Lactate oxidase variants and their use in lactate detection

Abstract

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

Description

[Background technology]

[0001] 1. Technical field

[0002] This disclosure relates to lactate oxidase variants and their use in lactate detection. More specifically, this disclosure relates to lactate oxidase variants and the electrical detection and quantification of lactate in liquid samples.

[0003] 2. Explanation of related technologies

[0004] Because lactic acid is a key metabolite in cellular anaerobic metabolic pathways, and is a major metabolite in intracellular anaerobic metabolic pathways across various organisms, it can provide clues for monitoring multiple physiological processes in diverse organisms. Therefore, lactic acid concentration is widely used as an important parameter for assessing patient health status in clinical diagnostics and for ongoing monitoring in the food and fermentation industries.

[0005] In clinical diagnosis, sustained anaerobic metabolism leading to elevated lactate levels causes lactate accumulation, inevitably resulting in lactic acidosis, a symptom of severe sepsis. Therefore, a patient's blood lactate level serves as a warning signal of disease severity, facilitating the diagnosis and treatment of a wide range of conditions. Lactate is also an important element in sports medicine, particularly in assessing physical performance in athletics. Elevated blood lactate levels lead to a decrease in blood pH, ultimately causing fatigue. Blood lactate levels during exercise are used as an indicator of exercise training status and physical performance.

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

[0007] Various methods have been developed to measure lactate levels, with high-performance liquid chromatography (HPLC) being the most common. Other analytical methods such as fluorescence measurement, colorimetric analysis, chemiluminescence, and magnetic resonance spectroscopy are also commonly used. However, these approaches have drawbacks, including time-consuming processes, expensive equipment, and the need for trained personnel. To overcome these limitations, portable and disposable biosensors are currently being developed. Typically, biosensing methods offer the advantages of being simple and direct, combining rapid reaction, high specificity, cost-effectiveness, and ease of use. Ideally, lactate concentrations in biological fluids such as whole blood, sweat, and saliva should be measurable with biosensors. However, currently available biosensors only support lactate detection in blood, require invasive sample collection methods, and are not widely adopted by users, nor can they be used to detect lactate in samples other than blood.

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

[0009] Therefore, in the relevant technological fields, there is a need for improved tools and approaches for non-invasive, efficient, and continuous detection of lactic acid levels in human body fluids. [Overview of the project]

[0010] The following is a brief summary of the disclosures to provide readers with a basic understanding. This summary is not a comprehensive overview of the disclosures, nor does it identify the main / essential elements of the claimed invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as an introduction to the more detailed descriptions that will follow.

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

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

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

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

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

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

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

[0018] In some embodiments of the present disclosure, the lactate oxidase variant of the present disclosure has lower binding affinity for lactic acid than wild-type lactate oxidase.

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

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

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

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

[0023] According to some embodiments of the present disclosure, the method can detect lactic acid in the range of 0 to 300 mM in a liquid sample.

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

[0025] Reading the following detailed explanation in conjunction with the attached drawings will make the descriptions in this specification easier to understand.

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

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

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

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

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

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

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

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

[0034] [Figure 4B] Figure 4B is a plot showing the relationship between lactic acid concentration and current, measured according to the method of this disclosure using carboxymethylated S175C of this disclosure in PB solution (pH 7.0).

[0035] [Figure 5] Figure 5 is a line graph showing calibration curves of lactate concentration versus current obtained according to the method of this disclosure, performed using different amounts of A96C of this disclosure in synthetic sweat (pH 5.0).

[0036] [Figure 6] Figure 6 is a line graph showing calibration curves of lactate concentration versus current obtained according to the method of this disclosure, performed using A96C of this disclosure in synthetic sweat with various pH values ​​(pH 5.0-7.0).

[0037] [Figure 7] Figure 7 is a plot showing the currents corresponding to specific lactic acid concentrations (40 mM) detected using A96C of this disclosure in synthetic sweat with various salinity levels (0 to 800 mM) and pH values. [Modes for carrying out the invention]

[0038] The detailed description provided below in relation to the attached drawings is intended to describe this embodiment and is not intended to represent the only form in which this embodiment may be constructed or used. This description describes the function of the embodiment and the construction and operation procedures of the example. However, the same or equivalent functions and procedures may be realized by different embodiments.

