TWI867923B - Lactate oxidase variants and their uses for lactate detection - Google Patents

Abstract

Disclosed herein is directed to a lactate oxidase variant that exhibits reduced affinity to lactate as compared to a wild-type lactate oxidase. The lactate oxidase variant has one or more amino acid substitutions occurring at positions 95, 96, and/or 175 of the wild-type amino acid sequence. Also disclosed herein is a method for detecting and quantifying lactate in various liquid samples by using the present lactate oxidase variant. The method mainly includes steps of contacting the liquid sample with said lactate oxidase variant; measuring a current generated by the reaction between the lactate oxidase variant and the lactate in the liquid sample; and determining the concentration of lactate in the liquid sample by interpolating or extrapolating the current with that of a control sample having a known concentration of lactate.

Description

Lactate oxidase variants and their use in lactate detection

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.

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

In clinical diagnosis, the accumulation of lactic acid concentration is caused by continuous anaerobic metabolism, which inevitably leads to lactic acidosis and becomes one of the symptoms of severe sepsis. Therefore, the patient's blood lactate level can be used as a warning 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 exercise training status and physical fitness.

Lactic acid is also found in the food and fermentation industries. Lactic acid is produced during the food fermentation process 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 cured vegetables. In this way, lactic acid can be used as an indicator of food freshness and quality.

A variety of methods have been 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 aforementioned limitations, portable and disposable biosensors are currently being developed. In general, biosensing methods have the advantages of being simple, direct, fast-response, high specificity, economical, and easy to use. Ideally, the lactate concentration of biological fluids (such as whole blood, sweat, and saliva) can be measured by a biosensor; however, the biosensors currently available on the market are only suitable for blood lactate detection, which requires invasive sample collection methods, is not user-friendly, and cannot be used to detect samples other than blood.

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 80 mM after exercise, while the amount of lactate in blood only rises to 25 mM 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 detectors to detect sweat lactate concentration.

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 in human body fluids in a non-invasive and effective manner.

In order to provide readers with a basic understanding, a brief summary of the present disclosure is provided below. This disclosure is not an extensive overview of the present disclosure, nor is it intended to identify key/essential elements of the present disclosure or to outline the scope of the present disclosure. Its sole purpose is to present some concepts of the present disclosure in a simplified conceptual form as a preface to the more detailed description presented later.

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 SEQ ID NO: 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 SEQ ID NO: 1, or a combination thereof. In the lactate oxidase variant of the present disclosure, alanine (A) at position 95 of SEQ ID NO: 1 is substituted with aspartate (N) or glutamine (Q); alanine (A) at position 96 of SEQ ID NO: 1 is substituted with cysteine (C); and/or serine (S) at position 175 of SEQ ID NO: 1 is substituted with cysteine (C).

According to one embodiment of the present disclosure, the lactate oxidase variant comprises an amino acid sequence of SEQ ID NO: 1, wherein alanine (A) at position 95 of the sequence is substituted by aspartic acid (N).

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

According to another embodiment of the present disclosure, the alanine (A) at position 96 of SEQ ID NO: 1 is replaced by cysteine (C).

According to yet another embodiment of the present disclosure, the serine (S) at position 175 of SEQ ID NO: 1 is substituted by cysteine (C).

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

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

In some embodiments, the present disclosure provides that the lactate oxidase variant has a binding affinity for lactate that is lower than the binding affinity of wild-type lactate oxidase for lactate.

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 aforementioned lactate oxidase variant with a liquid sample; step (b): measuring the current generated by the reaction between the aforementioned 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.

According to some embodiments, the liquid sample has a pH value ranging from 4 to 9.

According to some embodiments, the liquid sample has a salinity between 0 and 1000 mM.

In certain preferred embodiments, the liquid sample is sweat.

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

After reading the following implementation methods, a person with 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.

In order to make the description of the disclosure more detailed and complete, the following provides 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 their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

1. Definition

For the convenience of explanation, the specific terms recorded in this specification, embodiments and the attached patent application are collectively explained here. Unless otherwise defined herein, all technical and scientific terms herein have the same meaning as the terms commonly known to those skilled in the art to which the present invention belongs.

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

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods.

Typically, the term "wild-type" is used to describe a gene or protein that exists in its natural, non-mutated (unaltered) 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 the full-length native lactate oxidase of 374 amino acids as shown in SEQ ID NO: 1.

