition metal complex includes a polymer backbone, a spacer, and a transition metal complex. Specifically, a redox polymer is a polymer that contains a 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 in 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).

[0109] According to certain embodiments, when the redox mediator comprises osmium, the redox mediator may be an osmium transition metal complex having one or more ligands, each of which has a nitrogen-containing heterocycle, such as 2,2'-bipyridine, 1,10-phenanthroline, 1-methyl,2-pyridylbiimidazole, or a derivative thereof. The redox mediator may also have one or more ligands covalently attached in a polymer, each of which has at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or a derivative thereof. An example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups, and (b) an osmium cation complexed with two ligands, each of which contains 2,2'-bipyridine, 1,10-phenanthroline, or a derivative thereof, and the two ligands are not necessarily identical. Some derivatives of 2,2'-bipyridine for complexing with osmium cations include, but are not limited to, mono-, di-, and polyalkoxy-2,2'-bipyridines, including 4,4'-dimethyl-2,2'-bipyridine and 4,4'-dimethoxy-2,2'-bipyridine. Derivatives of 1,10-phenanthroline for complexing with osmium cations include, but are not limited to, mono-, di-, and polyalkoxy-1,10-phenanthrolines, such as 4,7-dimethyl-1,10-phenanthroline and 4,7-dimethoxy-1,10-phenanthroline. Polymers for complexing with osmium cations include, but are not limited to, polymers and copolymers of poly(1-vinylimidazole) and poly(4-vinylpyridine). Suitable copolymer substituents of poly(1-vinylimidazole) include acrylonitrile, acrylamide, and electron transfer agents having osmium complexed to a polymer or copolymer of substituted or quaternary N-vinylimidazole, e.g., poly(1-vinylimidazole). An example of a redox polymer is poly(1-vinylimidazole) or Os 3+ / 2+ , Ru 3+ / 2+ , and Fe 3+ / 2+The copolymer is derived from a copolymer of (1-vinylimidazole) bonded to a metal ion selected from the group consisting of:

[0110] The term "transition metal" refers to elements whose atoms can give rise to cations with partially filled or incomplete d sub-shells. Transition metals are thus the elements in the d block of the periodic table, as well as the lanthanides and actinides.

[0111] Non-limiting examples of transition metal complexes include complexes containing titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, 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 coordinated to one or more ligands that are usually monodentate, bidentate, tridentate, or tetradentate.

[0112] Non-limiting examples of transition metal complexes include complexes containing lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0113] Non-limiting examples of transition metal complexes include complexes containing actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, and lawrencium.

[0114] Sandwich compounds are chemical compounds that feature a metal bound to two arene ligands by haptic covalent bonds. The arene has the formula C n H n , substituted derivatives (e.g. Cn (CH3) n ) and heterocyclic derivatives (e.g. BC n H n+1 ). A special type of sandwich complex is the metallocene. Metallocenes contain a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure, i.e., the two cyclopentadienyl anions lie in parallel planes with equal bond length and strength. Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1,1'-dimethylferrocene [DMF], and ferrocene monocarboxylic acid.

[0115] Organic redox compounds, also referred to herein as organic redox mediators, are organic molecules capable of acting as redox mediators. Non-limiting examples of organic redox mediators are organic molecules such as quinones, compounds with quinoid structures such as benzoquinone or phenanthroline quinone, phenazines such as 1-methoxyphenazine methosulfate, tetracyanoquinodimethane (TCNQ), N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivatives of these molecules.

[0116] According to one embodiment of the present invention, the soluble redox mediator is a freely diffusing compound dissolved in the solution phase that is capable of transferring electrons between the enzyme and the electrode by changing its redox state.

[0117] According to one embodiment of the present invention, the insoluble redox mediator is a compound that is not dissolved in the solution phase and is immobilized on the electrode surface that can transfer electrons between the enzyme and the electrode by changing its redox state.

[0118] According to one embodiment of the present invention, the wired redox mediator is a polymer with several covalently attached redox mediator units that limit their diffusion. According to one embodiment of the present invention, the LDH is immobilized on the electrode by adsorption, physical entrapment in a polymer, complexation, preferably via an additional complexing linker, covalent, especially crosslinking, or ionic bonding, and / or the immobilized LDH can be crosslinked, especially by bifunctional agents, to increase its stability or activity. Crosslinking agents are, for example, dialdehydes such as glutaraldehyde.

[0119] According to another embodiment of the present invention, the oxygen interference of the electrode is less than 10%. More specifically, the oxygen interference of the electrode is less than 9, 8, 7, 6, 5, 4, 3, 2, 1% or even undetectable.

[0120] The term "oxygen interference" as used herein refers to a phenomenon occurring in second generation biosensors that rely on mediated electron transfer from an enzyme to an electrode. If the enzyme in this system is able to transfer electrons to oxygen and the redox mediator used for mediated electron transfer, some of the electrons will be transferred to oxygen and not to the redox mediator. Since accurate determination of the concentration of an analyte depends on the transfer of electrons acquired from the substrate to the redox mediator, fluctuations in oxygen levels lead to signal fluctuations and thus erroneous results. In other words, oxygen competes with the redox mediator at the active site of the enzyme, thereby "stealing" electrons from the detectable electron flow, leading to a reduction in the sensor signal that depends on the available oxygen concentration, which may vary from sample to sample.

[0121] Oxygen interference can be measured by measuring the sensor at high and low oxygen concentrations in the measurement solution in the presence of a redox mediator, which can be adjusted by bubbling the solution with nitrogen or argon gas. Differences in the recorded current response of the sensor can be used to quantify the interference.

[0122] Alternatively, oxygen interference can be measured at the enzyme level, whereby the electron transfer of LDH to oxygen (oxidase activity) and to other electron acceptors (dehydrogenase activity) can be determined by various enzyme assays. The ratio of oxidase activity to dehydrogenase activity can be determined from the specific activities determined by these assays.

[0123] According to one embodiment of the present invention, the oxygen interference at the enzyme level is determined by determining the oxidase activity using Amplex Red (AR) assay and the dehydrogenase activity using DCIP assay.Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / DCIP) is less than 0.1 or less than 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or even less than 0.01.Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / DCIP) may also be impossible to determine due to undetectable oxidase activity.Amplex Red and DCIP assays are described in the following sections.

[0124] According to another embodiment of the present invention, the oxygen interference at the enzyme level is determined by determining the oxidase activity using Amplex Red (AR) assay and the dehydrogenase activity using FcPF6 assay.Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / FcPF6) is less than 0.1 or less than 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or even less than 0.01.Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / FcPF6) may also be impossible to determine due to undetectable oxidase activity.Amplex Red and FcPF6 assays are described in the following sections.

