Lactate oxidase variants and uses thereof in lactic acid detection

Abstract

The present disclosure relates to a lactate oxidase variant having a lower affinity for lactic acid (or lactate) than that of a wild-type lactate oxidase for lactic acid (or lactate). The lactate oxidase variant has one or more amino acid substitutions at the 95th, 96th and / or 175th positions of a wild-type amino acid sequence. In addition, methods of detecting and quantifying lactic acid (or lactate salts) in various liquid samples by the lactate oxidase variants of the present disclosure are also disclosed herein. The method mainly comprises the steps of: contacting the lactate oxidase variant with a liquid sample; measuring a current generated by a reaction between the lactate oxidase variant and lactic acid in the liquid sample; and determining the lactic acid concentration of the liquid sample by interpolating or extrapolating the measured current to the current of the control sample having the known lactic acid concentration.

Description

Background of the Invention 1. Technical Field

[0002] The present disclosure relates to lactate oxidase variants and their use in lactate detection, and more particularly to lactate oxidase variants and their use in electrical detection and quantification of lactic acid (or lactate) in liquid samples. 2. Background technology

[0004] Lactate is a key metabolite of the anaerobic metabolic pathway in cells and can therefore be used as a clue to monitor a variety of physiological processes in various organisms. Therefore, lactate concentration has become an important parameter and is widely used in clinical diagnosis to assess patient health status and for continuous monitoring in the food and fermentation industries.

[0005] In clinical diagnosis, the accumulation of lactic acid concentration is caused by the accumulation of lactic acid due to continuous anaerobic metabolism, which inevitably leads to lactic acidosis, becoming one of the symptoms of severe sepsis. Therefore, the patient's blood lactate level can be used as an alarm signal for the severity of the disease to improve the diagnosis and treatment of a wide range of diseases. Lactate is also a significant factor in sports medicine, especially for measuring the physical fitness of athletes. An increase in blood lactate will lead to a decrease in blood pH, which is one of the causes of fatigue. Therefore, the blood lactate level during exercise can be used as an indicator to evaluate the status of exercise training and physical fitness.

[0006] Lactic acid estimation can also be found in the food and fermentation industry. Lactic acid is produced during food fermentation and can be used to detect the presence of microbial fermentation in fermented foods such as fermented dairy products, wine, cured meats and fish, and pickled vegetables. Thus, lactic acid can be used as an indicator of food freshness and quality.

[0007] There are many methods developed to detect lactate levels; high performance liquid chromatography (HPLC) is the most common technique. Other analytical methods including fluorescence, colorimetry, chemiluminescence, and magnetic resonance spectroscopy are also common. However, these methods have some drawbacks, such as time-consuming analysis and expensive equipment and trained manpower. To overcome the above limitations, portable and disposable biosensors are currently being developed. In general, biosensing methods have the advantages of being simple and direct, with rapid response and high specificity, and being economical and easy to use. Ideally, the lactate concentration of biological fluids (such as whole blood, sweat, and saliva) can be measured by biosensors; however, the biosensors currently on the market are only suitable for blood lactate detection and require invasive sample collection methods, which is not popular with users and cannot be used to detect samples other than blood.

[0008] Taking human sweat as an example, the range of lactate concentration is wider than that in blood, and the variation is greater (in some cases, sweat lactate can rise to 80mM after exercise, while the amount of lactate in blood only rises to 25mM during exercise), resulting in poor accuracy of conventional lactate meters for sweat lactate (or lactate). Moreover, all current biosensors require a large volume of liquid to operate, and the difference in pH between blood and sweat also makes it difficult for blood lactate meters to detect sweat lactate concentration.

[0009] In view of the above, there is a need in the prior art to provide an improved tool and method for continuously detecting the lactate content of human body fluids in a non-invasive and effective manner. Summary of the invention

[0010] In order to provide the reader with a basic understanding of the present invention, a brief summary of the present disclosure is provided below. This summary is not an extensive overview of the present disclosure, and is not intended to identify the key / essential components of the present invention or to outline the scope of the present invention. Its sole purpose is to present some concepts of the present disclosure in a simplified conceptual form as a preface to a more detailed description presented later. Page 2 of 29 (Invention Specification)

[0011] As embodied and broadly described herein, one aspect of the present disclosure is a lactate oxidase variant derived from the wild-type lactate oxidase of sequence number: 1, which exhibits reduced lactate affinity but results in a greater detectable range, wherein the lactate oxidase variant comprises an amino acid substitution at position 95, 96 or 175 of sequence number: 1 or a combination thereof. In the lactate oxidase variant of the present disclosure, the alanine (A) at position 95 of sequence number: 1 is substituted with asparagine (N) (or asparagine) or glutamine (Q); the alanine (A) at position 96 of sequence number: 1 is substituted with cysteine ​​(C); and / or the serine (S) at position 175 of sequence number: 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 sequence number: 1, wherein the alanine (A) at position 95 of the sequence is substituted by 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 of the sequence is substituted by glutamine (Q).

[0014] According to another embodiment of the present disclosure, the alanine (A) at position 96 of sequence number: 1 is replaced by cysteine ​​(C).

[0015] According to yet another embodiment of the present disclosure, the serine (S) at position 175 of SEQ ID NO: 1 is replaced by 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 an amino acid sequence of sequence number: 2, 3, 4, 5 or 6.