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-translational modified (PTM) and/or chemically modified to increase the functional diversity of the proteosome. Types of PTMs include, but are not limited to, phosphorylation, methylation, acetylation, ubiquitination, hydroxylation, succinylation, glycosylation, and ubiquitination; and exemplary chemical modifications include, but are not limited to, carboxymethylation. In the present disclosure, the modification occurring on the amino acid is carboxymethylation. Commonly known and commonly used names are used interchangeably herein to refer to the same mutation occurring in a peptide sequence. For example, a substitution from alanine (A) to aspartate (N) at position 95 may be represented by 95A, A95, A95N, or Ala95Asp according to the present disclosure.

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 mutant variant thereof). The affinity of an enzyme for a substrate is generally considered to be related to the Michaelis constant ( _Km ), which describes the substrate concentration at which half of the active sites of the enzyme are occupied by the substrate. The smaller the _Km , the stronger the binding affinity for the substrate. Generally, the binding affinity of a mutant form may or may not be altered compared to the wild-type enzyme, depending on the location at which the mutation occurs. 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.

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. The liquid sample used in the present disclosure may be a biological sample with an organism's metabolites (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 fluids); and fermented liquids produced by microorganisms (e.g., fermented foods and sour 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. Additionally, liquid samples have various measurable parameters, including but not limited to pH and salinity.

2. Specific implementation methods

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 being able to detect concentrated lactate with high sensitivity without being affected by pH or salinity. In addition, in the presence of an electric field, the enzyme (i.e., lactate oxidase) and substrate (i.e., lactate, lactate, or a combination thereof) react to generate an electric current, and the present invention surprisingly found that there is a linear relationship between high concentrations of lactate (e.g., >20 mM) and the current, so that the current can be used as an indicator of lactate detection.

2.1 Lactate oxidase variants

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 at positions 95, 96 and/or 175 of SEQ ID NO: 1 are substituted. Specifically, the alanine (A) at position 95 of SEQ ID NO: 1 is substituted by asparagine (N) or glutamine (Q); the alanine (A) at position 96 of SEQ ID NO: 1 is substituted by cysteine (C); and/or the serine (S) at position 175 of SEQ ID NO: 1 is substituted by cysteine (C).

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

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 aspartic acid (N). Accordingly, the lactate oxidase A95N variant has the amino acid sequence of SEQ ID NO: 2.

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 substituted with glutamine (Q). Accordingly, the lactate oxidase A95Q variant has the amino acid sequence of SEQ ID NO: 3.

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.

According to 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.

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 from natural bacteria (e.g., Aerococcus viridans ) can be obtained from public databases such as UniProtKB (Aerococcus viridans ATCC 11563; accession code: D4YFm2). The amino acid sequence of wild-type lactate oxidase is shown in sequence number: 1.

The lactate oxidase variants disclosed herein 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 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 appropriate 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, the expression vector is an expression cassette in which the polynucleotide encoding the lactate oxidase variant of the present invention is transferred to a promoter and/or other transcriptional or translational control elements that are operably associated with the nucleic acid encoding the 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 the pRSET expression vector and conventional tools well known in the art.

The lactate oxidase variants of the present invention may alternatively or optionally be subjected 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 ubiquitination. Exemplary chemical modifications include, but are not limited to, carboxymethylation. PTMs and/or chemical modifications may be performed on proteins by any method and tool known in the art, depending on actual needs and desired purposes. According to an embodiment of the present disclosure, the lactate oxidase variant of the present invention is carboxymethylated by reacting with iodoacetate (IA) or iodoacetic acid (IAA), wherein iodoacetate or iodoacetic acid covalently binds to the thiol group of cysteine (C) to generate a carboxyl-methyl group. In a specific embodiment, the lactate oxidase variant S175C of the present disclosure is chemically modified by reacting with iodoacetic acid to generate a carboxymethylated cysteine residue. Accordingly, the carboxymethylated lactate oxidase S175C has the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the amino acid substitutions and modifications made to the wild-type lactate oxidase result in a reduction in the binding affinity of the enzyme (i.e., lactate oxidase) to a substrate (i.e., lactate/lactic acid) 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., _Km and _Vmax of the Michaelis-Menten equation) can be determined by following well-established procedures known in the art.

2.2 Method for detecting and quantifying lactate

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 previous 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 with a known lactate concentration.

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 by means 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 lactate 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 a working electrode and/or a 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 may be a saturated calomel electrode or a silver/silver chloride electrode. The electrode system may be made with any of the above-listed specified materials 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 a 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.

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 medium. According to a specific embodiment, the lactate oxidase variant of the present invention and the redox medium are mixed in a specified ratio to form a mixture, which is then fixed on the surface of the SPCE by electrodeposition or drop-casting, thereby producing a lactate sensor suitable for the method of the present invention.