[0039] 1.Definition

[0040] For convenience, the specific terms used herein, in the examples and in the appended claims are set forth herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by an ordinary person skilled in the art to which the invention pertains.

[0041] In this specification, the singular forms "a," "and," and "the" are used to include plural referents unless the context explicitly indicates otherwise.

[0042] While the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values ​​shown in the specific examples are as accurate as possible. However, these values ​​inherently contain a certain degree of error due to the standard deviation in each test measurement.

[0043] Typically, the term “wild-type” is used to describe a gene or protein when it is found in nature in an unmutated (unaltered) state. In this specification, the term “wild-type lactate oxidase” refers to the form of lactate oxidase protein that normally occurs in nature (i.e., bacteria) without genetic, structural, and / or functional changes. Specifically, the wild-type form of lactate oxidase is the full-length, naturally occurring lactate oxidase with 374 amino acids, as shown in Sequence ID No. 1.

[0044] In this specification, the term “lactate oxidase variant” is intended to encompass one or more forms of lactate oxidase polypeptides resulting from wild-type lactate oxidase by substitutions in which at least one amino acid in the wild-type lactate oxidase sequence is replaced by another amino acid. Alternatively, or optionally, the term “lactate oxidase variant” refers to a form of lactate oxidase peptide in which one or more residues have been subjected to post-translational modifications (PTMs) and / or chemical modifications to enhance the functional diversity of the proteome. Examples of PTMs include, but are not limited to, phosphorylation, methylation, acetylation, ubiquitination, hydroxylation, succinylation, glycosylation, and SUMOylation. Exemplary chemical modifications include, but are not limited to, carboxymethylation. In this disclosure, the modification performed on an amino acid residue is carboxymethylation. Well-known and commonly used notations may be used interchangeably in this specification to indicate the same mutation occurring in the peptide sequence. According to this disclosure, for example, a substitution of alanine (A) to asparagine (N) at position 95 may be represented as 95A, A95, A95N, or Ala95Asp.

[0045] In this specification, the term “binding affinity” refers to the sum of the 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 its variants). The affinity of an enzyme to a substrate is typically expressed by the Michaelis constant (K), which represents the substrate concentration at which half of the enzyme’s active site is occupied by the substrate. m It is thought to be related to K. m The smaller the value, the stronger the binding affinity to the substrate. Typically, the binding affinity of the mutant may or may not change compared to the wild-type enzyme, depending on the site of the mutation. According to this disclosure, the binding affinity of the lactate oxidase mutant of this disclosure to lactate and / or lactic acid is reduced compared to wild-type lactate oxidase.

[0046] In this specification, the term “liquid sample” refers to a sample taken and / or obtained from the natural environment or from a man-made product in liquid form, which may or may not contain lactate and / or lactic acid, and whose solvent is primarily water. A liquid sample as used in this disclosure may be a biological sample containing biological metabolites (i.e., lactate and / or lactic acid). Examples of biological samples suitable for use in this disclosure include mammalian, more preferably human, bodily fluids (e.g., sweat, urine, saliva, blood, and interstitial fluid) and fermentation liquids produced by microorganisms (e.g., fermented and spoiled foods). A liquid sample may contain one or more substances, including but not limited to minerals, trace elements, metal ions and / or heavy metal ions, metabolites, excrement, microplastics, micronekton, and microorganisms. Furthermore, a liquid sample may have a variety of measurable parameters, including but not limited to pH and salinity.

[0047] 2. Detailed Description of Preferred Embodiments

[0048] This disclosure is based, at least in part, on the discovery that some lactate oxidase mutants have a lower binding affinity to lactate / lactic acid than wild-type lactate oxidase, and therefore can detect high concentrations of lactate with high sensitivity without being affected by pH or salinity. Furthermore, it was unexpectedly discovered that an electric current is generated by the reaction of the enzyme (i.e., lactate oxidase) and substrate (i.e., lactate, lactic acid, or a combination thereof) in the presence of an electric field, and that a linear relationship exists between high concentrations of lactate (e.g., >20 mM) and this current, and therefore, the above-mentioned current may be useful as an indicator of lactate detection.

