Lactate oxidase variants and their use in lactate detection

Abstract

Disclosed herein are lactate oxidase variants that have reduced affinity for lactate compared to wild-type lactate oxidase. The lactate oxidase variants have one or more amino acid substitutions at positions 95, 96, and / or 175 of the wild-type amino acid sequence. Also disclosed are methods for detecting and quantifying lactate in various liquid samples using the disclosed lactate oxidase variants. The methods primarily involve contacting a liquid sample with the lactate oxidase variant, measuring a current generated by the reaction of the lactate oxidase variant with lactate in the liquid sample, and determining the lactate concentration in the liquid sample by interpolating or extrapolating the current value using a current value from a control sample having a known lactate concentration.

Description

[Background technology]

[0001] 1.Technical Field

[0002] The present disclosure relates to lactate oxidase variants and their use for detecting lactate. More particularly, the present disclosure relates to lactate oxidase variants and the electrical detection and quantification of lactate in liquid samples.

[0003] 2. Description of Related Technology

[0004] Since lactate is a key metabolite in the anaerobic metabolic pathways of cells, it may provide clues for monitoring multiple physiological processes in various organisms, and therefore lactate concentration is widely used as a key parameter for assessing the health status of patients in clinical diagnosis and for continuous surveillance in the food and fermentation industries.

[0005] In clinical diagnosis, elevated lactate concentrations due to sustained anaerobic metabolism lead to the accumulation of lactate, inevitably resulting in lactic acidosis, one of the symptoms of severe sepsis. Therefore, a patient's blood lactate concentration serves as a warning signal of the severity of the disease and facilitates the diagnosis and treatment of a wide range of diseases. Lactate is also an important factor in sports medicine, particularly in assessing physical performance in athletic competitions. Elevated blood lactate levels lead to a decrease in blood pH, ultimately causing fatigue. Blood lactate levels during exercise are used as an indicator of exercise training status and physical performance.

[0006] Lactic acid analysis is also used in the food and fermentation industries: since lactic acid is produced during food fermentation, it is used to detect the presence of microbial fermentation in fermented foods such as fermented dairy products, wine, cured meats and fish, and pickles, and is an indicator of food freshness and quality.

[0007] Various methods for measuring lactate levels have been developed, with high-performance liquid chromatography (HPLC) being the most common. Other analytical methods, such as fluorometry, colorimetry, chemiluminescence, and magnetic resonance spectroscopy, are also commonly used. However, these approaches have drawbacks, such as time-consuming processes and the need for expensive equipment and trained personnel. Currently, portable and disposable biosensors have been developed to overcome these limitations. Typically, biosensing methods have the advantages of being simple and direct, combining rapid response with high specificity, economy, and ease of use. Ideally, lactate concentrations in biological fluids such as whole blood, sweat, and saliva should be measurable with biosensors. However, currently commercially available biosensors are limited to detecting lactate in blood and require invasive sample collection methods, making them unpopular with users and unable to be used to detect lactate in samples other than blood.

[0008] For example, the lactate concentration range in human sweat is wider and more variable than that in blood (blood lactate levels can rise to 25 mM during exercise, while in some cases they can reach 80 mM after exercise), making traditional lactate meters less accurate. Furthermore, all current biosensors require large amounts of liquid to operate, and the difference in pH between blood and sweat makes it difficult to use blood lactate meters to measure sweat lactate concentrations.

[0009] In view of the above, there is a need in the related art for improved tools and approaches for non-invasively, efficiently and continuously detecting lactate levels in human body fluids. Summary of the Invention

[0010] The following is a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an extensive overview of the disclosure, and it does not identify key / critical elements or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0011] As specifically and broadly described herein, one aspect of the disclosure relates to a lactate oxidase variant derived from the wild-type lactate oxidase of SEQ ID NO: 1, which has reduced affinity for lactate but provides a broader detection range, the lactate oxidase variant comprising an amino acid substitution at position 95, 96, or 175 of SEQ ID NO: 1, or a combination thereof. In the lactate oxidase variant of the disclosure, the alanine (A) at position 95 of SEQ ID NO: 1 is substituted with asparagine (N) or glutamine (Q), the alanine (A) at position 96 of SEQ ID NO: 1 is substituted with cysteine ​​(C), and / or the serine (S) at position 175 of SEQ ID NO: 1 is substituted with cysteine ​​(C).

[0012] According to one embodiment of the present disclosure, the lactate oxidase variant comprises the amino acid sequence of SEQ ID NO: 1 in which the alanine (A) at position 95 is substituted with asparagine (N).

[0013] According to an alternative embodiment of the present disclosure, the lactate oxidase variant comprises the amino acid sequence of SEQ ID NO: 1, wherein the alanine (A) at position 95 is substituted with glutamine (Q).

[0014] According to another embodiment of the present disclosure, the alanine (A) at position 96 of SEQ ID NO: 1 is substituted with a cysteine ​​(C).

[0015] According to yet another embodiment of the present disclosure, the serine (S) at position 175 of SEQ ID NO: 1 is substituted with a cysteine ​​(C).

[0016] Alternatively or optionally, the cysteine ​​(C) at position 175 of SEQ ID NO: 1 may be carboxymethylated.

[0017] According to a preferred embodiment of the present disclosure, the lactate oxidase variant has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, or 6.

[0018] In some embodiments of the present disclosure, the lactate oxidase variants of the present disclosure have a lower binding affinity for lactate than the wild-type lactate oxidase.

[0019] Another aspect of the present disclosure relates to a method for detecting and quantifying lactate in a liquid sample, comprising the steps of: (a) contacting the liquid sample with the lactate oxidase variant described above; (b) measuring a current generated by the reaction of the lactate oxidase variant with lactate in the liquid sample; and (c) determining the lactate concentration in the liquid sample by interpolating or extrapolating the current measured in step (b) using current values ​​of control samples having known lactate concentrations.

[0020] According to some embodiments of the present disclosure, the liquid sample has a pH value in the range of 4 to 9.