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 sulfonate), poly(pyrrole), poly(pyrrole)-poly(vinyl sulfonate), poly(vinylpyrrolidone), poly(1-vinylimidazole, PVIm), ferricyanide salts, ferrocyanide salts, cobalt phthalocyanine, hydroxymethyl ferrocyanide, ferrocene), zirconium (Os) complex, [7-(dimethylamino)-4-nitrophenothiazin-3-ylidene]-dimethylazanium chloride, benzo[a]phenoxazin-9-ylidene(dimethyl)azanium, tetrathiafulvalene, and copolymers or combinations thereof. In a specific embodiment, the redox medium is a copolymer of poly(1-vinylimidazole) and a zirconium complex (PVImQOs); in other specific embodiments, the redox medium is potassium ferricyanide (K ₃ [Fe(CN) ₆ ]).

To establish a calibration curve, a control sample with a known concentration of lactic acid or lactate salt can be brought into contact 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 fixed on the electrode and lactic acid can be detected by any means known 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 lactic acid concentration. In some embodiments of the present disclosure, the known lactic acid 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, 50, 56.6, 60, 65. 4, 70, 74.1, 80, 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 a specific 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 lactate 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 lactate 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 lactate concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, and 200 mM. According to the implementation mode of the present disclosure, the control sample is a simulation of 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 simulates the pH value, osmotic pressure and ion concentration of human body fluids; preferably, the control sample is a phosphate buffer.

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 electrochemical analyzer mentioned above.

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 embodiment, the liquid sample has a pH 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, 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM. In other 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 can be derived from the natural environment (e.g., an animal body) or a man-made product (e.g., a food). Examples of liquid samples suitable for the method of the present invention include, but are not limited to, liquid, urine, saliva, blood, interstitial fluid, and fermentation fluid produced by microorganisms. In a specific embodiment, the liquid sample is sweat.

In the last 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) to obtain the lactic acid concentration of the liquid sample.

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 lactate in a range from 0 to 300 mM; for example, from 0 to 280 mM, from 0 to 260 mM, from 0 to 250 mM, from 0 to 200 mM, from 0 to 150 mM, from 0 to 130 mM, from 0 to 120 mM, from 0 to 110 mM, from 0 to 100 mM, from 0.2 to 5 mM, from 0.2 to 10 mM, from 0.2 to 166 mM, from 0.2 to 180 mM, from 0.2 to 260 mM, from 0.5 to 90 mM, from 0.5 to 100 mM, from 0.5 to 110 mM, from 0.5 to 120 mM or from 5 to 100 mM. In certain preferred embodiments, the present invention is capable of detecting lactic acid at a concentration exceeding 100 mM in a liquid sample.

By virtue of the above characteristics, the disclosed method can detect and quantify lactic acid concentration, especially for high amounts of lactic acid in liquid environments that cannot be detected by conventional detection methods. In addition, the disclosed method can detect lactic acid in aqueous samples containing a variety of substances, and thus can be applied to a variety of liquid sources.

Embodiment

Materials and methods

Gene synthesis and mutation induction

Escherichia coli BL21 (DE3) was transformed with expression vectors expressing wild-type and mutant lactate oxidase, respectively. The lactate oxidase gene was cloned based on the wild-type sequence of Aerococcus viridans (American Type Culture Collection, ATCC) strain No. 11563 (Accession No.: D4YFm2) retrieved from the database UniProtKB, wherein the wild-type sequence was inserted into the multiple 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 mutation induction kit (Quikchange, Agilent, US) and the routine protocol provided therein with the primer pairs listed in Table 1. The plasmid sequence was confirmed using a DNA analyzer (Model: 3730XL; Supplier: Thermo Fisher Scientific, USA).

Table 1: Primer sequences Introduction name Nucleotide sequence (5'→3') Serial Number A95N-F CCGTTTATTATGGCACCGATTAACGCACATGGTCTGGCACATAC 7 A95N-R GTATGTGCCAGACATGTGCGTTAATCGGTGCCATAATAAACGG 8 A95Q-F CGTTTATTATGGCACCGATTCAAGCACATGGTCTGGCACAT 9 A95Q-R ATGTGCCAGACCATGTGCTTGAATCGGTGCCATAATAAACG 10 A96C-F TTATGGCACCGATTGCATGCCATGGTCTGGCACATACCAC 11 A96C-R GTGGTATGTGCCAGACCATGGCATGCAATCGGTGCCATAA 12 S175C-F TATTCTGACCGCAGATTGCACCGTTAGCGGTAATC 13 S175C-R GATTACCGCTAACGGTGCAATCTGCGGTCAGAATA 14