[0049] 2.1 Lactate oxidase variants

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

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

[0052] According to some embodiments of the present disclosure, a lactate oxidase variant of the present disclosure, named A95N, 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 with asparagine (N). Thus, the lactate oxidase A95N variant has the amino acid sequence of SEQ ID NO: 2.

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

[0054] According to other embodiments of the present disclosure, a lactate oxidase variant of the present disclosure, named A96C, may have an amino acid sequence identical to that of SEQ ID NO: 1 by at least 99.8%, wherein the alanine (A) at position 96 of SEQ ID NO: 1 is replaced with cysteine ​​(C). Thus, the lactate oxidase A96C variant has the amino acid sequence of SEQ ID NO: 4.

[0055] According to yet another embodiment of the present disclosure, a lactate oxidase variant of the present disclosure, named S175C, 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 with a cysteine ​​(C). Thus, the lactate oxidase S175C variant has the amino acid sequence of SEQ ID NO: 5.

[0056] The lactate oxidase variants described herein can be prepared by substitution or modification using genetic or chemical methods well known in the art. Genetically, these may include site-directed mutagenesis of coding DNA sequences, polymerase chain reaction (PCR), gene synthesis, and CRISPR / cas9 gene editing. The exact nucleotide changes may be confirmed, for example, by sequencing. The nucleotide sequences of eubacteria (e.g., Aerococcus viridans) lactate oxidase are available 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 SEQ ID NO: 1.

[0057] The lactate oxidase variants of this disclosure may be produced, for example, by solid-phase peptide synthesis or recombinant production. In recombinant production, one or more polynucleotides encoding the lactate oxidase variant are isolated and inserted into a suitable vector for further cloning and / or expression in host cells, mainly Escherichia coli. Such polynucleotides may be readily isolated and sequenced by conventional methods. An expression vector containing the coding sequence of the lactate oxidase variant of this disclosure, along with a suitable transcription / translation regulatory signal, may be constructed using methods well known to those skilled in the art. Examples of these methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. The expression vector may be a plasmid, a viral portion, or a nucleic acid fragment. Typically, the expression vector is an expression cassette cloned with the polynucleotide encoding the lactate oxidase variant of the present invention operably ligated to a promoter and / or other transcription or translation regulatory elements, and may be operably ligated to a nucleic acid encoding a polypeptide if the promoter can effectively transcribe its nucleic acid. According to some embodiments of the present disclosure, site-directed mutagenesis of natural lactate oxidase is expressed and carried out using a pRSET expression vector and standard tools well known in the art.

[0058] Alternatively, the lactate oxidase variants of the present disclosure may undergo further post-translational modifications (PTMs) and / or chemical modifications, in which case additional functional groups are introduced to the residues. Typical PTMs include, but are not limited to, phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, acetylation, hydroxylation, succinylation, and SUMOylation. Typical chemical modifications include, but are not limited to, carboxymethylation. PTMs and / or chemical modifications of proteins can be carried out by any method and tools known in the art, as appropriate to the actual needs and purposes. According to one aspect of the present disclosure, the lactate oxidase variants of the present disclosure are carboxymethylated by reaction with iodoacetate (IA) or iodoacetic acid (IAA), covalently bonding with the thiol group of cysteine ​​(C) to produce a carboxymethyl group. In one embodiment, the lactate oxidase mutant S175C of this disclosure undergoes chemical modification by reacting with iodoacetic acid, generating a carboxymethylated cysteine ​​residue. Therefore, carboxymethylated lactate oxidase S175C has the amino acid sequence of SEQ ID NO: 6.

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

[0060] 2.2 Methods for the detection and quantification of lactic acid

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

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

[0063] For the purpose of detecting lactic acid, the lactate oxidase variant of this disclosure is attached to the surface of the electrode system described above (e.g., SPCE) in the presence of a redox mediator. According to a feasible embodiment, a lactic acid sensor suitable for use in this method is manufactured by mixing the lactate oxidase variant of this disclosure and a redox mediator in a predetermined ratio to form a mixture, which is then immobilized on the surface of the SPCE by electrodeposition or dropwise deposition.