[0021] According to some embodiments of the present disclosure, the liquid sample has a salinity between 0 and 1000 mM.

[0022] In some preferred embodiments, the liquid sample is sweat.

[0023] According to some embodiments of the present disclosure, the method is capable of detecting lactate in a range of 0 to 300 mM in a liquid sample.

[0024] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0025] The following detailed description will be better understood when read in conjunction with the accompanying drawings, in which:

[0026] [Figure 1A] FIG. 1A is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the mutant lactate oxidase A95Q of the present disclosure in a phosphate buffer (PB) solution (pH 5.0).

[0027] [Figure 1B] FIG. 1B is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the mutant lactate oxidase A95Q of the present disclosure in PB solution (pH 7).

[0028] [Figure 2A] FIG. 2A is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the mutant lactate oxidase A96C of the present disclosure in PB solution (pH 5.0).

[0029] [Figure 2B] FIG. 2B is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the mutant lactate oxidase A96C of the present disclosure in PB solution (pH 7.0).

[0030] [Figure 2C] FIG. 2C is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the mutant lactate oxidase A96C of the present disclosure in synthetic sweat (pH 5.0).

[0031] [Figure 3A] FIG. 3A is a plot showing the relationship between lactate concentration and current measured according to the method of the present disclosure carried out using the mutant lactate oxidase S175C of the present disclosure in PB solution (pH 5.0).

[0032] [Figure 3B] FIG. 3B is a plot showing the relationship between lactate concentration and current measured according to the method of the present disclosure carried out using the mutant lactate oxidase S175C of the present disclosure in PB solution (pH 7.0).

[0033] [Figure 4A] FIG. 4A is a plot showing the relationship between lactate concentration and current measured according to the method of the present disclosure carried out using carboxymethylated S175C of the present disclosure in PB solution (pH 5.0).

[0034] [Figure 4B] FIG. 4B is a plot showing the relationship between lactate concentration and current measured according to the method of the present disclosure carried out using carboxymethylated S175C of the present disclosure in PB solution (pH 7.0).

[0035] [Figure 5] FIG. 5 is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using different amounts of A96C of the present disclosure in synthetic sweat (pH 5.0).

[0036] [Figure 6] 6 is a line graph showing a calibration curve of lactate concentration versus current obtained according to the method of the present disclosure carried out using the A96C of the present disclosure in synthetic sweat having various pH values ​​(pH 5.0-7.0);

[0037] [Figure 7] FIG. 7 is a plot showing the current corresponding to a particular lactate concentration (40 mM) detected using the A96C of the present disclosure in synthetic sweat having various salinities (0 to 800 mM) and pH values. DETAILED DESCRIPTION OF THE INVENTION

[0038] The detailed description provided below in connection with the accompanying drawings is intended to be illustrative of the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or used. The description sets forth the functions of the embodiment and procedures for construction and operation of the example. However, the same or equivalent functions and procedures may be accomplished by different embodiments.

[0039] 1.Definition

[0040] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] The singular forms "a," "and," and "the" are used herein to include plural referents unless the context clearly dictates otherwise.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, they inherently contain certain errors resulting from the standard deviation found in their respective testing measurements.

[0043] Typically, the term "wild-type" is used to describe a gene or protein when that gene or protein is found in nature in its unmutated (unaltered) state. As used herein, the term "wild-type lactate oxidase" refers to the form of the lactate oxidase protein that normally occurs in nature (i.e., bacteria) without genetic, structural, and / or functional alterations. Specifically, the wild-type form of lactate oxidase is the full-length native lactate oxidase having 374 amino acids set forth as SEQ ID NO:1.

[0044] As used herein, the term "lactate oxidase variant" is intended to encompass one or more forms of a lactate oxidase polypeptide derived from a wild-type lactate oxidase by substitution, in which at least one amino acid in the wild-type lactate oxidase sequence is replaced with another amino acid. Alternatively or optionally, the term "lactate oxidase variant" refers to a form of a lactate oxidase peptide in which one or more residues have been subjected to post-translational modification (PTM) and / or chemical modification to increase the functional diversity of the proteome. Types of PTM include, but are not limited to, phosphorylation, methylation, acetylation, ubiquitination, hydroxylation, succinylation, glycosylation, and sumoylation. Exemplary chemical modifications include, but are not limited to, carboxymethylation. In the present disclosure, the modification performed on an amino acid residue is carboxymethylation. Well-known and commonly used designations may be used interchangeably herein to indicate the same mutation occurring in a peptide sequence. According to the present disclosure, for example, an alanine (A) to asparagine (N) substitution at position 95 may be designated as 95A, A95, A95N, or Ala95Asp.

[0045] As used herein, the term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a substrate (i.e., lactate and / or lactic acid) and an enzyme (e.g., lactate oxidase or a variant thereof). The affinity of an enzyme for a substrate is typically measured using the Michaelis constant (K), which represents the substrate concentration at which half of the enzyme's active sites are occupied by the substrate. m ) is thought to be related to K m The smaller the Λ, the stronger the binding affinity for the substrate. Typically, the binding affinity of the mutant compared to the wild-type enzyme may or may not change depending on the position where the mutation occurs. According to the present disclosure, the binding affinity of the lactate oxidase variant of the present disclosure to lactate and / or lactic acid is reduced compared to the wild-type lactate oxidase.

[0046] As used herein, the term "liquid sample" refers to a sample collected and / or obtained from a natural environment or an artificial source in a liquid form where the solvent is primarily water and may or may not contain lactate and / or lactic acid. A liquid sample as used in the present disclosure may be a biological sample containing metabolic products of an organism (i.e., lactate and / or lactic acid). Examples of biological samples suitable for use in the present disclosure include mammalian, more preferably human, bodily fluids (e.g., sweat, urine, saliva, blood, and interstitial fluid) and fermentation liquids produced by microorganisms (e.g., fermented and spoiled foods). A liquid sample may contain one or more substances, including, but not limited to, minerals, trace elements, metal and / or heavy metal ions, metabolites, excrement, microplastics, micronekton, and microorganisms. Furthermore, a liquid sample may have various measurable parameters, including, but not limited to, pH and salinity.