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

E. coli BL21 (hereinafter referred to as DE3) was transformed with plasmids containing wild-type and lactate oxidase mutants, respectively, and the transformed DE3 was inoculated into LB 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 removed and inoculated into the main medium: 500 mL ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM KH ₂ PO ₄ , 25 mM (NH ₄ ) ₂ SO ₄ , 50 mM Na ₂ HPO ₄ and 1 mM MgSO ₄ ) containing 100 µg/mL ampicillin. The culture medium was then incubated with shaking at 37°C for 8 hours. The supernatant was removed by centrifugation (4°C, 5000 g) for 20 minutes, and the cell pellet was collected and stored at -20°C.

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

E coli cells transformed 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 recombinant histidine-tagged (His-tag) protein, was purified as a crude cell extract using a nickel affinity column and a protein purification kit (supplier: ÄKTA 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), and 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. The fractions containing the target protein were dialyzed and concentrated using a dialysis membrane (10 kDa) after SDS-PAGE analysis. After replacement with 50 mM Tri-HCl solution (pH 7.0), the purified enzyme solution was freeze-dried and stored at -20°C.

Carboxylmethylation of mutant lactate oxidase

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 min.

Enzyme assay

Determination of protein concentration

The analyte (20 μL) was mixed with 200 μL of protein stain (Bradford reagent) for 5 minutes to form a mixture, and the O.D. 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 O.D. values.

Protein activity assay

1. Lactic acid oxidation activity

50 μL of the diluted enzyme was added to each well of the microtiter plate, followed by a mixture of 0.25 mg/mL 3, 3', 5, 5'-tetramethylbenzidine (TMB), 5 U/mL horseradish peroxidase (HRP) and 50 μL of 10 mM lactic acid, and the mixture was allowed to react at 30°C with intermittent shaking for five minutes. The reaction was then blocked by adding 50 μL of 1 M HCl, the molar attenuation coefficient was set to 59 cm ^-1 ·mmol ^-1 , and the absorbance (OD) of the solution was measured at a wavelength of 450 nm. The activity of lactate oxidase at ₃₀ °C, pH 7.0 (i.e., 1 U) was calculated using the following equation: Activity (U/mL) = [Δ OD ( ODtest − ODblank ) × 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 pathlength (cm), and Vs is the enzyme volume.

2. Dehydrogenase activity

Phenazine methosulfate (PMS, 0.5 mM) and 2,6-dichlorophenol-indophenol (DCIP, 1.2 mM) were mixed in potassium phosphate buffer (PPB, 20 mM, pH 7.0) 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 factor was set to 16.3 cm ^-1 ·mmol ^-1 , and 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, pH 7.0 was calculated using the following equation: Activity (U/mL) = [Δ OD ( ODtest − ODblank ₎ × Vt × df ] / (ε× t × l × Vs ), where Vt is the total volume, df is the dilution _rate , ε is the volume molar attenuation coefficient, t is the reaction time, l is the optical pathlength (cm), and Vs is the enzyme volume.

3. Binding affinity

By reacting the lactate oxidase variant 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), the enzyme kinetic parameters K _m , K _cat and V _max of the wild-type or mutant lactate oxidase were determined, and the reaction rate was determined based on the slope of the linear graph of the time (i.e., reaction interval) and the absorbance (OD) at a wavelength of 450 or 600 nm detected every 3 seconds during the reaction interval. The K _m , K cat and V max values were obtained by plotting a calibration curve between lactate concentration (on the x-axis) and reaction rate (on the y-axis) under the Michaelis - _{Menten equation} .

Preparation of lactate sensor with modified electrodes

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 fixed on the surface of the working electrode by electrodeposition or drip coating as described above.

Electrodeposition

The outer surface of SPCE was washed with ddH ₂ O and then coated 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). Surface modification was achieved by cyclic voltammetry of SPCE at 37°C with a preset potential of -1.0 V to 0.0 V and a preset scan rate of 200 mV/s for 50 cycles. The modified SPCE was placed in PPB solution (pH 5.0, 100 mM) and cyclic voltammetry was performed again for 20 cycles at a second preset potential between -300 mV and 600 mV and a preset scan rate of 60 mV/s to remove residual PVImQOs. After drying at room temperature, 4 μL of protective agent containing 1% polyglucosamine and 0.075% genipin was further added to the modified SPCE.