[0018] In some embodiments, the lactate oxidase variants of the present disclosure have a binding affinity for lactate that is lower than the binding affinity of wild-type lactate oxidase for lactate. Page 3 of 29 pages (Invention Specification)

[0019] Another aspect of the present disclosure is a method for detecting and quantifying lactic acid and / or lactate in a liquid sample. The method comprises step (a): contacting the lactate oxidase variant with the liquid sample; step (b): measuring a current generated by a reaction between the lactate oxidase variant and lactic acid in the liquid sample; and step (c): determining the lactic acid concentration of the liquid sample by interpolating or extrapolating the current measured in step (b) with the current of a control sample with a known lactic acid concentration.

[0020] According to some embodiments, the liquid sample has a pH value between 4 and 9.

[0021] According to certain embodiments, the liquid sample has a salt concentration between 0 and 1,000 mM.

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

[0023] According to certain embodiments of the present disclosure, the method is capable of detecting lactic acid in a liquid sample at a concentration ranging from 0 to 300 mM.

[0024] After referring to the following embodiments, a person having ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation modes adopted by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

[0026] Figure 1AThe line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A95Q of the present invention and performing the method of the present disclosure in phosphate (hereinafter referred to as PB) buffer (pH 5.0). Page 4 of 29 (Invention Specification)

[0027] Figure 1B The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A95Q of the present invention and performing the method of the present disclosure in PB buffer (pH 7.0).

[0028] Figure 2A The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A96C of the present invention and performing the method of the present disclosure in PB buffer (pH 5.0).

[0029] Figure 2B The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A96C of the present invention and performing the method of the present disclosure in PB buffer (pH 7.0).

[0030] Figure 2C The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A96C of the present invention and performing the method of the present disclosure in artificial sweat (pH 5.0).

[0031] Figure 3A The correlation between the lactate concentration and the measured current obtained by performing the method of the present disclosure in PB buffer (pH 5.0) using the mutant lactate oxidase S175C of the present invention is shown.

[0032] Figure 3B The correlation between the lactate concentration and the measured current obtained by performing the method of the present disclosure in PB buffer (pH 7.0) using the mutant lactate oxidase S175C of the present invention is shown.

[0033] Figure 4A The correlation between the lactate concentration and the measured current obtained by performing the method of the present disclosure in PB buffer (pH 5.0) using the mutant lactate oxidase S175C of the present invention is shown.

[0034] Figure 4B The correlation between the lactate concentration and the measured current obtained by performing the method of the present disclosure in PB buffer (pH 7.0) using the mutant lactate oxidase S175C of the present invention is shown.

[0035] Figure 5The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A96C of the present invention and performing the method of the present disclosure in artificial sweat (pH 5.0).

[0036] Figure 6 The line graph depicts a calibration curve of lactate concentration vs. current established by using the mutant lactate oxidase A96C of the present invention and performing the method of the present disclosure in artificial sweat having various pH values ​​(pH 5.0-7.0). Page 5 of 29 (Invention Specification)

[0037] Figure 7 The graph shows the detected current corresponding to a specific lactate concentration (40 mM) in artificial sweat samples with various salinities (0 to 800 mM) and pH values ​​by using the A96C of the present invention. DETAILED DESCRIPTION

[0038] In order to make the description of the content of this disclosure more detailed and complete, the following is an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

[0039] 1. Definition

[0040] For the sake of convenience, specific terms described in this specification, embodiments and the appended patent claims are collectively described here. Unless otherwise defined herein, all technical and scientific terms herein have the same meaning as those commonly known to those skilled in the art to which the present invention belongs.

[0041] As used herein, the singular forms "a," "an," and "the" include plural forms as well, unless the context clearly indicates otherwise.

[0042] Although the numerical ranges and parameters defining the broader scope of the invention are approximations, the numerical values ​​in the specific embodiments are presented as precisely as possible. However, any numerical value inherently contains inevitably the standard deviation resulting from the respective testing methods.

[0043] Typically, the term "wild-type" is used to describe a gene or protein that exists in its natural, non-mutated (unchanged) form. As used herein, the term "wild-type lactate oxidase" refers to a form of lactate oxidase protein that is commonly found in nature (i.e., bacteria) without genetic, structural and / or functional changes. Specifically, the wild-type form of lactate oxidase has a full-length native lactate oxidase of 374 amino acids as shown in SEQ ID NO: 1.

[0044] As used herein, "lactate oxidase variants" are intended to encompass one or more lactate oxidase polypeptide forms derived from wild-type lactate oxidase by substitution, where substitution refers to the replacement of at least one amino acid in the wild-type lactate oxidase sequence with another amino acid. The term "lactate oxidase variant" alternatively or optionally refers to a form of lactate oxidase in which one or more residues are post-translationally modified (PTM) and / or chemically modified to increase the functional diversity of the protein body. Types of PTMs include phosphorylation, methylation, acetylation, ubiquitination, hydroxylation, succinylation, glycosylation, and ubiquitination, but are not limited thereto; and exemplary chemical modifications include, but are not limited to, carboxymethylation. In the present disclosure, the modification occurring on the amino acid is carboxymethylation. The commonly known and commonly used names may be used interchangeably herein to indicate the same mutation occurring on the peptide sequence. According to the present disclosure, for example, substitution from alanine (A) to asparagine (N) at position 95 may be represented by 95A, A95, A95N, or Ala95Asp.

[0045] The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site for a substrate (i.e., lactate and / or lactic acid) and an enzyme (e.g., lactate oxidase or a mutant variant thereof). The affinity of an enzyme for a substrate is generally considered to be related to the Michaelis constant (K m ), which describes the substrate concentration at which half of the enzyme's active sites are occupied by the substrate. m The smaller the size, the stronger the binding affinity for the substrate. Generally, the binding affinity of the mutant form may or may not be changed compared to the wild-type enzyme, depending on the location of the mutation. According to the present disclosure, the lactate oxidase variants of the present invention have a decreased binding affinity for lactate and / or lactic acid compared to the wild-type lactate oxidase.