[0047] 2. Detailed Description of the Preferred Embodiments

[0048] The present disclosure is based, at least in part, on the discovery that some lactate oxidase variants have lower lactate / lactic acid binding affinity than wild-type lactate oxidase, thereby enabling sensitive detection of high concentrations of lactate without being affected by pH or salinity. Furthermore, it has been unexpectedly discovered that the reaction of the enzyme (i.e., lactate oxidase) and substrate (i.e., lactate, lactic acid, or a combination thereof) in the presence of an electric field generates a current, and that a linear relationship exists between high concentrations of lactate (e.g., >20 mM) and the current; therefore, the current may serve as an indicator for lactate detection.

[0049] 2.1 Lactate oxidase mutants

[0050] A first aspect of the present disclosure relates to a lactate oxidase variant comprising at least one amino acid mutation and having reduced lactate / lactic acid binding affinity compared to wild-type lactate oxidase. The lactate oxidase variant has an amino acid sequence derived from wild-type lactate oxidase as set forth in SEQ ID NO: 1, with one or more amino acid substitutions at positions 95, 96, and / or 175 of SEQ ID NO: 1. Specifically, the alanine (A) at position 95 of SEQ ID NO: 1 is substituted with asparagine (N) or glutamine (Q), the alanine (A) at position 96 of SEQ ID NO: 1 is substituted with cysteine ​​(C), and / or the serine (S) at position 175 of SEQ ID NO: 1 is substituted with cysteine ​​(C).

[0051] According to embodiments of the present disclosure, the lactate oxidase of the present disclosure may have an amino acid sequence at least 99% identical to SEQ ID NO:1, for example, having 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% sequence identity to SEQ ID NO:1, preferably an amino acid sequence at least 99.2% identical to SEQ ID NO:1, more preferably an amino acid sequence at least 99.8% identical to SEQ ID NO:1, wherein at least one amino acid substitution occurs at position 95, 96, or 175 of SEQ ID NO:1, and wherein such amino acid substitution is selected from the group consisting of A95N, A95Q, A96C, S175C, and combinations thereof.

[0052] According to some embodiments of the present disclosure, a lactate oxidase variant of the present disclosure, designated A95N, may have an amino acid sequence at least 99.8% identical to SEQ ID NO: 1, with an asparagine (N) replacing alanine (A) at position 95 of SEQ ID NO: 1. Thus, the lactate oxidase A95N variant has the amino acid sequence of SEQ ID NO: 2.

[0053] According to another embodiment of the present disclosure, the lactate oxidase variant of the present disclosure, designated A95Q, may have an amino acid sequence at least 99.8% identical to SEQ ID NO: 1, with a substitution of glutamine (Q) for alanine (A) at position 95 of SEQ ID NO: 1. Thus, the lactate oxidase A95Q variant has the amino acid sequence of SEQ ID NO: 3.

[0054] According to another embodiment of the present disclosure, the lactate oxidase variant of the present disclosure, designated A96C, may have an amino acid sequence at least 99.8% identical to SEQ ID NO: 1, with alanine (A) at position 96 of SEQ ID NO: 1 substituted with cysteine ​​(C). Thus, the lactate oxidase A96C variant has the amino acid sequence of SEQ ID NO: 4.

[0055] According to yet another embodiment of the present disclosure, the lactate oxidase variant of the present disclosure, designated S175C, may have an amino acid sequence at least 99.8% identical to SEQ ID NO: 1, with a substitution of cysteine ​​(C) for serine (S) at position 175 of SEQ ID NO: 1. Thus, the lactate oxidase S175C variant has the amino acid sequence of SEQ ID NO: 5.

[0056] The lactate oxidase variants of the present disclosure can be prepared by substitution or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the coding DNA sequence, polymerase chain reaction (PCR), gene synthesis, CRISPR / cas9 gene editing, etc. The exact nucleotide changes may be confirmed, for example, by sequencing. The nucleotide sequence of eubacterial (e.g., Aerococcus viridans) lactate oxidase is available from public databases such as UniProtKB (Aerococcus viridans ATCC 11563, accession code: D4YFm2). The amino acid sequence of wild-type lactate oxidase is set forth in SEQ ID NO: 1.

[0057] The lactate oxidase variants of the present disclosure may be produced, for example, by solid-phase peptide synthesis or recombinant production. In recombinant production, one or more polynucleotides encoding the lactate oxidase variants are each isolated and inserted into an appropriate vector for further cloning and / or expression in a host cell, primarily E. coli. Such polynucleotides may be readily isolated and sequenced using conventional methods. Methods well known to those skilled in the art may be used to construct expression vectors containing the coding sequence of the lactate oxidase variants of the present disclosure along with appropriate transcriptional / translational control signals. Examples of these methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. The expression vector may be a plasmid, part of a virus, or a nucleic acid fragment. Typically, the expression vector is an expression cassette into which a polynucleotide encoding the lactate oxidase variant of the present disclosure is cloned in operably linkage with a promoter and / or other transcriptional or translational control elements; the promoter may be operably linked to a nucleic acid encoding a polypeptide, provided that the promoter is capable of effectively directing the transcription of that nucleic acid. According to some embodiments of the present disclosure, site-directed mutagenesis of native lactate oxidase is expressed and performed using the pRSET expression vector and standard tools well known in the art.

[0058] Alternatively or optionally, the lactate oxidase variants of the present disclosure may be further subjected to post-translational modification (PTM) and / or chemical modification, in which case additional functional groups are introduced into residues. Representative PTMs include, but are not limited to, phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, acetylation, hydroxylation, succinylation, and sumoylation. Representative chemical modifications include, but are not limited to, carboxymethylation. PTM and / or chemical modifications of proteins can be performed by any method and tool known in the art, depending on actual needs and purposes. According to one aspect of the present disclosure, the lactate oxidase variants of the present disclosure are carboxymethylated by reacting with iodoacetate (IA) or iodoacetic acid (IAA), which covalently bonds to the thiol group of cysteine ​​(C) to generate a carboxymethyl group. In one example, the lactate oxidase variant S175C of the present disclosure is chemically modified by reaction with iodoacetic acid to generate a carboxymethylated cysteine ​​residue, and the carboxymethylated lactate oxidase S175C thus has the amino acid sequence of SEQ ID NO: 6.