Drip coating method i. A mixed solution (1 μL) of enzyme solution (1 to 8 U/μL), potassium ferrocyanide (K ₃ [Fe(CN) ₆ ], 25 to 50 mM) and 0.1% Triton X-100 was dripped onto the electrode surface and dried at room temperature. ii. Alternatively, 5 μL of PVImQOs (10 mg/mL) and 2.5 μL of enzyme solution were dripped onto the electrode area of SPCE separately and sequentially. After drying at 4°C, a protective agent (4 μL) containing 1% polyglucosamine and 0.075% genipin was further added to the surface of SPCE. The modified SPCE was dried and stored at room temperature.

Electrochemical evaluation

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

Lactate detection

Sample preparation

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 was prepared by adjusting the amount of monobasic and dibasic potassium phosphate (obtained from the supplier) and its pH value.

Create a calibration curve

Lactic acid solutions of different 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 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 60 seconds 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, thereby establishing a calibration curve of current vs. lactate concentration.

Embodiment 1 : Preparation of lactate oxidase variants of the present invention

Five lactate oxidase variants of the present invention were generated by the method described in the above "Materials and Methods" section. 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.

Table 2: Lactate oxidase variants of the present invention Lactate oxidase variants Amino acid sequence Serial Number A95N MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPI N AHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHS GLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAPGSFDTTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY 2 A95Q MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPI Q AHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHS GLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAPGSFDTTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY 3 A96C MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIA C HGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHSG LPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAPGSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLLFDNPYGYEY 4 S175C MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTAD C TVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHSGLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAP GSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY 5 S175C- carboxymethylation MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTAD C* TVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHSGLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAP GSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY 6 *: Cysteine modified with carboxymethyl group

Embodiment 2 : Characteristic analysis of lactate oxidase variants of the present invention

2.1 Oxidase and dehydrogenase activities

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.

Table 3: Quantitative results of oxidase and dehydrogenase activities of wild-type (WT) and mutant lactate oxidase Oxidase activity (U/mg) Dehydrogenase activity (U/mg) WT 58.18 9.16 A95N ND 0.07 A95Q 0.93 0.49 A96C 17.76 1.07 S175C 18.33 1.21 *ND: Not Measured

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.

2.2 Enzyme kinetics

This experiment evaluated whether the binding affinity of lactate oxidase variants is 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 _m and K _cat values of the enzyme were determined according to the process described in "Materials and Methods". The K _m and K _cat values are listed in Table 4.

Table 4: _Km and _Kcat values of lactate oxidase variants and wild-type (WT) enzyme of the present invention pH 5 pH 7 Chemical reaction K _m (mM) K _cat (S ^-1 ) K _cat /K _m (mM ^-1 S ^-1 ) K _m (mM) K _cat (S ^-1 ) K _cat /K _m (mM ^-1 S ^-1 ) Oxidation WT 2.47 10.47 4.25 0.45 10.56 23.5 A95N ND ND ND ND ND ND A95Q 34.07 0.32 0.01 12.24 0.48 0.04 A96C 22.65 4.63 0.2 10.44 7.84 0.75 S175C 17.22 9.16 0.53 2.38 5.56 2.33 Dehydrogenation WT 1.51 2.45 1.63 0.29 3.23 11.1 A95N 551.5 0.91 0.0017 1464 4.72 0.0032 A95Q 6.63 0.045 0.0068 10.31 0.17 0.02 A96C 17.94 0.67 0.038 8.81 1.48 0.17 S175C 5.94 1.00 0.17 3.07 4.43 1.44 *ND: Not Measured

As a result, it was found that the K _m values of the lactate oxidase variants of the present invention were significantly higher than that of the wild-type enzyme regardless of whether the pH value was changed (i.e., pH 5.0 or pH 7.0), indicating that each of the lactate oxidase variants of the present invention has a lower lactate binding affinity.

Embodiment 3 : Lactate detection by using the lactate oxidase variant of the present invention

This experiment evaluated 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 precipitating 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 onto the surface of SPCE; while LS-II was prepared by immobilizing the enzyme and potassium ferrocyanide (K3[Fe(CN)6]) onto the surface of SPCE. LS-I and LS-II were used to detect lactate in designated samples, respectively.

3.1 Binding affinity

This experiment evaluates whether the binding affinity of the lactate oxidase variants of the present invention is affected by pH. To this end, the reaction of lactic acid (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 process described in the "Materials and Methods" section, and the results are summarized in Table 5.