[0046] The "liquid sample" referred to herein refers to a sample collected and / or obtained from a natural environment or an artificial product, which may or may not contain lactate and / or lactic acid in the form of a liquid, and the solvent is mostly water. Page 7 of 29 (Invention Specification) The liquid sample used in the disclosure may be a biological sample having an organic metabolite (i.e., lactic acid and / or lactate). Examples of biological samples suitable for the present disclosure include mammalian body fluids, preferably human body fluids (e.g., sweat, urine, saliva, blood, and interstitial fluid); and fermented liquids produced by microorganisms (e.g., fermented foods and rancid foods). The liquid sample may contain one or more substances, including but not limited to minerals, trace elements, metal ions and / or heavy metal ions, metabolites, excrement, microplastics, microswimmers, and microorganisms. In addition, the liquid sample has various measurable parameters, including but not limited to pH and salinity.

[0047] 2. Detailed description of preferred embodiments

[0048] The present disclosure is based, at least in part, on the discovery that certain lactate oxidase variants have lower lactate / lactate binding affinity than wild-type lactate oxidase, thereby enabling the detection of concentrated lactate with high sensitivity without being affected by pH or salinity. In addition, in the presence of an electric field, an enzyme (i.e., lactate oxidase) and a substrate (i.e., lactate, lactic acid, or a combination thereof) react to generate an electric current, and the present invention unexpectedly discovered that there is a linear relationship between high concentrations of lactate (e.g., >20 mM) and the current, such that the current can be used as an indicator of lactate detection.

[0049] 2.1 Lactate oxidase variants

[0050] The first aspect of the present disclosure belongs to a lactate oxidase variant, which comprises at least one amino acid mutation, which results in a reduced binding affinity for lactate / lactic acid compared to the wild-type lactate oxidase. The lactate oxidase variant has an amino acid sequence derived from the wild-type lactate oxidase as described in SEQ ID NO: 1, wherein one or more amino acids are substituted 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 replaced by asparagine (N) or glutamine (Q). generation; the alanine (A) at position 96 of sequence number: 1 is replaced by cysteine ​​(C); and / or the serine (S) at position 175 of sequence number: 1 is replaced by cysteine ​​(C).

[0051] According to an embodiment of the present disclosure, the lactate oxidase of the present invention may have an amino acid sequence that is at least 99% identical to that of sequence number: 1, for example, it has a sequence similarity of 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% and 99.9% to sequence number: 1; preferably, it has an amino acid sequence that is at least 99.2% identical to that of sequence number: 1; more preferably, it has an amino acid sequence that is at least 99.8% identical to that of sequence number: 1, and at least one amino acid substitution occurs at position 95, 96 or 175 of sequence number: 1, and the amino acid substitution is selected from the group consisting of A95N, A95Q, A96C, S175C and a combination thereof.

[0052] According to some embodiments of the present disclosure, the lactate oxidase variant of the present invention is called A95N, which may have an amino acid sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the alanine (A) at position 95 of SEQ ID NO: 1 is replaced by asparagine (N). Accordingly, the lactate oxidase A95N variant has the amino acid sequence of SEQ ID NO: 2.

[0053] According to other embodiments of the present disclosure, the lactate oxidase variant named A95Q of the present invention may have an amino acid sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the alanine (A) at position 95 of SEQ ID NO: 1 is replaced by glutamine (Q). Accordingly, the lactate oxidase A95Q variant has the amino acid sequence of SEQ ID NO: 3.

[0054] According to other embodiments of the present disclosure, the lactate oxidase variant of the present invention is called A96C, which may have an amino acid sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the alanine (A) at position 96 of SEQ ID NO: 1 is replaced by cysteine ​​(C). Accordingly, the lactate oxidase A96C variant has the amino acid sequence of SEQ ID NO: 4. Page 9 of 29 (Invention Specification)

[0055] According to yet another embodiment of the present disclosure, the lactate oxidase variant S175C of the present invention may have an amino acid sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the serine (S) at position 175 of SEQ ID NO: 1 is replaced by cysteine ​​(C). Accordingly, 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 known in the art. Genetic methods may include site-specific mutagenesis, polymerase chain reaction (PCR), gene synthesis, CRISPR / cas9 gene editing, and the like of the coding DNA sequence. For example, sequencing can be used to ensure the correct nucleotide changes. The nucleotide sequence of lactate oxidase of natural bacteria (e.g., Aerococcus viridans) can be obtained from public databases such as UniProtKB (Aerococcus viridans ATCC11563; accession code: D4YFm2). The amino acid sequence of wild-type lactate oxidase is shown in sequence number: 1.

[0057] The lactate oxidase variants of the present disclosure can be produced, for example, by solid-state peptide synthesis or recombinant production. For recombinant production, one or more polynucleotides encoding the lactate oxidase variants are each isolated and inserted into a suitable vector for further cloning (screening clones) or expression in a host cell (most often Escherichia coli). Such polynucleotides can be easily isolated and sequenced using conventional procedures. Methods well known to those of ordinary skill in the art can be used to construct expression vectors containing the coding sequence of the lactate oxidase variants of the present invention and suitable transcription / translation regulatory signals. Examples of these methods include, but are not limited to, in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. The expression vector can be a part of a plasmid, a virus, or can be a nucleic acid fragment. Typically, an expression vector is an expression cassette in which a polynucleotide encoding the lactate oxidase variant of the present invention is colonized (transgenic) to be operably associated with a promoter and / or other transcription or translation control components, wherein the promoter and / or other transcription or translation control Page 10 of 29 pages (Invention Specification) The promoter is operably associated with a nucleic acid encoding a polynucleotide, provided that the promoter is capable of affecting the transcription of the nucleic acid. According to some embodiments of the present disclosure, site-directed mutagenesis of natural lactate oxidase is expressed and performed by using pRSET expression vector and conventional tools well known in the art.