[0125] According to another embodiment of the present invention, the oxygen interference at the enzyme level is determined by determining the oxidase activity using Amplex Red (AR) assay and the dehydrogenase activity using 1-4-BQ assay. Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / 1-4-BQ) is less than 0.1 or less than 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or even less than 0.01. Specifically, the ratio of oxidase activity to dehydrogenase activity (AR / 1-4-BQ) may also be impossible to determine due to undetectable oxidase activity. Amplex Red and 1-4-BQ assays are described in the following sections.

[0126] The enzymatic activity of LDH or its variants was measured, for example, using 2,6-dichlorophenol-indophenol sodium salt hydrate (DCIP) (molar extinction coefficient ε ε ) at 30°C and 520 nm or 600 nm as previously described (WJ Bao, S.N. et al. (1993), Krondorfer I. et al. (2014), Harreither, W. et al. (2011)). 520nm =6.8mM -1 cm -1 ; Molar extinction coefficient ε 600nm =8.98mM -1 cm -1The enzymatic activity can be determined by the DCIP assay, which evaluates the enzymatic activity from the colorimetric reduction of . The assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCl, 3 mM KCl, and contains 10 mM lactate and 120 μM DCIP acting as an electron acceptor. The DCIP assay thereby provides a measure of the efficiency of electron transfer between LDH to an external electron acceptor and provides an indication of the response of the enzyme on the electrode. One unit of enzymatic activity is defined as the amount of enzyme (U / mg) that oxidizes 1 μmol of lactate per minute under the assay conditions. Two electrons are gained per lactate molecule and transferred to a single DCIP molecule, so the lactate:DCIP reaction stoichiometry is 1:1. For detection of activity with other substrates, lactate can be exchanged for other compounds.

[0127] The enzymatic activity of the LDH variants was also determined by the addition of 500 μM 1,4-benzoquinone (1-4-BQ) (molar extinction coefficient ε 290nm =2.24mM -1 cm -1 ) or 160 μM ferrocenium hexafluorophosphate (FcPF6) (molar extinction coefficient ε 300nm =4.3mM -1 cm -1 ) can be determined by assessing the colorimetric reduction of ferrocenium hexafluorophosphate. The assay mixture is formulated as described for the DCIP assay, but contains 500 μM 1,4-benzoquinone or 160 μM ferrocenium hexafluorophosphate instead of DCIP. Two electrons are gained per lactate molecule and transferred to a single molecule of 1,4-benzoquinone, so the reaction stoichiometry of lactate:1,4-benzoquinone is 1:1. Two electrons are gained per lactate molecule and transferred to two individual molecules of ferrocenium hexafluorophosphate, so the reaction stoichiometry of lactate:ferrocenium hexafluorophosphate is 1:2. For detection of activity with other substrates, lactate can be exchanged for other compounds (Brugger D et al. (2014), Sygmund, C. et al. (2011)).

[0128] The measurement of the enzymatic activity of LDH or its variants can also be determined with respect to oxygen as an electron acceptor. Specifically, the oxidase activity of LDH is measured. If the ability of LDH to transfer electrons to oxygen is very low, the oxidase activity may be undetectable. As an example of a suitable method, the Amplex Red assay can be used. The oxidase activity is measured by measuring the oxidase activity of LDH with 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM Amplex Red (resorufin: ε 560nm =54.0mM -1 cm -1 ), which is present at ambient concentrations of approximately 250 μM (Kadowaki, MAS et al. (2020)).

[0129] Specifically, the recombinant LDH described herein contains at least 1 U / mg of enzyme activity as measured by the DCIP assay described herein. One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of lactate per minute under each condition. Specific activity is given in "U / mg" or "U per mg".

[0130] The term "activity" as used herein refers to a functionally active molecule, for example, in the context of enzyme activity. A functional enzyme is specifically characterized by a catalytic center that recognizes an enzyme substrate and catalyzes the conversion of the substrate to a conversion product. For LDH, the main substrate is L-lactate, and the conversion product is pyruvate. An enzyme variant is considered functional when determining its enzyme activity in a standard test system, for example, where the enzyme activity is at least 50%, or at least any of 60%, 70%, 80%, 90%, 100%, or even more than 100% of the activity of the parent (unmodified or wild-type enzyme).

[0131] According to one embodiment of the present invention, the electrode of the present invention is part of a biosensor.Therefore, a particular use of the electrode of the present invention is to provide a biosensor, more specifically a second generation lactate biosensor, using mediated electron transfer properties (MET) to detect lactate and / or measure lactate concentration. 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. In particular, the biosensor may be suitable for 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, 2°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.

[0132] According to another embodiment, the biosensor may have one or more electrodes that include LDH. In a further embodiment, the lactate biosensor includes a working electrode that includes a conductive material, where the LDH is in close 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 / standard electrodes.

[0133] As described in Rocchitta G. et al., 2016, biosensors can be used as offline, in vivo, or online devices. In offline devices, the target analyte is measured in a collected biological sample. For example, offline biosensors can be used to measure lactate in food or blood samples. In in vivo sensors, the biosensor is embedded and continuously detects extracellular changes in the concentration of the analyte. In online devices, the biosensor is integrated with a sampling device embedded in the body or in biological materials.

[0134] The particular configuration of a biosensor may depend on the use for which the biosensor is intended and the conditions under which the biosensor will operate. In certain embodiments of the invention, the biosensor may be a single-use biosensor for the detection of lactate, whereby the biosensor may be a biosensor strip.

[0135] In certain embodiments of the present invention, the biosensor is a biosensor that is placed in vivo in a subject or a transcutaneously placed biosensor that is configured for placement in vivo. In one example, at least a portion of the sensor may be placed in subcutaneous tissue to test lactate concentration in interstitial fluid. In another example, at least a portion of the sensor may be placed in dermal tissue to test analyte concentration in dermal fluid.

[0136] In certain embodiments of the invention, the biosensor may be used for continuous measurement of lactate in vivo. In certain embodiments of the invention, the biosensor may be an implantable biosensor. The biosensor may be placed subcutaneously in the subject.

[0137] In certain embodiments of the present invention, the biosensor may be a microneedle-based sensor that is recognized by a series of multiple needles placed subcutaneously in a subject. The subject may be a human subject.

[0138] According to one embodiment of the invention, the electrodes of the invention are part of a device, which may be a wearable device such as a smart watch, a fitness tracker, a heart rate monitor belt, or a device connecting the electrodes to a fixed or portable analytical instrument.