Table 5: Km and Vmax values of wild-type (WT) and mutant lactate oxidase of the present invention pH 5.0 pH 7.0 K _m (mM) V _max (μA) K _m (mM) V _max (μA) WT 1.84 25.29 2.33 41.14 A95N 6.87 0.087 255.5 3.45 A95Q 24.12 0.019 9.47 0.22 A96C 3.28 0.095 2.08 0.99 S175C 1.67 6.16 1.31 11.78 S175C** 3.27 0.27 17.16 1.39 ** Modification of cysteine at position 175 with a carboxymethyl group

As a result, it was found that the lactate oxidase variants of the present invention had a higher K _m value than the wild-type enzyme, as summarized in Table 5. The data in Table 5 are consistent with the evaluation of the previous Example 2, both of which indicate that the lactate oxidase variants of the present invention have a lower binding affinity for lactate than the wild-type, especially in a low pH environment.

3.2 Lactate detection using the lactate oxidase variants of the present invention

3.2.1  A95Q , A96C or S175C

This experiment investigated the detection efficiency and limitation 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 medium 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 (0-300 mM) was applied to each lactate sensor, and the generated current was measured and recorded according to the process described in the "Materials and Methods" section. The results are presented in Figures 1 to 3.

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. The results presented in Figures 1A and 1B respectively indicate that the A95Q variant of lactate oxidase of the present invention can detect a wide range of lactic acid in liquid samples.

As for A96C, it was found that A96C could detect lactate concentrations ranging from about 0.5 mM to 90 mM in either pH 5.0 (FIG. 2A) or pH 7.0 (FIG. 2B) buffer. Consistent results were also observed in lactate detection in artificial sweat (pH 5.0) (FIG. 2C). In artificial sweat, the detectable lactate concentration ranged from 0.5 mM to 90 mM, indicating that the lactate oxidase A96C of the present invention is capable of detecting lactate in a simulated biological fluid environment.

As for S175C, it was found that trace lactic acid 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 (Figures 3A and 3B). These results indicate that the lactate oxidase variant S175C of the present invention can detect small amounts of lactic acid (e.g., less than 10 mM) in aqueous samples without being affected by different pH values.

3.2.2 Carboxymethylation S175C

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 depicted in Figures 4A and 4B.

As shown in the figure, carboxylation significantly improved the detection range of lactate by the S175C variant compared to the control variant. Specifically, the maximum concentration of lactate that the carboxylation variant could detect in acidic buffer (pH 5.0) and neutral buffer (pH 7.0) was 166.7 mM and 259.3 mM, respectively.

3.3 The universality of the lactate oxidase variants of the present invention for detecting lactate

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 the salinity of the buffer. The results are presented in Figures 5 to 7.

The results showed that the maximum lactate amount that could be detected by sensors with 1, 1.5 or 2 units (U) of A96C was over 100 mM. In particular, the lactate sensor with 1.5U of A96C (i.e., A96C-LS-II-1.5U) showed the strongest detection ability, which could detect the lactate concentration of artificial sweat up to 120 mM (Figure 5). In addition, the sensor with 1 unit of A96C (i.e., A96C-LS-II-1U) showed the highest resolution and smaller variability, which means that only a small amount of mutant enzyme is needed to achieve the desired detection efficiency.

As for the effect of pH on lactate detection, the experiment found that the mutant lactate oxidase A96C of the present invention was able to detect lactate in the range of 0 to 200 mM in artificial sweat samples ( FIG. 6 ).

As for the effect of salinity, the results showed 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 in different pH values and/or salinity (Figure 7). The data presented in Figure 7 confirm that the detection performance of the lactate oxidase A96C of the present invention is not affected or interfered by the salinity of artificial sweat.

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 more widely applicable lactate detection.

It should be understood that the above description of the embodiments is given only in the form of embodiments, and various modifications can be made by those with ordinary knowledge in the art to which the present invention belongs. The above specification, embodiments and experimental results provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not used to limit the present invention. Those with ordinary knowledge in the art to which the present invention belongs can make various changes and modifications 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 attached patent application.

without

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the attached drawings are described as follows:

FIG. 1A is a line graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A95Q of the present invention and performing the method of the present disclosure in phosphate (PB) buffer (pH 5.0).

FIG. 1B is a line graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A95Q of the present invention and performing the method of the present disclosure in PB buffer (pH 7.0).

FIG. 2A is a line graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A96C of the present invention and performing the method of the present disclosure in PB buffer (pH 5.0).

FIG. 2B is a line graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A96C of the present invention and performing the method of the present disclosure in PB buffer (pH 7.0).

FIG. 2C is a line graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A96C of the present invention and performing the method of the present disclosure in artificial sweat (pH 5.0).

FIG. 3A shows the correlation between lactate concentration and measured current obtained by performing the method of the present disclosure in PB buffer (pH 5.0) using the lactate oxidase mutant S175C of the present invention.