[0059] In certain embodiments, the amino acid substitutions and / or modifications made to wild-type lactate oxidase may reduce the binding affinity of the enzyme (i.e., lactate oxidase) for the substrate (i.e., lactate / lactic acid) by at least 50%, e.g., at least 30%, 25%, 20%, 10%, 7%, 5%, 2%, or 1%. The binding affinity of the lactate oxidase variants of the present disclosure for the substrate can be measured or determined by various assays well known in the art, such as colorimetric assays, and the kinetic parameters (e.g., K in the Michaelis-Menten equation) of each lactate oxidase variant can be determined by following established procedures well known in the art. m and V max ) is determined.

[0060] 2.2 Lactic acid detection and quantification method

[0061] Another aspect of the present disclosure relates to a method for detecting and quantifying lactate in a liquid sample, the method comprising at least the following steps: (a) contacting a liquid sample with the lactate oxidase variant described in the preceding paragraph; (b) measuring the current generated by the reaction of the lactate oxidase variant of the present disclosure with lactate in a liquid sample; and (c) determining the concentration of lactate in the liquid sample by interpolating or extrapolating the current measured in step (b) using the current of a control sample having a known lactate concentration.

[0062] According to the present disclosure, the current generated by the reaction of the lactate oxidase variant of the present disclosure with lactate in a liquid sample in step (a) can be measured using an electrode system. In this regard, it is preferable to prepare a standard calibration curve for lactate detection by measuring the current generated between lactate oxidase and various known concentrations of lactate before starting the present method. The electrode system typically includes a working electrode, a counter electrode, and an optional reference electrode. Typical materials suitable for constructing the working electrode and / or counter electrode include, but are not limited to, carbon (e.g., pyrolytic carbon, graphite, graphene, glassy carbon, carbon paste, perfluorocarbon (PFC), etc.) and metal (e.g., platinum, gold, silver, nickel, palladium, etc.). Furthermore, a typical reference electrode may be a saturated calomel electrode or a silver / silver chloride electrode. The electrode system can be fabricated from any of the specified materials exemplified above by methods well known in the art, such as photolithographic deposition, sputtering, or printing (e.g., screen printing, gravure printing, flexographic printing, etc.). In one embodiment, the electrode system of the present disclosure is a screen-printed carbon electrode (SPCE) made of graphite and graphene.

[0063] For the purpose of detecting lactate, the lactate oxidase variant of the present disclosure is attached to the surface of the electrode system (e.g., SPCE) described above in the presence of a redox mediator. According to a possible embodiment, a lactate oxidase variant of the present disclosure and a redox mediator are mixed in a predetermined ratio to form a mixture, which is then immobilized on the surface of an SPCE by electrodeposition or dropwise application, thereby producing a lactate sensor suitable for use in the present method.

[0064] Examples of redox mediators suitable for use in the methods of the present disclosure include, but are not limited to, poly(aniline)-poly(acrylate), poly(aniline)-poly(vinylsulfonic acid), poly(pyrrole), poly(pyrrole)-poly(vinylsulfonic acid), poly(vinylpyrrolidone), poly(1-vinylimidazole) (PVIm), ferricyanide salts, ferrocyanide salts, cobalt phthalocyanine, hydroxymethylferrocene, osmium (Os) complexes, [7-(dimethylamino)-4-nitrophenothiazin-3-ylidene]-dimethylazanium chloride, benzo[a]phenoxazin-9-ylidene(dimethyl)azanium, tetrathiafulvalene, and copolymers or combinations thereof. In one embodiment, the redox mediator is a copolymer of poly(1-vinylimidazole) and an osmium complex (PVImQOs), and in another embodiment, the redox mediator is potassium ferricyanide (K3[Fe(CN)6]).

[0065] To prepare a calibration curve, a control sample containing a known concentration of lactate or lactic acid is contacted with a lactate sensor to which a fixed potential is applied, and the current generated by the electrochemical reaction between lactate and the lactate oxidase variant of the present disclosure immobilized on the electrode can be detected by any means known in the art, specifically, by an electrochemical analyzer. Thus, a standard calibration curve can be prepared based on the detection currents corresponding to known concentrations of lactate. In some embodiments of the present disclosure, the known lactate concentrations range from about 0 to 300 mM, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 9.9, 10, 14.8, 19.6, 20, 29.1, 30, 38.5, 40, 47.6, 50, 56.6, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 1 In one example, the calibration curve is generated using lactic acid at concentrations of 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 38.5, 47.6, 56.6, 65.4, 74.1, 82.6, 90, 90.9, 100, 110, 120, 130, 130.4, 140, 150, 160, 166.7, 170, 180, 190, 200, 210, 220, 230, 240, 250, 259.3, 260, 270, 280, 290, or 300 mM. In another example, the calibration curve is prepared using lactic acid at concentrations of 0.2, 0.5, 1, 2, 5, 9.9, 19.6, 29.1, 47.6, 90.9, 130.4, 166.7, 200, and 259.3 mM. In yet another example, the calibration curve is prepared using lactic acid at concentrations of 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 10 mM. In yet another example, the calibration curve is prepared using lactic acid at concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, and 200 mM. According to an embodiment of the present disclosure, the control sample simulates human sweat. Preferably, the control sample used in the present method is synthetic sweat prepared according to international standards.Alternatively or optionally, the control sample used in the method specifically mimics the pH, osmolality and ionic concentration of human body fluids. Preferably, the control sample is a phosphate buffer.

[0066] In step (a) of the method, a liquid sample (e.g., human sweat, buffer solution, etc.) is contacted with the lactate oxidase variant of the present disclosure immobilized on the surface of an electrode system for a time sufficient to generate an electrochemical current resulting from the reaction between the enzyme and the substrate, i.e., at least 5, 10, 20, or 60 seconds, after which, in step (b), the current is measured and determined by any means known in the art, such as the electrochemical analyzer described above.