[0139] According to another embodiment of the present invention, a kit for detecting and / or quantifying lactate comprises the electrode of the present invention, or the biosensor of the present invention, or the device of the present invention, and further comprises an instruction manual. The kit may also comprise auxiliary substances such as buffers, and containers such as sample holding means and / or lactate standards. The lactate standards may be used to calibrate the assay. The kit may also comprise a reader for signals, particularly electrochemical signals such as a potentiostat, a computer readable memory device with software for calibration and / or measurement calculation.

[0140] According to one embodiment of the present invention, the LDH can be recombinantly expressed by methods commonly known in the art. For example, the LDH of the present invention can be expressed using standard methods for cloning, transformation, and recombinant production in Escherichia coli or Pichia pastoris.

[0141] According to a particular invention, the recombinant LDH of the invention comprises the amino acid GP at the N-terminus. This specific N-terminus is generated by N-terminal addition of a tag encoding the amino acid sequence GSSHHHHHHGLEVLFQGP (SEQ ID NO: 250), or alternatively a tag with a greater or lesser number of His residues. Specifically, this tag is added in each amino acid sequence between the initiation codon encoding the N-terminal M and the second codon encoding the second amino acid. This tag comprises an HRV 3C protease cleavage site that leaves the N-terminal GP after digestion with HRV 3C protease. Due to the addition of such a tag and the specific cleavage site of the protease, the LDH has a defined N-terminus that comprises GP. Surprisingly, this defined N-terminus results in an increase in the specific activity of lactate dehydrogenase compared to the uncleaved N-terminus without the defined N-terminal GP. Specifically, the LDH with GP at the N-terminus has at least a 4-fold increased specific activity compared to the undigested N-terminus. Specifically, said increase in specific activity can be 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or even more compared to the undigested N-terminus without the defined N-terminal GP.

[0142] The terms "increased activity", "increased activity", and the like, as used herein, may refer to a detectable increase in the activity of an enzyme. The terms "increased activity" or "increased activity", as used herein, may mean that a modified enzyme, such as an LDH variant with a defined N-terminus including a leader sequence GP at the N-terminus as described herein, exhibits higher activity than a comparable enzyme of the same type, such as an enzyme without a particular modification. As another example, a modified LDH may include a sequence change in the polypeptide or nucleotide sequence encoding the LDH. For example, the activity of a modified or engineered enzyme may be about 5% or higher, about 10% or higher, about 15% or higher, about 20% or higher, about 30% or higher, about 50% or higher, about 60% or higher, about 70% or higher, or about 100% or higher than the activity of a non-engineered enzyme of the same type, such as a wild-type enzyme. The activity of a particular protein or enzyme in a recombinant or engineered cell may be 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 than the activity of the same type of protein or enzyme in a parent cell, e.g., a non-engineered cell. Increased activity of an enzyme or protein in a cell may be verified by any method known in the art.

[0143] The term "functional variant" or "functionally active variant" also encompasses naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, allelic variants, also referred to as homologs, are alternative forms of nucleic acids or peptides characterized by having one or more nucleotide or amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the nucleic acid or polypeptide. Specifically, functional variants may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residue substitutions, deletions, and / or additions, or combinations thereof. Specifically, the substitutions, deletions, and / or additions may be conservative modifications. Specifically, the substitutions, deletions, and / or additions do not reduce the specific activity of the enzyme. Specifically, a functionally active variant of LDH as described herein comprises a specific enzyme activity on acetate of at least 1 U / mg as determined by a DCIP assay as described herein.

[0144] Specifically, functional variants as described herein include those that have 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; The variants may comprise deletions and / or additions or substitutions, deletions and / or additions of 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 acids. In particular, these modifications may be conservative modifications. In particular, these modifications do not reduce the specific activity of the enzyme. In particular, the functionally active variants as described herein comprise substitutions, deletions and / or additions of up to 15, preferably up to 10 or 5, amino acids. In particular, these modifications may be conservative modifications. In particular, these modifications do not reduce the specific activity of the enzyme.

[0145] Specifically, the functionally active variants described herein comprise at least 40, 50, 60, 70, 80 or 90% or even higher enzymatic activity of the respective wild-type enzyme. According to a specific example, when the LDH described herein is derived from Pediococcus acidilactici LDH (PaLDH), said recombinant LDH is a functionally active variant when it comprises at least 40, 50, 60, 70, 80 or 90% or even higher enzymatic activity of the LDH comprising SEQ ID NO:2.

[0146] Functional variants may be obtained by sequence alterations in polypeptide or nucleotide sequences, for example by one or more point mutations, where the sequence alterations preserve or improve the enzyme's characteristics, such as its stability or activity. Such sequence alterations may include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions. Conservative substitutions are made 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, acidic side chains, non-polar aliphatic side chains, non-polar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc.

[0147] A point mutation is understood in particular as a manipulation of a polynucleotide which results in the expression of an amino acid sequence which differs from the unmanipulated amino acid sequence in one or more single (non-contiguous) or double substitutions or exchanges, deletions or insertions of amino acids for different amino acids.

[0148] According to certain embodiments, the LDH described herein comprises one or more tag sequences, specifically N-terminal tag sequences.Specifically, such tag sequences are C-terminal to the N-terminal methionine of the recombinant LDH described herein.Such tag sequences can comprise any number of amino acids, from more than 2, 4, 5, 6 or 10 amino acids, and up to 20 or 50 or more amino acids.Specifically, the tag sequence used herein can be any tag sequence known to those skilled in the art.Specifically, the tag sequence used herein is selected from affinity tag, solubility enhancement tag, or monitoring tag.

[0149] Affinity tags are, for example, amino acid sequences that can be used for the purification of proteins to which they are attached. These affinity tags have high affinity for suitable ligands of solid supports, such as chromatography resins, or directly for resins. The selective binding of proteins with affinity tags to specific resins allows the proteins to be purified very effectively with only one chromatography step. According to certain embodiments, the affinity tag sequences used herein are selected from histidine (His) tags, specifically polyhistidine tags, polyarginine tags, FLAG tags, Strep tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, S tags, HA tags, c-Myc tags, and SUMO tags, or any other tags known to be useful for efficient purification of proteins to which they are fused. Preferably, the tag is a His tag containing one or more H, specifically a hexahistidine tag. In particular, proteins that contain poly- or hexahistidine tags (His tags) can be captured and purified using chromatography, for example, by immobilized metal affinity chromatography (IMAC).