FIG. 3B shows the correlation between lactate concentration and measured current obtained by performing the method of the present disclosure in PB buffer (pH 7.0) using the lactate oxidase mutant S175C of the present invention.

FIG. 4A shows the correlation between lactate concentration and measured current obtained by performing the method of the present disclosure in PB buffer (pH 5.0) using the lactate oxidase mutant S175C of the present invention.

FIG. 4B shows the correlation between lactate concentration and measured current obtained by performing the method of the present disclosure in PB buffer (pH 7.0) using the lactate oxidase mutant S175C of the present invention.

FIG. 5 is a graph showing a calibration curve of lactate concentration vs. current established by using the lactate oxidase mutant A96C of the present invention and performing the method of the present disclosure in artificial sweat (pH 5.0).

FIG. 6 is a line graph showing 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).

FIG. 7 shows the detection current corresponding to a specific lactic acid concentration (40 mM) in artificial sweat samples having various salinity (0 to 800 mM) and pH values using the A96C of the present invention.

              Sequence Listing <![CDATA[<110> PolyShuo Biotech Co., Ltd.]]> <![CDATA[<120> Lactate oxidase variants and their use in lactate detection ]]> <![CDATA[<130> P4263-TW]]> <![CDATA[<160> 14 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Aerococcus viridans ]]> <![CDATA[<400> 1]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Ala Ala 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Ser Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ile Glu Glu Ile Ala Ala His Ser Gly Leu Pro Val Phe Val 225 230 235 240 Lys Gly Ile Gln His Pro Glu Asp Ala Asp Met Ala Ile Lys Ala Gly 245 250 255 Ala Ser Gly Ile Trp Val Ser Asn His Gly Ala Arg Gln Leu Tyr Glu 260 265 270 Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365 Pro Tyr Gly Tyr Glu Tyr 370 <![CDATA[<210> 2]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Single substitution at position 95 of SEQ ID NO:1 ]]> <![CDATA[<400> 2]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Asn Ala 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Ser Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ile Glu Glu Ile Ala Ala His Ser Gly Leu Pro Val Phe Val 225 230 235 240 Lys Gly Ile Gln His Pro Glu Asp Ala Asp Met Ala Ile Lys Ala Gly 245 250 255 Ala Ser Gly Ile Trp Val Ser Asn His Gly Ala Arg Gln Leu Tyr Glu 260 265 270 Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp 3]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Gln Ala 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Ser Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365 Pro Tyr Gly Tyr Glu Tyr 370 <![CDATA[<210> 4]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> SEQ ID NO:1 Single substitution at position 96]]> <![CDATA[<400> 4]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Ala Cys 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Ser Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ile Glu Glu Ile Ala Ala His Ser Gly Leu Pro Val Phe Val 225 230 235 240 Lys Gly Ile Gln His Pro Glu Asp Ala Asp Met Ala Ile Lys Ala Gly 245 250 255 Ala Ser Gly Ile Trp Val Ser Asn His Gly Ala Arg Gln Leu Tyr Glu 260 265 270 Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365 Pro Tyr Gly Tyr Glu Tyr 370 <![CDATA[<210> 5]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Single substitution at position 175 of SEQ ID NO:1]]> <![CDATA[<400> 5]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Ala Ala 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Cys Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ile Glu Glu Ile Ala Ala His Ser Gly Leu Pro Val Phe Val 225 230 235 240 Lys Gly Ile Gln His Pro Glu Asp Ala Asp Met Ala Ile Lys Ala Gly 245 250 255 Ala Ser Gly Ile Trp Val Ser Asn His Gly Ala Arg Gln Leu Tyr Glu 260 265 270 Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365 Pro Tyr Gly Tyr Glu Tyr 370 <![CDATA[<210> 6]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> The cysteine (C) at position 175 of SEQ ID NO:5 is carboxymethylated]]> <![CDATA[<220>]]> <![CDATA[<221> UNSURE]]> <![CDATA[<222> (175)..