[0058] The lactate oxidase variants of the present invention may alternatively or optionally be more susceptible to post-translational modifications (hereinafter referred to as PTMs) and / or chemical modifications, wherein additional functional groups are introduced into their residues. Exemplary post-translational modifications include, but are not limited to, phosphorylation, glycosylation, ubiquitination, s-nitrosylation, methylation, acetylation, hydroxylation, succinylation, and ubiquitinylation. Exemplary chemical modifications include, but are not limited to, carboxymethylation. Depending on actual needs and desired purposes, PTMs and / or chemical modifications may be performed on proteins by any methods and tools known in the art. According to one embodiment of the present disclosure, the lactate oxidase variants of the present invention are carboxymethylated by reacting with iodoacetate (IA) or iodoacetic acid (IAA), wherein iodoacetate or iodoacetic acid covalently binds to the thiol group of cysteine ​​(C), thereby producing a carboxyl-methyl group. In one embodiment, the lactate oxidase variant S175C of the present disclosure is chemically modified by reacting with iodoacetic acid to produce a carboxymethylated cysteine ​​residue. Accordingly, the carboxymethylated lactate oxidase S175C has the amino acid sequence of SEQ ID NO:6.

[0059] In a specific embodiment, the amino acid substitutions / modifications of the wild-type lactate oxidase result in a reduction in the binding affinity of the enzyme (i.e., lactate oxidase) to the substrate (i.e., lactate / lactate) to 50%, such as at least 30%, 25%, 20%, 10%, 7%, 5%, 2% or even 1%. The binding affinity of the lactate oxidase variants of the present invention to their substrates can be measured by various methods known in the art, such as colorimetric assays, wherein the kinetic parameters of each lactate oxidase variant (e.g., K of the Michaelis-Menten equation) are measured. m and V max ) can be determined by following well-established procedures known in the art. Page 11 of 29 pages (Invention Specification)

[0060] 2.2 Methods for detecting and quantifying lactic acid

[0061] Another aspect of the present disclosure is a method for detecting and quantifying lactic acid and / or lactate in a liquid sample. The method comprises at least the following steps: (a) contacting the liquid sample with the lactate oxidase variant of the present invention as described in the preceding paragraph; (b) measuring the current generated by the reaction between the lactate oxidase variant of the present invention and lactic acid in the liquid sample; and (c) determining the lactate concentration of the liquid sample by interpolating or extrapolating the current measured in step (b) with the current of a control sample having a known lactate concentration.

[0062] According to the present disclosure, the current generated by the reaction between the lactate oxidase variant of the present invention and the lactic acid in the liquid sample in step (a) can be measured with the aid of an electrode system. In view of this, before starting the method of the present invention, it is best to construct a standard calibration curve for lactic acid detection by measuring the current generated between lactate oxidase and various known concentrations of lactic acid. The structure of the electrode system itself usually includes a working electrode and a counter electrode, and optionally a reference electrode. Exemplary materials suitable for constructing the working electrode and / or the counter electrode include, but are not limited to: carbon (e.g., pyrolytic carbon, graphite, graphene, glass graphite, carbon paste, perfluorocarbon (PFC) or the like) and metal (e.g., platinum, gold, silver, nickel, palladium or the like). In addition, an exemplary reference electrode can be a saturated calomel electrode or a silver / silver chloride electrode. The electrode system can be made of any of the specified materials listed above by methods well known in the art, such as photolithography vapor deposition, sputtering or printing (e.g., screen printing, gravure printing, quick-dry printing, etc.). In one specific embodiment, the electrode system of the present disclosure is a screen-printed carbon electrode (hereinafter referred to as SPCE) made of graphite and graphene. Page 12 of 29 (Invention Specification)

[0063] For the purpose of detecting lactic acid, the lactate oxidase variant of the present invention is deposited on the surface of the aforementioned electrode system (e.g., SPCE) in the presence of a redox mediator. According to a specific embodiment, the lactate oxidase variant of the present invention and the redox mediator are mixed in a specified ratio to form a mixture, which is then immobilized on the surface of the SPCE by electrodeposition or drop-casting, thereby preparing a lactate sensor suitable for the method of the present invention.

[0064] Examples of redox mediators suitable for use in the present invention include, but are not limited to, poly(aniline)-poly(acrylate), poly(aniline)-poly(vinyl sulfonic acid), poly(pyrrole), poly(pyrrole)-poly(vinyl sulfonic acid), poly(vinyl pyrrolidone), poly(1-vinylimidazole), ferrocyanide salts, ferrocyanide salts, cobalt phthalocyanine, hydroxymethylferrosin, osmium (Os) complex, [7-(dimethylamino)-4-nitrophenothiazin-3-ylidene]-dimethylammonium chloride, benzo[a]phenol -9-ylidene (dimethyl) ammonium, tetrathiafulvene and copolymers or combinations thereof. In a specific embodiment, the redox mediator is a copolymer of poly (1-vinylimidazole) and an osmium complex (PVImQOs); in other specific embodiments, the redox mediator is potassium ferrocyanide (K3[Fe(CN)6]).