[0150] Solubility enhancing tag can be fused to the LDH described herein at the N-terminus.Solubility enhancing tag can increase the titer of soluble protein when expressed in host cell, for example in the cytoplasm of P. pastoris, compared to the expression of untagged protein.In addition, according to a specific embodiment, the solubility enhancing tag sequence used herein is 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 fused thereto, for example during expression in host cell. Specifically, the solubility enhancing 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.

[0151] According to further particular embodiments, 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 the production steps including fermentation, isolation and purification by simple in-situ, in-line, online or at-line detectors such as UV, IR, Raman, fluorescence, etc.

[0152] The term "sequence identity" as used herein is understood as the relationship between two amino acid sequences or two nucleotide sequences, and is described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or ortholog compared to a parent nucleotide sequence 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 corresponding positions to some extent, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at corresponding positions to some extent, up to 100%.

[0153] Sequence similarity searching is an effective and reliable strategy for identifying homologs with excessive (e.g., at least 50%) sequence identity. Frequently used sequence similarity search tools are, for example, BLAST, FASTA, and HMMER.

[0154] Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects a common ancestry. Homologs can include orthologs, understood herein as variants of the same protein in different organisms, e.g., such as variants of such proteins in different organisms or species.

[0155] To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence, and then the % complementarity can be calculated as the % identity between the first sequence and the second converted sequence using the algorithm described above.

[0156] "Percentage (%) identity" with respect to amino acid sequences, homologs and orthologs described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific polypeptide sequence after aligning the sequences and introducing gaps as necessary, without considering any conservative substitutions as part of sequence identity.Those skilled in the art can determine the appropriate parameters for alignment, including any algorithm, required to achieve the highest scoring alignment over the full length of the sequences being compared.In the case of the percentage determined for sequence identity, it is possible that arithmetic decimal places that are not possible for full nucleotides or amino acids may occur.In this case, the percentage shall be rounded up to the full nucleotide or amino acid.

[0157] For the purposes described herein, sequence identity between two amino acid sequences is determined using standard methods, for example, using the NCBI BLAST program version 2.2.29 (January 6, 2014), or online using the multiple sequence alignment tool EMBL-EBI Clustal Omega (Sievers, F. et al., Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)).

[0158] For example, "percent (%) identity" for the nucleotide sequence of a nucleic acid molecule or a part thereof, particularly a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence after aligning the sequences and introducing gaps as necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent nucleotide sequence identity can be achieved in various ways that are within the scope of those skilled in the art, for example, using publicly available computer software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithm, required to achieve the highest scoring alignment over the full length of the sequences being compared.

[0159] Optimal alignment may be determined using 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., Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).

[0160] In structural alignment, the maximum set of corresponding pairs of amino acid residues that give a good structural fit when the structures are overlapped, i.e. superimposed, is identified. The positions of the protein backbone C-alpha atoms and / or the locations of the secondary structure elements are thereby taken into account in this alignment. Tools are available for performing structural alignment, for example the Protein Data Bank provides a tool for pairwise structural alignment. In particular, structural superposition is also a tool for determining corresponding amino acid positions in various enzymes. Structural superposition can be performed using the Molecular Graphics System PyMOL (Schrodinger) using the command "align".

[0161] The examples described herein are illustrative of the present invention and are not intended to be limitations on the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the present invention. Therefore, it should be understood that the examples are merely illustrative and are not limitations on the scope of the present invention. EXAMPLES

[0162] material Phosphate-buffered saline (PBS) 11 mM, pH 7.4 with 137 mM NaCl and 3 mM KCl was used as the standard buffer for all experiments unless otherwise stated. Britton-Robinson Universal Buffer (BRB) contains 40 mM phosphate, boric acid and acetate. Bacterial cultures were routinely performed in Luria-Bertani (LB) medium (10 g / L casein-derived peptone, 5 g / L yeast extract and 10 g / L NaCl) with 100 mg / L ampicillin. When culturing bacteria carrying the pNIC-CH plasmid for PaLDH, ampicillin was replaced with 50 mg / L kanamycin. General medium components were purchased from Carl Roth; L-sodium lactate, ferrocenium hexafluorophosphate (FcPF6), isopropyl bD-1-thiogalactopyranoside (IPTG), 2,6-dichlorophenol-indophenol sodium salt hydrate (DCIP), horseradish peroxidase (HRP), sodium glycolate, 1,4-benzoquinone (1,4-BQ), and R-2-hydroxybutyric acid were purchased from Sigma-Aldrich (Germany); S-2-hydroxyvaleric acid was purchased from BLD Pharmatech Ltd. (Shanghai); 2-hydroxypalmitic acid and 2-hydroxy-n-octanoic acid were purchased from TCI (Japan); (S)-2-hydroxybutyric acid and S-(+)-mandelic acid were purchased from Fluorochem. Ltd. (UK); 10-acetyl-3,7-dihydroxyphenoxazine (AmplexRed®) was purchased from Chemodex (Switzerland).

[0163] Example 1: Phylogenetic analysis Surprisingly, we found that there exists a group of enzymes closely related to, but separate from, the characterized LOx sequences. Furthermore, by an activity screening approach of new members of the LOx cluster that includes both groups of enzymes, we found that there is also a functional division of highly lactate specific oxidase versus dehydrogenase sequences. To get a more detailed view of the LOx cluster and its sequence / function distribution, a phylogenetic tree of all sequences in the cluster was inferred using RAxML and sequences with relative L-lactate activity from the screen were annotated to the tree (Figure 1). Clearly, a functional division of the tree into two major clades can be observed, one showing oxygen reactivity (LOx) and the other only showing dehydrogenase activity (LDH). Thus, the difference between the two groups found in the activity separating the LOx and LDH sequences is also supported by the phylogenetic information. Moreover, the two previously characterized enzymes AvLOx and PaLCTO fit well into the picture. AvLOx appears in the LOx clade, whereas PaLCTO appears in the LDH clade, which was surprising since PaLDH was previously reported not to utilize lactate by, for example, Ashok Y et al., 2020. Thus, PaLCTO can be considered as PaLDH. Besides PaLDH, no other previously characterized enzymes were found in the LDH clade, including membrane-bound LDH and NAD+-dependent LDH, as well as flavocytochrome b2, often referred to as LDH in the literature. This indicates that a new type of LDH, closely related to but separate from LOx, has indeed been invented.