[0067] According to some embodiments of the present disclosure, the liquid sample has a pH value between 4 and 9, for example, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9. In one example, the liquid sample has a pH value of 5.0, 6.0, or 7.0. According to alternative embodiments of the present disclosure, the liquid sample has a salinity of 0 to 1000 mM, for example, 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mM. In other examples, the liquid sample has a salinity of 0, 100, 200, 300, 400, 500, 600, 700, or 800 mM. Liquid samples suitable for use in the present method may be obtained from natural environments (e.g., animal bodies) or man-made sources (e.g., food). Examples of liquid samples suitable for use in the present method include, but are not limited to, sweat, urine, saliva, blood, interstitial fluid, and fermentation liquids produced by microorganisms. In one example, the liquid sample is sweat.

[0068] In the final step of the method, i.e., step (c), the lactate concentration in the liquid sample can be determined from the calibration curve by interpolation or extrapolation. Specifically, the current measured in step (b) is substituted into the calibration curve prepared before step (a) based on the known lactate concentrations in the control sample, thereby determining the lactate concentration in the liquid sample.

[0069] In summary, the present method includes at least steps (a) to (c) described above, and can detect lactate in any liquid sample containing trace or large amounts of lactate. According to the present disclosure, the present method can detect lactate in a range of 0 to 300 mM, for example, 0 to 280 mM, 0 to 260 mM, 0 to 250 mM, 0 to 200 mM, 0 to 150 mM, 0 to 130 mM, 0 to 120 mM, 0 to 110 mM, 0 to 100 mM, 0.2 to 5 mM, 0.2 to 10 mM, 0.2 to 166 mM, 0.2 to 180 mM, 0.2 to 260 mM, 0.5 to 90 mM, 0.5 to 100 mM, 0.5 to 110 mM, 0.5 to 120 mM, or 5 to 100 mM. In some preferred embodiments, the present method can detect lactate in a liquid sample at levels greater than 100 mM.

[0070] Due to the above features, the present method can detect and quantify lactate concentration, especially abundant lactate in aqueous environments that cannot be detected by conventional detection methods. Furthermore, the present method can detect lactate in aqueous samples containing various substances, so it can be applied to a variety of liquid samples. [Example]

[0071] Example

[0072] Materials and Methods

[0073] Gene synthesis and mutagenesis

[0074] Expression vectors expressing wild-type and mutant lactate oxidases were introduced into Escherichia coli BL21(DE3). The lactate oxidase gene was cloned based on the wild-type sequence of Aerococcus viridans ATCC 11563 (accession code: D4YFm2) obtained from the UniProtKB database. The wild-type sequence was inserted into the multiple cloning site of the expression vector pRSET according to the preferred codon usage in E. coli. Site-directed mutagenesis was performed using the primer set listed in Table 1 using the standard protocol provided with a commercially available mutagenesis kit (Quikchange, Agilent, USA). Plasmid sequences were confirmed using a 3730XL DNA Analyzer (Thermo Fisher Scientific, USA).

[0075] Table 1 Primer sequences [Table 1]

[0076] Preparation of wild-type and mutant lactate oxidases of the present disclosure

[0077] E. coli BL21(DE3) was transformed with the wild-type and various lactate oxidase-containing plasmids. The transformed DE3 was inoculated into LB medium containing ampicillin and grown in a shaking incubator (250 rpm) at 37°C for 16 to 20 hours. One percent of the bacterial culture was removed and used to inoculate 500 mL of ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM KH2PO4, 25 mM (NH4)2SO4, 50 mM Na2HPO4, and 1 mM MgSO4) containing 100 μg / mL ampicillin as the main culture. The medium was then grown at 37°C for 8 hours with shaking. The supernatant was removed by centrifugation (4°C, 5000xg, 20 minutes), and the cell pellet was collected and stored at -20°C.

[0078] Purification of wild-type and mutant lactate oxidases of the present disclosure

[0079] E. coli cells transformed with wild-type or mutant lactate oxidase were resuspended in 50 mL of 50 mM potassium phosphate buffer (PPB, pH 7.0) containing 500 mM NaCl and 20 mM imidazole and homogenized using a NanoLyzer. The resulting cell suspension was centrifuged at 10,000 g for 1 hour at 4°C, and the supernatant was collected. The supernatant contained the recombinant His-tagged protein as a crude cell extract, which was purified using a nickel affinity column and a protein purification kit (A¨KTA start, Cytiva). The column was eluted with 5 volumes of buffer A (pH 7.0, 20 mM imidazole, 500 mM NaCl, and 50 mM PPB), and the crude cell extract was loaded onto the column. The column was then washed again with buffer A (3 volumes) and eluted with 20 volumes of 4 to 100% buffer B (pH 7.0, 500 mM imidazole, 500 mM NaCl, and 50 mM PPB). A total of 20 fractions were collected. After SDS-PAGE analysis, fractions containing the target protein were dialyzed and concentrated using a dialysis membrane (10 kDa). After replacing the solvent with 50 mM Tris-HCl (pH 7.0), the purified enzyme solution was lyophilized and stored at -20°C.

[0080] Carboxymethylation of mutant lactate oxidase

[0081] A solution of the purified lactate oxidase mutant (80 μL or 3.5 U) was mixed with 0.1 M iodoacetic acid (IAA) (20 μL) at 37° C. for 60 minutes.

[0082] Enzyme assay

[0083] Measurement of protein concentration

[0084] The analyte (20 μL) was mixed with 200 μL of protein dye (Bradford reagent) for 5 min to form a mixture, and the OD value of the mixture was measured at 595 nm. The protein concentration was determined based on a calibration curve constructed based on standard concentrations of bovine serum albumin (BSA) solution ranging from 0.05 to 0.3 mg / mL and their corresponding OD values.