(175)]]> <![CDATA[<223> Carboxymethylation]]> <![CDATA[<400> 6]]> Met Asn Asn Asn Asp Ile Glu Tyr Asn Ala Pro Ser Glu Ile Lys Tyr 1 5 10 15 Ile Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val 20 25 30 Val Pro His Gly Gly Phe Asn Tyr Ile Ala Gly Ala Ser Gly Asp Glu 35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60 Pro Arg Leu Ala Gln Asp Val Glu Ala Pro Asp Thr Ser Thr Glu Ile 65 70 75 80 Leu Gly His Lys Ile Lys Ala Pro Phe Ile Met Ala Pro Ile Ala Ala 85 90 95 His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val 100 105 110 Ser Glu Phe Gly Thr Ile Met Ser Ile Ser Ala Tyr Ser Gly Ala Thr 115 120 125 Phe Glu Glu Ile Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gln 130 135 140 Ile Tyr Met Ala Lys Asp Asp Gln Gln Asn Arg Asp Ile Leu Asp Glu 145 150 155 160 Ala Lys Ser Asp Gly Ala Thr Ala Ile Ile Leu Thr Ala Asp Cys Thr 165 170 175 Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro 180 185 190 Phe Gly Met Pro Ile Val Gln Arg Tyr Leu Arg Gly Thr Ala Glu Gly 195 200 205 Met Ser Leu Asn Asn Ile Tyr Gly Ala Ser Lys Gln Lys Ile Ser Pro 210 215 220 Arg Asp Ile Glu Glu Ile Ala Ala His Ser Gly Leu Pro Val Phe Val 225 230 235 240 Lys Gly Ile Gln His Pro Glu Asp Ala Asp Met Ala Ile Lys Ala Gly 245 250 255 Ala Ser Gly Ile Trp Val Ser Asn His Gly Ala Arg Gln Leu Tyr Glu 260 265 270 Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala Ile Ala Glu Arg Val Asn 275 280 285 Lys Arg Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Val Ser Gly Val Ala Ala Asp Ala Leu Gly Arg 305 310 315 320 Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gln Gly Ala Tyr Ser 325 330 335 Val Leu Asp Tyr Phe Gln Lys Asp Leu Thr Arg Val Met Gln Leu Thr 340 345 350 Gly Ser Gln Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365 Pro Tyr Gly Tyr Glu Tyr 370 <![CDATA[<210> 7]]> <![CDATA[<211> 44]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Forward primer for mutant lactate oxidase A95N]]> <![CDATA[<400> 7]]> ccgtttatta tggcaccgat taacgcacat ggtctggcac atac 44 <![CDATA[<210> 8]]> <![CDATA[<211> 44]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Reverse primer for mutant lactate oxidase A95N]]> <![CDATA[<400> 8]]> gtatgtgcca gaccatgtgc gttaatcggt gccataataa acgg 44 <![CDATA[<210> 9]]> <![CDATA[<211> 41]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Forward primer for mutant lactate oxidase A95Q]]> <![ CDATA[<400> 9]]> cgtttattat ggcaccgatt caagcacatg gtctggcaca t 41 <![CDATA[<210> 10]]> <![CDATA[<211> 41]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Reverse primer for mutant lactate oxidase A95Q]]> <![CDATA[<400> 10]]> atgtgccaga ccatgtgctt gaatcggtgc cataataaac g 41 <![CDATA[<210> 11]]> <![CDATA[<211> 40]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Forward primer of mutant lactate oxidase A96C]]> <![CDATA[<400> 11]]> ttatggcacc gattgcatgc catggtctgg cacataccac 40 <![CDATA[<210> 12]]> <![CDATA[<211> 40]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Reverse primer of mutant lactate oxidase A96C]]> <![CDATA[<400> 12]]> gtggtatgtg ccagaccatg gcatgcaatc ggtgccataa 40 <![CDATA[<210> 13]]> <![CDATA[<211> 35]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Forward primer of mutant lactate oxidase S175C]]> <![CDATA[<400> 13]]> tattctgacc gcagattgca ccgttagcgg taatc 35 <![CDATA[<210> 14]]> <![CDATA[<211> 35]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Reverse primer for mutant lactate oxidase S175C]]> <![CDATA[<400> 14]]> gattaccgct aacggtgcaa tctgcggtca gaata 35
        

Claims (6)

A lactate oxidase variant, wherein the amino acid sequence of the lactate oxidase variant is composed of the amino acid sequence of sequence number: 4. A method for detecting and quantifying lactic acid in a liquid sample, comprising: (a) contacting the liquid sample with a lactate oxidase variant as described in claim 1; (b) measuring a current generated by the reaction between the lactate oxidase variant as described in claim 1 and lactic acid in the liquid sample; and (c) measuring 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 having a known lactic acid concentration. The method as described in claim 2, wherein the pH value of the liquid sample is from 4 to 9. The method as described in claim 2, wherein the salinity of the liquid sample is between 0 and 1000 mM. The method as described in claim 2, wherein the liquid sample is sweat. The method of claim 2, wherein the method can detect lactic acid concentration in a liquid sample between 0 and 200 mM.

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