[0164] Based on these new insights into the phylogenetic and functional properties of the HAOx enzyme family, detailed sequence analysis revealed that LDH can be separated from the rest of the HAOx family members, and therefore from the LOx members, by specific features of the amino acid sequence of the LDH members. LDH is a member of the HAOx family that contains the sequence T-(X x ) n1 -F-(Xx ) n2 -X1-X2-(X x ) n3 -X3-X4 (SEQ ID NO: 1) appears to separate LOx from SEQ ID NO: 1. The amino acid sequence of the LOx enzyme does not include SEQ ID NO: 1 as defined herein. Surprisingly, other members of the HAOx family also do not include SEQ ID NO: 1 as defined herein, while sharing at least 50% sequence identity to SEQ ID NO: 2.

[0165] Thus, FMN-dependent LDHs having at least 50% sequence identity to SEQ ID NO:2 and including SEQ ID NO:1 as defined herein appear to form a distinct class of enzymes with the common functional and technical property of oxidizing lactate and transferring the electrons so acquired to an electron acceptor other than oxygen.

[0166] Selected representatives of a subclade of this distinct class of LDHs have been functionally expressed and characterized as described herein. Example 2: Analysis of structural motifs T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x ) n3 The sequence -X3-X4 (SEQ ID NO: 1) forms a structural motif in the three-dimensional structure of the folded enzyme. The sequence is located in or interacts with the lid of the active site of the enzyme. The active site lid of the LDH of the present invention is described in the crystal structure of PaLDH (Ashok et al., 2020). The crystal structure of PaLDH can be found in two conformations, referred to herein as the "open and closed" conformations of the active site lid, in the Protein Data Bank (pdb)-code 6RHT. In the crystal structure of PaLDH, the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x )n3 The α-carbons of amino acid residues T, F, X1 being N and X2 being L of -X3-X4 (SEQ ID NO: 1) are located within a distance of 12 Å from each other, and these amino acids do not differ substantially in the open and closed conformations. x ) n1 -F-(X x ) n2 -X1-X2-(X x ) n3 The position in the crystal structure where residues X3 is G and X4 is I in -X3-X4 (SEQ ID NO: 1) is different in the open and closed conformations. The relative distance in the crystal structure is calculated based on the residues of 6RHT in PaLDH, i.e., the sequence T-(X x ) n1 -F-(X x ) n2 -X1-X2-(X x ) n3 The α-carbon distance matrix for T122, F152, N196, L197, G209 and 1210 of -X3-X4 (SEQ ID NO: 1) is given in Table 3 below. Distances are given in Å. Distances for G and I are given relative to their positions in the open and closed conformations, respectively.

[0167] [Table 3]

[0168] For the five LDH sequences according to the present invention, structural models were calculated using the RoseTTAFold method on the Rosetta web server (https: / / robetta.bakerlab.org / submit.php) (Baek, Minkyung et al., 2021). The distance of every motif residue to every other motif residue in the structure was measured with PyMOL as the distance between the α-carbons of the residues in angstroms (Å).

[0169] The α-carbon distance matrix [Å] for the motif residues in LjLDH is given in Table 4 below.

[0170] [Table 4]

[0171] The α-carbon distance matrix [Å] for the motif residues in LhLDH is given in Table 5 below.

[0172] [Table 5]

[0173] The α-carbon distance matrix [Å] for the motif residues in SsLDH is given in Table 6 below.

[0174] [Table 6]

[0175] The α-carbon distance matrix [Å] for the motif residues in GbLDH is given in Table 7 below.

[0176] [Table 7]

[0177] The α-carbon distance matrix [Å] for the motif residues in EaLDH is given in Table 8 below.

[0178] [Table 8]

[0179] Example 3: Recombinant expression and characterization of LDH Plasmids and genes Genes encoding the selected LDH enzymes and AvLOx for comparison were ordered codon-optimized for E. coli expression in the pET-21(+) vector from Twist Bioscience (South San Francisco, USA). The LDH enzymes selected were Lactobacillus johnsonii LjLDH (SEQ ID NO: 59), Lactobacillus helsingborgensis LhLDH (SEQ ID NO: 13), Shigella sp. FC1655 SsLDH (SEQ ID NO: 74), Gilliamella bombicola GbLDH (SEQ ID NO: 73), Enterococcus avium EaLDH (SEQ ID NO: 155), and Pediococcus acidilactici PaLDH (SEQ ID NO: 2). An N-terminal purification tag (-GSS-HHHHHH-G-LEVLFQGP- (SEQ ID NO: 250)) was added between the initiating Met and the second amino acid with 146 bp and 110 bp overlap at the 5' and 3' ends, respectively, following the Gibson Assembly (New England Biolabs) protocol. The native form of PaLDH was cloned into the pNIC-CH vector as previously described (Ashok Y. et al., 2020). The predicted bacterial signal peptide of SsLDH was removed by amplifying the plasmid without the signal peptide coding region by PCR and religating the linearized plasmid using KLD Enzyme Mix (New England Biolabs). The plasmid was transformed into chemically competent E. coli BL21 (DE3) cells by heat shock transformation. All plasmid modifications and transformations were verified by Sanger sequencing (Microsynth, Austria).

[0180] Enzyme expression Expression of recombinant genes in E. coli BL21(DE3) was carried out at a scale of 40 or 250 mL of expression medium in baffled shake flasks. LB-amp (LB-kan for PaLDH) medium was incubated at 37 °C for 1 h at 600 nm (OD 600 Inoculate the bacterial culture until the cells reach an OD of 0.05. 600 The cultures were incubated at 37°C and 180 rpm until a pH of 0.45-0.50 was reached, where expression was induced with 100 μM IPTG (250 μM for 250 mL expression). The induced cultures were incubated at 20°C and 180 rpm overnight for 19 h. Cells were harvested by centrifugation at 4000 rpm for 20 min at 4°C and washed once with 50 mM potassium phosphate buffer (PPB) pH 6.5. The washed cell pellets were stored at -20°C before cell disruption.