[0085] Measurement of protein activity

[0086] 1. Lactate oxidase

[0087] 50 μL of the diluted enzyme was added to each well of a microplate, and 50 μL of a staining reagent prepared by mixing 0.25 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) and 5 U / mL horseradish peroxidase (HRP), and 50 μL of 10 mM lactic acid were added sequentially to each well. The reaction was carried out at 30°C for 5 minutes with intermittent shaking. After that, 50 μL of 1 M hydrochloric acid was added to stop the reaction, and the mixture was diluted with 59 cm -1 mmol -1 The absorbance (OD) of the solution was measured at a wavelength of 450 nm using a molar extinction coefficient (ε) of 1. The lactate oxidation activity (i.e., 1 U) was measured at 30°C and pH 7.0 using the following formula: Activity (U / mL) = [ΔOD(OD 試験 -OD ブランク )×V t × df] / (ε×t×l×Vs), where Vt is the total volume, df is the dilution factor, ε is the molar extinction coefficient, t is the reaction time, l is the optical path length (cm), and Vs is the enzyme volume.

[0088] 2. Dehydrogenase activity

[0089] Phenazine methosulfate (PMS, 0.5 mM) was mixed with 2,6-dichlorophenolindophenol (DCIP, 1.2 mM) in potassium phosphate buffer (PPB, 20 mM, pH 7.0) to form the reaction reagent. Enzyme solution (50 μL, in 50 mM PPB, pH 7.0) was added to each well of a microplate at a ratio of 50 μL per well. The reaction reagent (50 μL) and 10 mM lactic acid (50 μL) were then added sequentially to each well. The microplate was then stirred intermittently at 30°C for 5 minutes. The optical density (OD) at 600 nm in each well was measured, and the molar extinction coefficient (ε) was determined to be 16.3 cm. -1 mmol -1 Lactate dehydrogenase activity (i.e., 1 U) was determined at 30°C and pH 7.0 according to the following formula: Activity (U / mL) = [ΔOD(OD 試験 -OD ブランク )×V t × df] / (ε×t×l×Vs), where Vt is the total volume, df is the dilution factor, ε is the molar extinction coefficient, t is the reaction time, l is the optical path length (cm), and Vs is the enzyme volume.

[0090] 3.Binding affinity

[0091] Enzyme kinetic parameter K of wild-type or mutant lactate oxidase m , K. cat , and V max was determined by reacting the lactate oxidase variants of the present disclosure and the wild-type lactate oxidase with various concentrations of lactate (0.1 to 200 mM for oxidase, 0.1 to 900 mM for dehydrogenase) at pH 5.0 or pH 7.0, respectively, and the reaction rates were determined individually based on the slope of a linear plot of absorbance (OD) at a wavelength of 450 nm or 600 nm measured every 3 seconds within the reaction interval versus time (i.e., reaction interval). m , K. cat , and V max The value of was calculated a posteriori from the calibration curve between lactate concentration (x-axis) and reaction rate (y-axis) in the Michaelis-Menten equation.

[0092] Fabrication of lactate sensors using modified electrodes

[0093] For cyclic voltammetry (CV) analysis, a screen-printed carbon electrode (SPCE, TE100, Zensor) was used as a three-electrode system, and the enzyme of the present disclosure was immobilized on the surface of the working electrode by electrodeposition or the dropping method described below.

[0094] Electroplating

[0095] The exterior surface of the SPCE was washed with ddH2O and then covered with a mixture of 20 μL of PVImQOs (10 mg / mL) and 100 μL of an enzyme solution (4 U) of either wild-type lactate oxidase or a lactate oxidase variant of the present disclosure (i.e., A95N, A95Q, A96C, S175C, or modified S175C). The SPCE was then subjected to 50 cycles of cyclic voltammetry at a preset potential between -1.0 V and 0.0 V and a preset scan rate of 200 mV / s at 37 °C to achieve surface modification. The modified SPCE was then immersed in a PPB solution (pH 5.0, 100 mM) and subjected to 20 cycles of cyclic voltammetry at a second preset potential between -300 mV and 600 mV and a scan rate of 60 mV / s to remove residual PVImQOs. After drying at room temperature, 4 μL of a protective agent containing 1% chitosan and 0.075% genipin was further added to the modified SPCE.

[0096] Dripping method i. A mixture of enzyme solution (1 to 8 U / μL), potassium ferricyanide (K3[Fe(CN)6], 25 to 50 mM), and 0.1% Triton X-100 (1 μL) was dropped onto the electrode surface to cover it, and the electrode was allowed to dry at room temperature. Alternatively, 5 μL of PVImQOs (10 mg / mL) and 2.5 μL of enzyme solution were sequentially dropped onto the electrode area of ​​the SPCE. After drying at 4°C, 4 μL of a protective agent containing 1% chitosan and 0.075% genipin was added to the surface of the SPCE. The modified SPCE was then dried and stored at room temperature.

[0097] Electrochemical evaluation

[0098] The SPCE was connected to an electrochemical analyzer (ACIP100) in combination with a CS100 electrode stand (Zensor) with an ECP100 (Zensor) cable connector. Voltammograms and results were recorded and analyzed with the on-device software of the ACIP100 (Zensor).

[0099] Lactic acid detection

[0100] Sample preparation

[0101] Synthetic sweat was prepared according to the international standard ISO 3160-2, and its pH was set to 5, 6, or 7 by adjusting the amount of sodium hydroxide. Potassium phosphate buffer (0.1 M) and its pH were prepared by adjusting the amounts of monobasic and dibasic potassium phosphate obtained from a commercial supplier (Merck).