[0065] To establish a calibration curve, a control sample with a known concentration of lactic acid or lactate can be contacted with a lactate sensor to which a fixed potential is applied, so that the current generated by the electrochemical reaction between the lactate oxidase variant of the present invention and lactic acid fixed on the electrode can be detected by any conventional means in the art, in particular, by an electrochemical analyzer. Accordingly, a standard calibration curve can be established based on the measured current and corresponding to the known lactate concentration. In some embodiments of the present disclosure, the known lactate concentration ranges 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, .6, 74.1, 82.6, 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 one embodiment, the calibration curve is constructed with lactate 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, 90.9, 130.4 and 166.7 mM. In other embodiments, the calibration curve is constructed with lactic acid 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 still other embodiments, the calibration curve is constructed with lactic acid 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 still other embodiments, the calibration curve is constructed with lactic acid 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 is a simulated human sweat; preferably, the control sample used in the method of the present invention is artificial sweat prepared according to international standards. Alternatively or optionally, the control sample used in the method of the present invention specifically mimics the pH, osmotic pressure and ion concentration of human body fluids; preferably the control sample is a phosphate buffer.

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

[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 a specific embodiment, the liquid sample has a pH value of 5.0, 6.0 or 7.0. According to an alternative embodiment of the present disclosure, the liquid sample has a salinity of 0 to 1000 mM; for example, page 14 of 29 (invention specification) For example, 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM salinity. In other specific embodiments, the salinity of the liquid sample is 0, 100, 200, 300, 400, 500, 600, 700 or 800 mM. Liquid samples suitable for use in the present invention may be derived from natural environments (e.g., animal bodies) or man-made products (e.g., food). Examples of liquid samples suitable for the methods of the present invention include, but are not limited to, liquid, urine, saliva, blood, interstitial fluid, and fermentation broth produced by microorganisms. In specific embodiments, the liquid sample is sweat.

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

[0069] In summary, the method of the present invention comprises at least the aforementioned steps (a) to (c), wherein the method of the present invention is capable of detecting lactic acid in any liquid sample having a trace amount or a large amount of lactic acid. According to the present disclosure, the method of the present invention can detect lactic acid ranging from 0 to 300mM; for example, from 0 to 280mM, from 0 to 260mM, from 0 to 250mM, from 0 to 200mM, from 0 to 150mM, from 0 to 130mM, from 0 to 120mM, from 0 to 110mM, from 0 to 100mM, from 0.2 to 5mM, from 0.2 to 10mM, from 0.2 to 166mM, from 0.2 to 180mM, from 0.2 to 260mM, from 0.5 to 90mM, from 0.5 to 100mM, from 0.5 to 110mM, from 0.5 to 120mM or from 5 to 100mM. In certain preferred embodiments, the present invention can detect lactic acid in a concentration exceeding 100mM in a liquid sample.

[0070] By virtue of the above-mentioned features, the method of the present disclosure can detect and quantify the concentration of lactic acid, especially for high amounts of lactic acid in a liquid environment that cannot be measured by conventional detection methods. In addition, the present disclosure, page 15 of 29 pages (invention specification) The method is capable of detecting lactic acid in aqueous samples containing a variety of substances and can therefore be applied to a variety of fluid sources.

[0071] Example

[0072] Materials and Methods

[0073] Gene synthesis and mutagenesis

[0074] Escherichia coli BL21 (DE3) was transfected with expression vectors expressing wild-type and mutant lactate oxidase, respectively. The lactate oxidase gene was cloned (screened and cloned) based on the wild-type sequence of Aerococcus viridans (American Type Culture Collection, ATCC) No. 11563 strain (Accession No.: D4YFm2) retrieved from the database UniProtKB, wherein the wild-type sequence was inserted into the multiple cloning (screening and cloning) site of the expression vector pRSET, and the codons preferentially used by Escherichia coli were used. Site-directed mutagenesis was performed using a commercially available mutagenesis induction kit (Quikchange, Agilent, US) and the routine procedures provided therein with the primer pairs listed in Table 1. The plastid sequence was confirmed using a DNA analyzer (Model: 3730XL; Supplier: Thermo Fisher Scientific, USA).

[0075] Table 1: Primer sequences

[0076] Preparation of wild-type and mutant lactate oxidase of the present invention

[0077] E. coli BL21 (hereinafter referred to as DE3) was transfected with plasmids containing wild-type and lactate oxidase variants, respectively. The transfected DE3 was inoculated into LB culture medium containing ampicillin and cultured in a shaking incubator (250 rpm) at 37°C for 16 to 20 hours. 1% of the bacterial culture was taken out and inoculated into the main culture medium: 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. The culture medium was then cultured at 37°C with shaking for 8 hours. The supernatant was removed by centrifugation (4°C, 5000g) for 20 minutes, and the cell pellet was collected and stored in a -20°C environment.

[0078] Purification of wild-type and mutant lactate oxidase of the present invention

[0079] E coli cells transfected with wild-type or mutant lactate oxidase were resuspended in 50 mL of potassium phosphate buffer (50 mM) (PPB, pH 7.0) containing 500 mM NaCl and 20 mM imidazole, and treated with a homogenizer (NanoLyzer). The obtained cell fluid was centrifuged at 10,000 g for 1 hour at 4°C, and the supernatant was collected. The collected supernatant, containing the recombinant histidine-tagged (His-tag) protein, was used as a crude cell extract and purified by a nickel affinity column and a protein purification kit (supplier: start, Cytiva). The column was eluted with five volumes of buffer A (pH 7.0, 20 mM imidazole, 500 mM NaCl and 50 mM PPB), the crude cell extract was loaded into the column, washed with buffer A (3 volumes), then eluted with 20 volumes of 4 to 100% buffer B (pH 7.0, 500 mM imidazole, 500 mM NaCl and 50 mM PPB), and finally 20 fractions were collected. After SDS-PAGE analysis, the fractions containing the target protein were dialyzed and concentrated using a dialysis membrane (10 kDa). After replacement with 50 mM Tri-HCl solution (pH 7.0), the purified enzyme solution was lyophilized and stored at -20°C.