[0181] Purification and protein concentration determination Frozen cell pellets were thawed, resuspended in 50 mM PPB, 500 mM NaCl, 50 mM imidazole pH 6.5, and disrupted in a French press for 4-5 passages. Cell debris was removed by centrifugation (3000 rcf for 30 min at 4 °C) and the resulting supernatant was filtered through a 0.22 μm membrane filter and loaded onto a 2 × 5 mL IMAC HisTrap™ FF column (Cytiva, USA) using an Akta FPLC system (GE Healthcare, USA). His-tagged proteins were eluted with a linear imidazole gradient (50-750 mM) in 50 mM PPB, 500 mM NaCl, pH 6.5, and fractions were pooled according to activity and elution peak measured at 280 nm and 450 nm. Pooled fractions were concentrated on Amicon centrifugal filters (MWCO 10 kDa), rebuffered in 11 mM PBS, pH 7.4, and stored at 4° C. Enzyme homogeneity was assessed by SDS-PAGE, and protein concentrations were determined based on the theoretical extinction coefficients as determined by amino acid sequence (48360, 41830, 24870, 29910, 28420, 25900, and 51340 M for LjLDH, LhLDH, SsLDH, GbLDH, EaLDH, PaLDH, and AvLOx, respectively).-1 cm -1 ) were calculated from their absorbance at 280 nm assuming the ExPASy tool ProtParam ( Gasteiger et al., 2005 ). Purified PaLDH was produced using E. coli BL21(DE3) expression, His-tag purification, followed by cleavage of the tag by TEV protease digestion, and size-exclusion chromatography as previously described ( Ashok et al., 2020 ).

[0182] Enzyme activity measurement Spectrophotometric enzyme activity assays were recorded in at least triplicate in 96-well microtiter plates using an EnSpire multimode plate reader (PerkinElmer) or an Infinite M Quant plate reader (Tecan) at 30° C. Volumetric activity was calculated from the linear correlation of absorbance change over time (ΔAbs / Δt) at the monitored wavelengths.

[0183] Activity assays contained the electron donor substrate L-lactate at a concentration of 10 mM dissolved in buffer unless otherwise stated. Oxidase activity was measured using 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM AmplexRed (10-acetyl-3,7-dihydroxyphenoxazine, resorufin:E 560nm =54.0mM -1 cm -1 Dehydrogenase activity was monitored using a peroxidase-coupled reaction containing 120 μM (300 μM for substrate screening) DCIP (2,6-dichlorophenol-indophenol sodium salt hydrate, ε 520nm =6.8mM -1 cm -1 or ε 600nm =8.98mM -1 cm -1 ), 500 μM 1,4-BQ (1,4-benzoquinone, ε 290nm =2.24mM -1 cm-1 ) or 160 μM FcPF6 (ferrocenium hexafluorophosphate, ε 300nm =4.3mM -1 cm -1 ) was measured by a direct dye-mediated assay containing 1,4-BQ. The relative substrate specificity and specific activity of FcPF6 and 1,4-BQ were measured in 50 mM potassium phosphate buffer (PPB) pH 6.5. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of α-hydroxy acid per minute at 30°C.

[0184] The apparent steady-state kinetic constants were calculated using 11 different concentrations of L-lactate ranging from 0.125 to 64 mM (4 to 500 mM for PaLDH) with a constant concentration of DCIP of 120 μM and iterative least-squares regression fitting using the Microsoft Excel Solver plug-in, using the Michaelis-Menten model (v = (v max * [S] / (K m +[S]))) was determined by fitting the observed data. Turnover rates were calculated based on the monomer mass of each enzyme.

[0185] The effect of pH on L-lactate oxidation activity with O2 and DCIP was evaluated by varying the pH from 4.5 to 10 in 0.5 increments in 40 mM Britton-Robinson universal buffer (BRB).

[0186] Thermal stability measurement Enzyme samples were diluted to a concentration of 1 mg / mL and incubated in duplicate for 30 min at temperatures ranging from 30 to 60 °C (43 to 73 °C for SsLDH and EaLDH). After cooling the heat-treated samples on ice for 15 min and centrifugation, the residual activity with L-lactate and DCIP was measured in duplicate. Heat inactivation temperatures (T 50) was estimated using an iterative least squares regression sigmoid curve fit (min+(max-min) / (1+10^(n * (log 10 (℃)-log 10 (EC 50 ))))).

[0187] HRV 3C protease digestion Purification tag cleavage was performed on a small scale for 22 h at 20 °C using 30 µg of enzyme and 1 µg of HRV 3C protease (produced in-house; SwissProt ID: sp|P03303|1538-1719). Alternatively, HRV 3C protease may be purchased (HRV-3C-Protease N-Terminal His-tagged recombinant protein, aqueous solution, 0.8-1.2 mg / mL|Sigma-Aldrich). Activity determination was performed in quadruplicate using L-lactate and DCIP. Blinds were incubated with buffer instead of HRV 3C.

[0188] result Expression, purification and UV-Vis spectrum of LDH Five LDH sequences were expressed, purified and characterized. The genes for the enzymes from L. johnsonii (L / LDH), L. helsingborgensis (LhLDH), Shigella species FC1655 (SsLDH), Guillaumelas bombicola (GbLDH) and Enterococcus avium (EaLDH) along with AvLOx were modified with an N-terminal purification tag consisting of 6×His and a recognition site for the 3C protease of human rhinovirus (HRV 3C). These six genes were then expressed in E. coli BL21(DE3) shake flask cultures and purified using immobilized metal affinity chromatography (IMAC). PaLDH was expressed and purified in the same way with an additional step of size exclusion chromatography as previously described (Ashok et al., 2020). Expression of LjLDH, LhLDH, SsLDH, GbLDH and EaLDH produced significantly more recombinant protein than expression of AvLOx (9, 20, 18, 119 and 7 times higher, respectively) when comparing expression yields normalized by the amount of harvested cell pellet (mg of purified enzyme / g of wet cell pellet) (Table 9). UV-Vis absorption spectra showed a typical peak for a flavoprotein with one maximum at approximately 278 nm and two FMN-dependent maxima at approximately 374 nm and 458 nm, which flattened out due to the reduction of the cofactor upon addition of 10 mM L-lactate (Figure 2).

[0189] [Table 9]

[0190] Substrate specificity of LDH Spectrophotometric assays were used to determine the specific activities for all LDHs with the electron acceptors O2 (air), dichlorophenol-indophenol (DCIP), 1,4-benzoquinone (1,4-BQ) and ferrocenium hexafluorophosphate (FcPF6) using 10 mM L-lactate (Table 10). The highest dehydrogenase specific activities were measured with DCIP for LjLDH, LhLDH, SsLDH and EaLDH (72, 152, 166 and 146 U / mg, respectively), with FcPF6 for GbLDH (207 U / mg) and with 1,4-BQ for PaLDH (25 U / mg). Oxygen reactivity was highest with 18 U / mg for AvLOx. This value was consistent with the V max This is in good agreement with the previously studied N-terminally tagged AvLOx ( Taurino et al., 2013 ) which showed 21.81 U / mg. The selected LDHs showed oxygen reactivity ranging from 0.07 to 0.71 U / mg, accounting for 0.37, 0.47, 0.07, 0.03, 0.05 and 0.37% of their maximum dehydrogenase activities for LjLDH, LhLDH, SsLDH, GbLDH, EaLDH and PaLDH, respectively.