[0102] Creating a calibration curve

[0103] Lactate solutions of various concentrations (i.e., 0, 0.2, 0.4, 0.5, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9.9, 10, 14.8, 19.6, 20, 29.1, 30, 38.5, 40, 47.6, 50, 56.6, 60, 65.4, 70, 74.1, 80, 82.6, 90, 90.9, 100, 120, 130.4, 166.7, 200, or 259.3 mM lactate in synthetic sweat or phosphate buffer) were applied to the enzyme-immobilized lactate sensor, and the current generated between the electrodes was measured at a fixed potential of 300 or 400 mV. The current value at 60 seconds of the reaction between a blank sample (i.e., 0 mM lactate) and the enzyme (i.e., the mutant lactate oxidase of the present disclosure) was recorded as background, and then buffer solutions containing the above concentrations of lactate were applied to the lactate sensor. Each reaction lasted approximately 20 to 60 seconds, and the current for each reaction was recorded every 5 or 10 seconds, allowing the construction of a calibration curve of current versus lactate concentration.

[0104] Example 1 Production of lactate oxidase variants of the present disclosure

[0105] The five lactate oxidase variants of the present disclosure were produced as described in the "Materials and Methods" section. The lactate oxidase variants thus obtained and their amino acid sequences are listed in Table 2, with the indicated amino acid substitutions and modifications in bold.

[0106] Table 2. Lactate oxidase variants of the present disclosure [Table 2] JPEG2025527991000004.jpg133164*: Cysteine ​​modified with a carboxymethyl group.

[0107] Example 2 Characterization of lactate oxidase variants of the present disclosure

[0108] 2.1 Oxidase and dehydrogenase activities

[0109] In this example, the oxidase and dehydrogenase activities of the lactate oxidase variants of the present disclosure were characterized. To this end, the wild-type and mutant lactate oxidases of the present disclosure were mixed and reacted with lactate (10 mM), respectively, and their protein activities were then calculated individually according to the procedures described in the "Materials and Methods" section. The quantitative results are shown in Table 3.

[0110] Table 3. Quantitative results of oxidase and dehydrogenase activities of wild-type (WT) and mutant lactate oxidases [Table 3] *ND: Not detectable.

[0111] The data in Table 3 reveal that the oxidase and dehydrogenase activities of the mutant lactate oxidases are significantly reduced compared to the wild-type protein, and in particular, the oxidase and dehydrogenase activities of the A95Q mutant are 98.4% and 95% lower, respectively, than those of the wild-type lactate oxidase.

[0112] 2.2 Enzyme kinetics

[0113] In this example, we investigated whether the binding affinity of the lactate oxidase variants of the present disclosure is affected by pH. To this end, we reacted the lactate oxidase variants of the present disclosure with various concentrations of lactate at pH 5.0 or pH 7.0 to measure the K m and K. cat The values ​​were determined according to the procedure described in the Materials and Methods section. m and K. cat The values ​​are shown in Table 4.

[0114] Table 4. K of lactate oxidase variants and wild-type (WT) enzyme of the present disclosure m and K. cat value [Table 4] *ND: Not detectable.

[0115] The K of the lactate oxidase variants of the present disclosure, regardless of changes in pH (i.e., pH 5.0 or pH 7.0), m The values ​​were significantly higher than that of the wild-type enzyme, indicating that each of the lactate oxidase variants of the present disclosure has a low binding affinity for lactate.

[0116] Example 3 Detection of Lactate Using the Lactate Oxidase Variants of the Disclosure

[0117] In this example, we evaluated the sensitivity and versatility of the disclosed lactate oxidase variants for detecting lactate in various samples. To this end, two lactate sensors (LS-I and LS-II) based on different types of redox mediators immobilized on a screen-printed carbon electrode (SPCE) were fabricated according to the procedures described in the "Materials and Methods" section. Specifically, LS-I and LS-II were fabricated by attaching a mixture of enzyme solutions (i.e., the disclosed lactate oxidase variants A95Q, A96C, S175C, and carboxymethylated S175C) and a polymeric mediator PVImQOs to the SPCE surface. LS-II was fabricated by immobilizing the enzyme and potassium ferricyanide (K3[Fe(CN)6]) on the SPCE surface. LS-I and LS-II were used to detect lactate in the designated samples.

[0118] 3.1 Binding affinity

[0119] In this experiment, we investigated whether the binding affinity of the lactate oxidase variants of the present disclosure is affected by pH. To this end, the reaction between lactate (various concentrations) and the lactate oxidase variants of the present disclosure was measured using the lactate sensor LS-I at pH 5.0 or pH 7.0. The K of the enzyme (i.e., the lactate oxidase variants of the present disclosure) was measured. m and V max The values ​​were determined according to the procedures described in the Materials and Methods section, and the results are summarized in Table 5.

[0120] Table 5. K of wild-type (WT) and mutant lactate oxidases of the present disclosurem value and V max value [Table 5] **Cysteine ​​at position 175 is modified with a carboxymethyl group.

[0121] As summarized in Table 5, the lactate oxidase variants of the present disclosure have a higher K than the wild-type enzyme. m The data in Table 5 are consistent with the previous evaluation in Example 2, and all indicate that the lactate oxidase variants of the present disclosure have lower lactate binding affinity than wild-type lactate oxidase, especially in low pH environments.

[0122] 3.2 Detection of Lactate Using the Lactate Oxidase Variants of the Disclosure

[0123] 3.2.1 A95Q, A96C, or S175C

[0124] In this experiment, we investigated the detection efficiency and limitations of the disclosed mutant lactate oxidases A95Q, A96C, and S175C. To this end, we independently immobilized the disclosed mutant lactate oxidases A95Q, A96C, or S175C within the mediator PVImQOs on a SPCE surface to fabricate type I lactate sensors (hereafter referred to as A95Q-LS-I, A96C-LS-I, and S175C-LS-I). Next, various concentrations of lactate (0-300 mM) in phosphate buffer (pH 5 and pH 7) or artificial sweat (pH 5) were applied to each lactate sensor, respectively, and the generated current was measured and recorded according to the procedures described in the "Materials and Methods" section. The results are shown in Figures 1 to 3.

[0125] Regardless of the pH of the buffer, the A95Q enzyme of the present disclosure was found to be able to successfully detect different concentrations of lactate, with the minimum and maximum concentrations being approximately 0.2 mM and 166 mM, respectively. Collectively, the data shown in Figures 1A and 1B demonstrated that the lactate oxidase A95Q variant of the present disclosure can detect a wide range of lactate concentrations in liquid samples.