[0080] Carboxylmethylation of mutant lactate oxidase

[0081] The purified lactate oxidase variant solution (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] Determination of protein concentration

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

[0085] Protein activity assay

[0086] 1. Lactic acid oxidation activity

[0087] The diluted enzyme was added to the microplate at 50 μL per well, followed by a mixture of 0.25 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) and 5 U / mL horseradish peroxidase (HRP) and 50 μL 10 mM lactic acid to each well, and the mixture was allowed to react at 30°C with intermittent shaking for five minutes. Then 50 μL 1M HCl was added to block the reaction, and the molar attenuation coefficient was set to 59 cm -1 ·mmol -1 , the absorbance of the solution was measured at 450 nm wavelength (OD). The activity of lactate oxidase at 30°C, pH 7.0 (i.e., 1 U) was calculated using the following equation: Activity (U / mL) = [ΔO.D. (OD 测试 -OD 空白 )×Vt×df] / (ε×t×l×Vs), where Vt is the total volume, df is the dilution rate, ε is the volumetric molar attenuation coefficient, t is the reaction time, l is the optical path length (cm) and Vs is the enzyme volume.

[0088] 2. Dehydrogenase activity

[0089] Mix caffeine in potassium phosphate buffer (PPB, 20 mM, pH 7.0). Phenazinemethosulfate (PMS, 0.5 mM) and 2,6-dichlorophenol-indophenol (DCIP, 1.2 mM) were mixed to produce a reaction reagent. Diluted enzyme (50 μL dissolved in 50 mM PPB, pH 7.0) was added to the microplate at 50 μL per well, and the reaction reagent (50 μL) and 10 mM lactic acid (50 μL) were added to each well in sequence, followed by intermittent shaking of the microplate at 30°C for five minutes. The molar attenuation coefficient was set to 16.3 cm -1 ·mmol -1 The absorbance (OD) of each well was measured at a wavelength of 600 nm. The activity of lactate dehydrogenase (i.e., 1 U) at 30°C and pH 7.0 was calculated using the following equation: Activity (U / mL) = [ΔO.D. (OD 测试 -OD 空白 )×Vt×df] / (ε×t×l×Vs), where Vt is the total volume, df is the dilution rate, ε is the volumetric molar attenuation coefficient, t is the reaction time, l is the optical path length (cm), and Vs is the enzyme volume.

[0090] 3. Binding affinity

[0091] The enzyme kinetic parameter K of the wild-type or mutant lactate oxidase was determined by reacting the lactate oxidase variants of the present invention and the wild-type lactate oxidase at pH 5.0 or pH 7.0 with different concentrations of lactate (0.1 to 200 mM oxidase; 0.1 to 900 mM dehydrogenase). m , K cat and V max , thereby determining the reaction rate based on the slope of the linear graph of time (i.e., reaction interval) and the absorbance (OD) at 450 or 600 nm detected every 3 seconds during the reaction interval. K was obtained by plotting a calibration curve between lactate concentration (on the x-axis) and reaction rate (on the y-axis) under the Michaelis equation. m , K cat and V max value.

[0092] Preparation of lactate sensor with modified electrode

[0093] Cyclic voltammetry (CV) was performed using a screen-printed carbon electrode (hereinafter referred to as SPCE, supplier: TE100, Zensor) as a three-electrode system, wherein the enzyme of the present invention was immobilized on the surface of the working electrode by electrodeposition or drop coating as described above.

[0094] Electrodeposition

[0095] The outer 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 enzyme solution (4U), either wild-type lactate oxidase or lactate oxidase variants of the present invention (i.e., A95N, A95Q, A96C, S175C or modified S175C). The SPCE was subjected to cyclic voltammetry for 50 cycles at a preset potential of -1.0 V to 0.0 V and a default scan rate of 200 mV / s at 37°C to achieve surface modification. The modified SPCE was placed in a PPB solution (pH 5.0, 100 mM) and cyclic voltammetry was performed again for 20 cycles at a second default potential between -300 mV and 600 mV and a default scan rate of 60 mV / s to remove residual PVImQOs. After drying at room temperature, 4 μL of a protective agent containing 1% polyglucosamine and 0.075% genipin was further added to the modified SPCE.

[0096] Drip coating method i. Add a mixed solution of enzyme solution (1 to 8 U / μL), potassium ferrocyanide (K3[Fe(CN)6], 25 to 50 mM) and 0.1% Triton X-100 (1 μL) dropwise to cover the electrode surface and dry at room temperature. ii. Alternatively, 5 μL of PVImQOs (10 mg / ml) and 2.5 μL of enzyme solution were separately and sequentially dropped onto the electrode area of ​​the SPCE. After drying at 4°C, a protective agent (4 μL) containing 1% polyglucosamine and 0.075% genipin was further added to the surface of the SPCE. The modified SPCE was dried and stored at room temperature.

[0097] Electrochemical evaluation

[0098] The SPCE was connected to an electrochemical analyzer (ACIP100) with an ECP100 (supplier: Zensor) cable connector and a CS100 electrode lift. The voltammogram was recorded and analyzed by the built-in ACIP100 software.