[0191] [Table 10]

[0192] Michaelis-Menten kinetics The steady-state kinetics of L-lactate was measured for all LDHs using DCIP as the electron acceptor at pH 7.4 (Table 11). LhLDH was primarily due to its low K m The enzyme showed the highest activity for L-lactate as judged by catalytic efficiency due to its K value. m The values ​​found for PaLDH showed very large differences with respect to all other LDHs. The turnover numbers of the LDHs studied for L-lactate did not change very dramatically, with the highest k cat Values ​​were determined for GbLDH.

[0193] [Table 11]

[0194] Effect of pH on LDH and AvLOx activities Optimal pH measurements were performed for all LDHs and AvLOx using the dehydrogenase (DCIP as electron acceptor) and oxidase (O2 / air coupled with Amplex Red) assays in Britton-Robinson buffer (BRB) from pH 4.5 to 10.0 (Figure 4). The resulting pH profiles differed substantially between the two activity assays for some of the enzymes studied. In general, all LDHs tended to have their pH optimum with DCIP at a value lower than the optimum for O2. The pH ranges where the enzymes show 80-100% activity with DCIP and O2, respectively, are 4.5-6.0 and 7.0-7.5 for LjLDH, 4.5-7.0 and 8.0-8.5 for LhLDH, 7.5-8.0 and 8.5-10.0 for SsLDH, 5.5-7.0 and 6.5-10.0 for GbLDH, 6.0-7.0 and 7.0-7.5 for EaLDH, and 4.5-5.5 and 7.0-8.5 for PaLDH. Interestingly, AvLOx showed a pH profile with respect to DCIP that was shifted to higher values ​​when compared to O2, with 80-100% activity seen in the pH ranges of 8.5-9.0 for DCIP and 7.0-8.5 for O2. The pH profiles also revealed that for LjLDH, GbLDH, PaLDH and AvLOx, the activity at pH 7.4 only represents approximately 9, 35, 6 and 39% of the maximum DCIP activity of the enzyme. This must be taken into account when interpreting the specific activity of the enzyme measured at the physiologically relevant pH 7.4. Furthermore, the influence of different buffer types on the activity of the enzymes was tested. For this purpose, the activity in PBS at pH 7.4 and in BRB at pH 7.5 was compared, showing that for LjLDH, LhLDH and GaLDH, the activity in PBS was 7.8, 2.0 and 2.1 times higher than in BRB pH 7.5. The other tested enzymes were hardly affected in their activity when comparing the two buffers.

[0195] Effect of temperature on LDH activity The thermal stability of LDH was determined by incubating the enzyme at different temperatures for 30 min and determining its residual activity. The temperature of half-maximal activity (T 50 ) were obtained from an iterative sigmoidal fit of the observed data (Table 12). The two highest T 50 Values ​​were observed for SsLDH and EaLDH, while LhLDH had the lowest T 50 The values ​​were shown.

[0196] [Table 12]

[0197] Effect of N-terminal purification tags on LDH and AvLOx activity The added purification tag contained a 6xHis tag as well as a cleavage site for the HRV 3C protease, which left an N-terminal Gly-Pro after digestion and completely cleaved off the 6xHis tag. The purified enzymes were incubated overnight (22 h) at 20°C with and without HRV 3C and their specific activities with L-lactate and DCIP were determined (Figure 3). The results thus obtained were quite different. AvLOx and LhLDH showed an approximately 2-fold increase in activity after digestion compared to the undigested sample, while GbLDH and EaLDH showed an 18- and 4-fold increase, respectively. Thus, we were able to confirm that the nature of the N-terminus also influences the activity of LDH.

[0198] Example 4: Electrode measurements Electrochemical measurements were performed on a three-electrode screen-printed electrode (DRP-C110, Metrohm). The sensor was modified with 1 μL of enzyme (EaLDH) solution by drop coating and drying to promote adsorption (5 mg / mL enzyme in 10 mM, pH 7.0 PB containing 0.2% Triton). The sensor was then immersed in 50 mM phosphate buffer containing 50 μM 1,4-benzoquinone as a mediator and increasing concentrations of lactate were added at 37 °C (Figure 5). A clear correlation between increasing concentrations of lactate and the current response could be observed and was used to obtain the calibration function of the biosensor. Using the same procedure, electrodes were prepared and measured with LjLDH (Figure 6), SsLDH (Figure 7), EaLDH (Figure 8), and AvLOX (Figure 9).

[0199] To test for oxygen interference, the buffer solution was purged with nitrogen for 15 min before measurement, a calibration with 5 mM lactate was performed, and the current densities in the absence and presence of oxygen were compared (Figure 10). In a set of at least three independent sensor prototypes, all LDH-based sensors were not interfered with by oxygen, while the prototype built with LOx enzyme (AvLOx) showed a highly significant (p=0.004) signal deviation in dependence of oxygen availability.

[0200] [Table 13-1]

[0201] [Table 13-2]

[0202] [Table 13-3]

Claims

1. A method for detecting and / or quantifying lactate in a sample, a) A step of supplying an electrode containing lactate dehydrogenase (LDH), wherein the LDH is flavin mononucleotide (FMN) and the following sequence T-(X) ｘ ) ｎ１ -F-(X) ｘ ) ｎ２ -X １ -X ２ - (X) ｘ )n ３ -X ３ -X ４ (Arrangement number 1) (In the formula, T is either T122 in sequence number 2, or corresponds to T122. F is either F152 in sequence number 2, or corresponds to F152. X ｘ is any amino acid, n1 is an integer between 25 and 35. n2 is an integer between 40 and 45. n3 is an integer between 5 and 20. X １ is N or F, X ２ is L or F, X ３ is G, T, or S, X ４ (is I or V) A step comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50% sequence identity with respect to SEQ ID NO: 2, b) The step of bringing the sample into contact with the electrode, c) A step of detecting oxidation of lactate by LDH and The above method, comprising the oxidation of lactate in the presence of a redox mediator.

2. The method according to claim 1, wherein the sample is selected from the group consisting of food; beverages; fermented foods; fermented beverages; chemicals; water; soil; and one of the samples supplied by humans or animals, specifically from one of the following: body fluids, interstitial fluid, blood, plasma, skin fluid, urine, tears, sweat, saliva, skin, meat, tissue, eyeball, cornea, and gastric juice.