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

[0065] To create a calibration curve, a control sample containing a known concentration of lactate or lactic acid is brought into contact with a lactic acid sensor to which a fixed potential is applied, and the current generated by the electrochemical reaction between the lactic acid and the lactate oxidase variant of the present disclosure immobilized on the electrode can be detected by any means known in the art, specifically an electrochemical analyzer. Thus, a standard calibration curve can be created based on the detection current corresponding to known concentrations of lactic acid. In some embodiments of the present disclosure, the known lactic acid concentrations range from about 0 to 300 mM, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 9.9, 10, 14.8, 19.6, 20, 29.1, 30, 38.5, 40, 47.6, 50, 56.6, 60, 6 The concentrations are 5.4, 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 one example, calibration curves are prepared with lactic acid at concentrations of 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 38.5, 47.6, 56.6, 65.4, 74.1, 82.6, 90.9, 130.4, and 166.7 mM. In other embodiments, calibration curves are prepared with lactic acid at concentrations of 0.2, 0.5, 1, 2, 5, 9.9, 19.6, 29.1, 47.6, 90.9, 130.4, 166.7, 200, and 259.3 mM. In yet another embodiment, calibration curves are prepared with lactic acid at concentrations of 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 10 mM. In yet another embodiment, calibration curves are prepared with lactic acid at concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, and 200 mM. According to embodiments of this disclosure, the control sample mimics human sweat. Preferably, the control sample used in this method is synthetic sweat prepared according to international standards.Alternatively, or optionally, the control sample used in this method mimics, in particular, the pH, osmotic pressure, and ion concentration of human body fluids. Preferably, the control sample is a phosphate buffer.

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

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

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

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

[0070] Due to the above characteristics, this method can detect and quantify lactic acid concentration, particularly abundant lactic acid in aqueous environments that could not be detected by conventional detection methods. Furthermore, since this method can detect lactic acid in aqueous samples containing various other substances, it can be applied to a wide variety of liquid samples. [Examples]

[0071] Examples

[0072] Materials and methods

[0073] Gene synthesis and mutagenesis

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

[0075] Table 1 Primer sequences [Table 1]

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

[0077] Escherichia coli BL21(DE3) was transformed with wild-type cells and plasmids containing various lactate oxidases. The transformed DE3 cells were 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 500 mL of ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM KH2PO4, 25 mM (NH4)2SO4, 50 mM Na2HPO4, and 1 mM MgSO4) containing 100 μg / mL ampicillin as the main culture medium. The medium was then cultured with shaking at 37°C for 8 hours. The supernatant was removed by centrifugation (4°C, 5000 g, 20 minutes), the cell pellet was collected, and stored at -20°C.

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

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

[0080] Carboxymethylation of mutant lactate oxidase

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

[0082] Enzyme assay

[0083] Measurement of protein concentration

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

[0085] Measurement of Protein Activity

[0086] 1. Lactate Oxidase

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

[0088] 2. Dehydrogenase Activity

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

[0090] 3.Binding affinity

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

[0092] Fabrication of a lactate sensor using modified electrodes

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

[0094] Electroplating

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

[0096] Dripping method i. A mixed solution of enzyme solution (1 to 8 U / μL), potassium ferricyanide (K3[Fe(CN)6], 25 to 50 mM), and 0.1% Triton X-100 (1 μL) was dropped onto the electrode surface to cover it, and allowed to dry at room temperature. ii. Alternatively, 5 μL of PVImQOs (10 mg / mL) and 2.5 μL of enzyme solution were added to the electrode area of ​​the SPCE, one by one and sequentially. After drying at 4°C, 4 μL of a protective agent containing 1% chitosan and 0.075% genipine was 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) combined with a CS100 electrode stand (Zensor) with an ECP100 (Zensor) cable connector. The voltammogram and results were recorded and analyzed using the on-device software of the ACIP100 (Zensor).

[0099] Lactic acid detection

[0100] Sample preparation

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

[0102] Calibration curve creation

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

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

[0105] The five lactate oxidase variants described herein were produced by the method described in the "Materials and Methods" section. The lactate oxidase variants thus obtained and their amino acid sequences are listed in Table 2, and specified amino acid substitutions and modifications are shown in bold.