[0099] Lactate testing

[0100] Sample preparation

[0101] Artificial sweat was prepared according to the recipe of international standard ISO 3160-2, and the pH value was set to 5, 6 or 7 by adjusting the content of sodium hydroxide. The potassium phosphate buffer (0.1 M) used in the present invention and its pH value can be prepared by adjusting the amount of monobasic and dibasic potassium phosphate (obtained from suppliers).

[0102] Create a calibration curve

[0103] Different concentrations of lactic acid solutions (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 lactic acid in artificial sweat or phosphate buffer) were applied to the enzyme-immobilized lactate sensor, and the current generated between the electrodes at a fixed potential of 300 or 400 mV was measured. The current value of the reaction between the blank sample (i.e., 0 mM lactate) and the enzyme (i.e., the mutant lactate oxidase of the present invention) at the 60th second was recorded as the background value, and then the buffer solution containing the lactate concentration was added to the lactate sensor. Each reaction lasted for about 20 to 60 seconds, and the current of each reaction was recorded at 5 or 10 seconds to establish a calibration curve of current vs. lactate concentration.

[0104] Example 1: Preparation of lactate oxidase variants of the present invention

[0105] Five lactate oxidase variants of the present invention were generated by the method described in the above section "Materials and Methods". The generated lactate oxidase variants and their amino acid sequences are shown in Table 2, wherein the designated amino acid substitutions and modifications are indicated in bold.

[0106] Table 2: Lactate oxidase variants of the present invention *: Cysteine ​​modified with carboxymethyl group

[0107] Example 2: Characteristic Analysis of Lactate Oxidase Variants of the Present Invention

[0108] 2.1 Oxidase and dehydrogenase activities

[0109] This experiment mainly analyzes the oxidation and dehydrogenase activities of the lactate oxidase variants of the present invention. To this end, the wild-type and mutant lactate oxidases of the present invention were mixed with lactic acid (10 mM) and reacted, and the activities of these proteins were calculated according to the process described in the "Materials and Methods" section. The quantitative results are listed in Table 3.

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

[0111] The data in Table 3 show that compared with the wild-type protein, the oxidation and dehydrogenase activities of the mutant lactate oxidase are significantly reduced, especially the oxidase activity or dehydrogenase activity of the A95Q variant, which is 98.4% and 95% less than that of the wild-type lactate oxidase, respectively.

[0112] 2.2 Enzyme kinetics

[0113] This experiment evaluated whether the binding affinity of lactate oxidase variants was affected by pH. To this end, the lactate oxidase variants of the present invention were reacted with different concentrations of lactic acid at pH 5.0 or pH 7.0, and the K of the enzyme was determined according to the procedure described in "Materials and Methods". m and K cat Value. K m and K cat The values ​​are listed in Table 4.

[0114] Table 4: K values ​​of lactate oxidase variants and wild-type (WT) enzyme of the present invention m and K cat value *ND: Not Measured

[0115] As a result, it was found that regardless of whether the pH value was changed (i.e., pH 5.0 or pH 7.0), the K of the lactate oxidase variants of the present invention was m The values ​​are significantly higher than those of the wild-type enzyme, indicating that each lactate oxidase variant of the present invention has a lower lactate binding affinity.

[0116] Example 3: Lactate Detection by Using the Lactate Oxidase Variants of the Present Invention

[0117] This experiment evaluates the sensitivity and versatility of the lactate oxidase variants of the present invention for lactate detection in different samples. To this end, two lactate sensors (hereinafter referred to as LS-I and LS-II) were constructed based on different redox media immobilized on SPCE according to the process described in "Materials and Methods". Specifically, LS-I was prepared by depositing a mixture of an enzyme solution (i.e., lactate oxidase variants A95Q, A96C, S175C and carboxymethylated S175C of the present invention) and a polymer medium on the surface of SPCE; while LS-II was prepared by immobilizing the enzyme and potassium ferricyanide (K3[Fe(CN)6]) on the surface of SPCE. LS-I and LS-II were used to detect lactate in designated samples, respectively.

[0118] 3.1 Binding affinity

[0119] This experiment evaluated whether the binding affinity of the lactate oxidase variants of the present invention is affected by pH. To this end, the reaction of lactate (of various concentrations) with the lactate oxidase variants of the present invention at pH 5.0 or pH 7.0 was measured by the lactate sensor LS-I. The Km and Vmax values ​​of the enzyme (i.e., the lactate oxidase variants of the present invention) were determined based on the procedures described in the "Materials and Methods" section, and the results are summarized in Table 5.

[0120] Table 5: Km and Vmax values ​​of wild type (WT) and mutant lactate oxidase of the present invention **Cysteine ​​at position 175 modified with a carboxymethyl group

[0121] As summarized in Table 5, the lactate oxidase variants of the present invention have a higher K than the wild-type enzyme. m The data in Table 5 are consistent with the evaluation in Example 2, both indicating that the binding affinity of the lactate oxidase variants of the present invention to lactate is lower than that of the wild type, especially in a lower pH environment.

[0122] 3.2 Lactate Detection Using the Lactate Oxidase Variants of the Present Invention

[0123] 3.2.1A95Q, A96C or S175C

[0124] This experiment investigated the detection efficiency and limitations of the mutant lactate oxidase A95Q, A96C and S175C of the present invention. To this end, the mutant lactate oxidase A95Q, A96C or S175C of the present invention was immobilized on the surface of SPCE with the mediator PVImQOs, thereby manufacturing the first type of lactate sensor (hereinafter referred to as A95Q-LS-I, A96C-LS-I and S175C-LS-I). Then, phosphate buffer (pH 5 and pH 7) or artificial sweat (pH 5) containing different lactate concentrations (0-300mM) was applied to each lactate sensor, and the current generated was measured and recorded according to the process described in the "Materials and Methods" section. The results are presented in Figures 1 to 3.