3. The method according to claim 1, wherein LDH is immobilized on the electrode.

4. The method according to claim 3, wherein LDH is immobilized by adsorption or complex formation.

5. The method according to claim 4, wherein the complex formation is mediated by a complexing linker, covalent bonds, or ionic bonds.

6. The method according to claim 3, wherein LDH is crosslinked.

7. The method according to claim 6, wherein LDH is crosslinked with a bifunctional agent.

8. The method according to claim 1, wherein the redox mediator is selected from the group consisting 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 thereof.

9. The method according to claim 1, wherein the amino acid sequence of LDH includes a sequence having at least 70%, 80%, 90%, or 95% sequence identity with amino acids G35 to K360 of SEQ ID NO:

2.

10. The method according to claim 1, wherein LDH is selected from sequences having at least 70%, 80%, 90%, or 95% sequence identity with sequence number 2, sequence number 13, sequence number 59, sequence number 73, sequence number 74, or sequence number 155.

11. The method according to claim 1, wherein LDH is selected from sequences with sequence numbers 2 to 249, or sequences having at least 90% or 95% sequence identity with sequences with sequence numbers 2 to 249.

12. An electrode containing LDH, wherein the LDH is a flavin mononucleotide (FMN) and the following sequence T-(X) ｘ ) ｎ１ -F-(X) ｘ ) ｎ２ -X １ -X ２ - (X) ｘ )n ３ -X ３ -X ４ (Arrangement number 1) (In the formula, T is either T122 in sequence number 2, or corresponds to T122. F is either F152 in sequence number 2, or corresponds to F152. X ｘ is any amino acid, n1 is an integer between 25 and 35. n2 is an integer between 40 and 45. n3 is an integer between 5 and 20. X １ is N or F, X ２ is L or F, X ３ is G, T, or S, X ４ (is I or V) The electrode comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50% sequence identity with respect to SEQ ID NO:

2.

13. The electrode according to claim 12, further comprising an oxidation-reduction mediator.

14. The electrode according to claim 12, wherein LDH is immobilized on the electrode.

15. The electrode according to claim 14, wherein LDH is immobilized by adsorption or complex formation.

16. The electrode according to claim 15, wherein the complex formation is mediated by a complexing linker, covalent bonds, or ionic bonds.

17. The electrode according to claim 14, wherein LDH is crosslinked.

18. The electrode according to claim 17, wherein LDH is crosslinked with a bifunctional agent.

19. The electrode according to claim 12, wherein the redox mediator is selected from the group consisting of one of the following: 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 thereof.

20. The electrode according to claim 12, wherein the amino acid sequence of LDH includes a sequence having at least 70%, 80%, 90%, or 95% sequence identity with amino acids G35 to K360 of SEQ ID NO:

2.

21. The electrode according to claim 12, wherein the LDH is selected from sequences having at least 70%, 80%, 90%, or 95% sequence identity with sequence number 2, sequence number 13, sequence number 59, sequence number 73, sequence number 74, or sequence number 155.

22. The electrode according to claim 12, wherein the LDH is selected from sequences of sequence numbers 2 to 249, or sequences having at least 90% or 95% sequence identity with respect to sequences of sequence numbers 2 to 249.

23. A biosensor comprising the electrode according to claim 12.

24. A device comprising the electrode according to claim 12, or the biosensor according to claim 23.

25. An enzyme composition comprising LDH and an oxidation-reduction mediator, wherein the LDH is a flavin mononucleotide (FMN) and the following sequence T-(X) ｘ ) ｎ１ -F-(X) ｘ ) ｎ２ -X １ -X ２ - (X) ｘ )n ３ -X ３ -X ４ (Arrangement number 1) (In the formula, T is either T122 in sequence number 2, or corresponds to T122. F is either F152 in sequence number 2, or corresponds to F152. X ｘ is any amino acid, n1 is an integer between 25 and 35. n2 is an integer between 40 and 45. n3 is an integer between 5 and 20. X １ is N or F, X ２ is L or F, X ３ is G, T, or S, X ４ (is I or V) The enzyme composition comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50% sequence identity with respect to SEQ ID NO:

2.

26. The enzyme composition according to claim 25, wherein the redox mediator is selected from the group consisting of one of the following: 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.

27. The enzyme composition according to claim 25, wherein the amino acid sequence of LDH includes a sequence having at least 70%, 80%, 90%, or 95% sequence identity with amino acids G35 to K360 of SEQ ID NO:

2.

28. The enzyme composition according to claim 25, wherein LDH is selected from sequences having at least 70%, 80%, 90%, or 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 155, or SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO:

155.

29. The enzyme composition according to claim 25, wherein LDH is selected from sequences of sequence numbers 2 to 249, or sequences having at least 90% or 95% sequence identity with sequences of sequence numbers 2 to 249.

30. Recombinant LDH containing flavin mononucleotide (FMN), wherein the amino acid sequence of the LDH is from the N-terminus to the C-terminus. i. Dipeptide GP and, ii. Array T-(X ｘ ) ｎ１ -F-(X ｘ ) ｎ２ -X １ -X ２ - (X ｘ )n ３ -X ３ -X ４ (Sequence No. 1) (In the formula, T is either T122 in sequence number 2, or corresponds to T122. F is either F152 in sequence number 2, or corresponds to F152. X ｘ is any amino acid, n1 is an integer between 25 and 35. n2 is an integer between 40 and 45. n3 is an integer between 5 and 20. X １ is N or F, X ２ is L or F, X ３ is G, T, or S, X ４ (is I or V) An amino acid sequence having at least 50% sequence identity with SEQ ID NO: 2, or amino acids 2 to 373 of SEQ ID NO: 2, and The above recombinant LDH, including the above.

31. The recombinant LDH according to claim 30, wherein the amino acid sequence of LDH includes a sequence having at least 70%, 80%, 90%, or 95% sequence identity with amino acids G35 to K360 of SEQ ID NO:

2.

32. The recombinant LDH according to claim 30, wherein the LDH is selected from sequences having at least 70%, 80%, 90%, or 95% sequence identity with sequence number 2, sequence number 13, sequence number 59, sequence number 73, sequence number 74, or sequence number 155.

33. The recombinant LDH according to claim 30, wherein the LDH is selected from sequences of sequence numbers 2 to 249, or sequences having at least 90% or 95% sequence identity with respect to sequences of sequence numbers 2 to 249.

34. A kit for detecting and / or quantifying lactic acid or a salt thereof, comprising the electrode according to claim 12, the biosensor according to claim 23, or the device according to claim 24, further comprising instructions for use.

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