[0126] For A96C, we found that A96C could detect lactate in the range of approximately 0.5 mM to 90 mM in either pH 5.0 (FIG. 2A) or pH 7.0 (FIG. 2B) buffer solutions. Similar results were also obtained for the detection of lactate in synthetic sweat (pH 5.0, FIG. 2C). In synthetic sweat, the detectable lactate concentrations ranged from 0.5 mM to 90 mM, demonstrating that the lactate oxidase A96C of the present disclosure is capable of detecting lactate in simulated biological fluid environments.

[0127] For S175, trace concentrations of lactate (i.e., 0.2 mM to 10 mM) were found to be detected by the lactate oxidase variant S175C of the present disclosure, regardless of the pH value of the buffer (Figures 3A and 3B). This result indicates that the lactate oxidase variant S175C of the present disclosure can detect and quantify lactate in aqueous samples at small scales (e.g., less than 10 mM in the present disclosure) without being affected by different pH values.

[0128] 3.2.2 Carboxymethylated S175C

[0129] In this experiment, the S175C mutant was reacted with 0.1 M iodoacetic acid (IAA) to generate carboxymethylated S175C and its effect on lactate detection was investigated. The results are shown in Figures 4A and 4B.

[0130] As shown in the figure, carboxymethylation significantly improved the lactate detection range of the S175C mutant compared to the control mutant. Specifically, the maximum lactate concentrations detectable by the carboxymethylated mutant in acid buffer (pH 5.0) and neutral buffer (pH 7.0) increased to 166.7 mM and 259.3 mM, respectively.

[0131] 3.3 Versatility of the disclosed lactate oxidase variants in lactate detection

[0132] In this example, the versatility of the disclosed lactate oxidase variants in lactate detection was investigated by varying the amount of enzyme immobilized on the detection electrode, the pH value and / or salinity of the buffer, and the results are shown in Figures 5 to 7.

[0133] The maximum detectable lactate concentration in synthetic sweat was found to exceed 100 mM for sensors immobilized with 1, 1.5, and 2 units (U) of A96C. In particular, the lactate sensor immobilized with 1.5 U of A96C (i.e., A96C-LS-II-1.5U) showed the highest detection capability, with a detectable lactate concentration of 120 mM in synthetic sweat (Figure 5). Furthermore, the sensor immobilized with 1 unit of A96C (i.e., A96C-LS-II-1U) showed the highest resolution and less variability (Figure 5), indicating that only a small amount of the mutant enzyme was required to achieve the desired detection effect.

[0134] Regarding the effect of pH on lactate detection, it was found that the mutant lactate oxidase A96C of the present disclosure was able to detect 0 to 200 mM lactate in sweat samples regardless of the pH level (FIG. 6).

[0135] Regarding the effect of salinity, slight differences in the current generated by the reaction of lactate and the mutant lactate oxidase A96C of the present disclosure were observed at various pH values ​​and / or salinities ( FIG. 7 ). The data shown in FIG. 7 confirmed that the detection ability of the mutant lactate oxidase A96C of the present disclosure was not affected or hindered by the salinity of synthetic sweat.

[0136] In summary, the data in Examples 1 to 3 demonstrate that the lactate oxidase variants of the present disclosure have superior lactate detection ability, regardless of the pH value and / or salinity of liquid samples, enabling broad versatility in lactate detection.

[0137] The above description of the embodiments is provided by way of example only, and it will be understood that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. As noted above, while various embodiments of the invention have been described with certain specificity or with reference to one or more specific embodiments, those skilled in the art may make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. 1. A lactate oxidase variant derived from the wild-type lactate oxidase of SEQ ID NO: 1, wherein the lactate oxidase variant comprises an amino acid substitution at position 95, 96, or 175 of SEQ ID NO: 1, or a combination thereof; alanine (A) at position 95 of SEQ ID NO: 1 is substituted with asparagine (N) or glutamine (Q); The alanine (A) at position 96 of SEQ ID NO: 1 is substituted with a cysteine ​​(C), and / or The serine (S) at position 175 of SEQ ID NO: 1 is substituted with a cysteine ​​(C); Lactate oxidase mutants.

2. 2. The lactate oxidase variant according to claim 1, wherein the alanine (A) at position 95 of SEQ ID NO: 1 is substituted with asparagine (N).

3. 2. The lactate oxidase mutant of claim 1, wherein the alanine (A) at position 95 of SEQ ID NO: 1 is substituted with glutamine (Q).

4. 2. The lactate oxidase mutant according to claim 1, wherein the alanine (A) at position 96 of SEQ ID NO: 1 is substituted with cysteine ​​(C).

5. 2. The lactate oxidase mutant according to claim 1, wherein serine (S) at position 175 of SEQ ID NO: 1 is substituted with cysteine ​​(C).

6. 6. The lactate oxidase mutant according to claim 5, wherein the cysteine ​​(C) at position 175 of SEQ ID NO: 1 is carboxymethylated.

7. 2. The lactate oxidase variant of claim 1, wherein the lactate oxidase variant has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, or 6.

8. 2. The lactate oxidase variant according to claim 1, wherein the lactate oxidase variant has a lower binding affinity for lactate than wild-type lactate oxidase.

9. 1. A method for detecting and quantifying lactate in a liquid sample, comprising: (a) contacting the liquid sample with the lactate oxidase variant of claim 1; (b) measuring the current generated by the reaction of the lactate oxidase variant of claim 1 with lactate in a liquid sample; and (c) determining the concentration of lactate in the liquid sample by interpolating or extrapolating the current measured in step (b) using the current of a control sample having a known lactate concentration; A method comprising:

10. 10. The method of claim 9, wherein the liquid sample has a pH value in the range of 4 to 9.

11. 10. The method of claim 9, wherein the liquid sample has a salinity between 0 and 1000 mM.

12. The method of claim 9, wherein the liquid sample is sweat.

13. 10. The method of claim 9, wherein the method is capable of detecting lactate in a range of 0 to 300 mM in a liquid sample.

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