[0125] The results showed that the A95Q enzyme of the present invention could successfully detect different concentrations of lactic acid regardless of the pH value of the buffer, and the minimum and maximum concentrations were approximately 0.2 mM and 166 mM, respectively. Figure 1A and 1B The results respectively indicate that the lactate oxidase A95Q variant of the present invention can detect a wide range of lactic acid in liquid samples.

[0126] As for A96C, it was found that both at pH 5.0 ( Figure 2A ) or pH 7.0( Figure 2B ) buffer, A96C can detect lactate concentrations ranging from about 0.5mM to 90mM. Consistent results were also observed in the detection of lactate in artificial sweat (pH 5.0) ( Figure 2C ). In artificial sweat, the detectable lactic acid concentration ranges from 0.5 mM to 90 mM, which indicates that the lactate oxidase A96C of the present invention is capable of detecting lactic acid in a simulated biological fluid environment.

[0127] As for S175C, it was found that trace lactate concentrations (i.e., 0.2 mM to 10 mM) could also be detected by the lactate oxidase variant S175C of the present invention, regardless of the pH value of the buffer ( Figure 3A and Figure 3B These results indicate that the lactate oxidase variant S175C of the present invention can detect quantitative small-scale (eg, less than 10 mM in the present invention) lactate in aqueous samples without being affected by different pH values.

[0128] 3.2.2 Carboxymethylated S175C

[0129] In this experiment, the S175C variant was reacted with 0.1 M iodoacetic acid (IAA) to produce carboxymethylated S175C and its effect on lactate detection was evaluated. The results are described in Figure 4A and 4B .

[0130] As shown in the figure, carboxymethylation significantly improves the detection range of lactate by the S175C variant compared to the control variant. Specifically, the maximum concentration of lactate that can be detected by the carboxymethylated variant in acidic buffer (pH 5.0) and neutral buffer (pH 7.0) is 166.7 mM and 259.3 mM, respectively.

[0131] 3.3 Universality of the lactate oxidase variants of the present invention for detecting lactate

[0132] This experiment evaluates the versatility of the lactate oxidase variants of the present invention by varying the amount of enzyme immobilized on the detection electrode, the pH value and / or salinity of the buffer. The results are presented in Figures 5 to 7 .

[0133] The results showed that the maximum lactate amount that could be detected by the sensor fixed with 1, 1.5 or 2 units (U) of A96C exceeded 100 mM. In particular, the lactate sensor fixed with 1.5 U of A96C (i.e., A96C-LS-II-1.5U) showed the strongest detection ability and could detect the lactate concentration of artificial sweat up to 120 mM ( Figure 5 ). In addition, page 28 of 29 (Invention Specification) The sensor with 1 unit of A96C immobilized (ie, A96C-LS-II-1U) exhibited the highest resolution and smaller variability, indicating that only a small amount of the mutant enzyme was required to achieve the desired detection efficiency.

[0134] As for the effect of pH value on lactic acid detection, the experiment found that the mutant lactate oxidase A96C of the present invention can detect lactic acid in the range of 0 to 200 mM in artificial sweat samples ( Figure 6 ).

[0135] As for the effect of salinity, it was found that the current generated by the reaction of the lactate oxidase A96C variant of the present invention with lactic acid had only a very small difference at different pH values ​​and / or salinities ( Figure 7 ). Figure 7 The data presented demonstrate that the detection performance of lactate oxidase A96C of the present invention is not affected or interfered by the salinity of artificial sweat.

[0136] In summary, the results of Examples 1 to 3 collectively indicate that the lactate oxidase variants of the present invention have improved lactate detection performance and are not affected by the pH value and / or salinity of the liquid sample, thereby achieving a more universal lactate detection.

[0137] It should be understood that the foregoing description of the embodiments is given only by way of example, and various modifications may be made by those with ordinary knowledge in the art to which the present invention belongs. The above description, examples and experimental results provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the art to which the present invention belongs may make various changes and modifications to the same without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the accompanying patent application.

Claims

1. A lactate oxidase variant derived from wild-type lactate oxidase (SEQ ID NO: 1), wherein the lactate oxidase variant has at least one amino acid substitution at position 95, 96 or 175 of SEQ ID NO: 1, or a combination of these positions, wherein The sequence numbering: 1 Alanine (A) at position 95 is replaced by asparagine (N) or glutamine (Q); The sequence number: 1 the alanine (A) at position 96 is replaced by cysteine ​​(C); and / or The serine (S) at position 175 of the sequence number: 1 is replaced by cysteine ​​(C).

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

3. The lactate oxidase variant according to claim 1, wherein the alanine (A) at position 95 of SEQ ID NO: 1 is substituted by glutamine (Q).

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

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

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

7. The lactate oxidase variant of claim 1, wherein the lactate oxidase variant has an amino acid sequence of sequence number: 2, 3, 4, 5 or 6.

8. The lactate oxidase variant of claim 1, wherein the lactate oxidase variant has a binding affinity for lactate that is lower than the binding affinity of the wild-type lactate oxidase for lactate.

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

10. The method of claim 9, wherein the pH value of the liquid sample is between 4 and 9.

11. The method of claim 9, wherein the salinity of the liquid sample is between 0 and 1,000 mM.

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

13. The method of claim 9, wherein the method can detect lactic acid in a liquid sample at a concentration between 0 and 300 mM.

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