[0106] Table 2 Lactate oxidase variants of the present disclosure [Table 2] JPEG0007917937000003.jpg133164*: Cysteine ​​modified with a carboxymethyl group.

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

[0108] 2.1 Oxidase and dehydrogenase activity

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

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

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

[0112] 2.2 Enzyme reaction kinetics

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

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

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

[0116] Example 3 Detection of lactate using the lactate oxidase variant of the present disclosure

[0117] In this embodiment, the sensitivity and versatility of the lactate oxidase variants of this disclosure for detecting lactate in various samples were evaluated. For this purpose, two lactate sensors (LS-I and LS-II) based on different types of redox mediators immobilized on screen-printed carbon electrodes (SPCEs) were prepared according to the procedure described in the "Materials and Methods" section. Specifically, LS-I was prepared by adhering a mixture of enzyme solutions (i.e., the lactate oxidase variants A95Q, A96C, S175C, and carboxymethylated S175C of this disclosure) and a polymeric mediator PVImQOs to the SPCE surface, while LS-II was prepared by immobilizing the enzyme and potassium ferricyanide (K3[Fe(CN)6]) to the SPCE surface. LS-I and LS-II were used to detect lactate in specified samples, respectively.

[0118] 3.1 Binding affinity

[0119] This experiment investigated whether the binding affinity of the lactate oxidase variant of this disclosure is affected by pH. For this purpose, the reaction between lactate (various concentrations) and the lactate oxidase variant of this disclosure was measured at pH 5.0 or pH 7.0 using a lactate sensor LS-I. m and V max The values ​​were determined based on the procedure described in the "Materials and Methods" section, and the results are summarized in Table 5.

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

[0121] As summarized in Table 5, the lactate oxidase mutants of this disclosure have a larger K than the wild-type enzyme. m It was found that the values ​​were present. The data in Table 5 are consistent with the previous evaluation in Example 2, and all indicate that the lactate oxidase mutants of this disclosure have a lower binding affinity to lactate than wild-type lactate oxidase, particularly under low pH conditions.

[0122] 3.2 Detection of lactate using the lactate oxidase variants of this disclosure

[0123] 3.2.1 A95Q, A96C, or S175C

[0124] In this experiment, the detection efficiency and limits of the mutant lactate oxidases A95Q, A96C, and S175C of this disclosure were investigated. For this purpose, mutant lactate oxidases A95Q, A96C, or S175C of this disclosure were independently immobilized on the SPCE surface within the mediator PVImQOs to produce Type I lactate sensors (hereinafter referred to as A95Q-LS-I, A96C-LS-I, and S175C-LS-I). Next, various concentrations of lactate (0-300 mM) were applied to each lactate sensor in phosphate buffer (pH 5 and pH 7) or artificial sweat (pH 5), and the generated current was measured and recorded according to the procedure described in the "Materials and Methods" section. The results are shown in Figures 1 to 3.

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

[0126] Regarding A96C, it was found that A96C can detect lactic acid in a range of approximately 0.5 mM to 90 mM in either a pH 5.0 (Figure 2A) or pH 7.0 (Figure 2B) buffer solution. Similar results were obtained in the detection of lactic acid in synthetic sweat (pH 5.0, Figure 2C). In synthetic sweat, the detectable lactic acid concentration ranged from 0.5 mM to 90 mM, demonstrating that the lactate oxidase A96C of this disclosure is capable of detecting lactic acid in a simulated biological fluid environment.

[0127] Regarding S175, it was found that the lactate oxidase mutant S175C of this disclosure could detect trace concentrations of lactate (i.e., from 0.2 mM to 10 mM) regardless of the pH value of the buffer (Figures 3A and 3B). This result demonstrates that the lactate oxidase mutant S175C of this disclosure can detect and quantify lactate in aqueous samples at small concentrations (e.g., less than 10 mM in this disclosure) without being affected by different pH values.

[0128] 3.2.2 Carboxymethylated S175C

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

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

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

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

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

[0134] Regarding the effect of pH on lactic acid detection, it was found that the mutant lactate oxidase A96C of this disclosure can detect lactic acid in sweat samples from 0 to 200 mM regardless of the pH level (Figure 6).

[0135] Regarding the effect of salinity, slight differences in the current generated by the reaction between lactic acid and the mutant lactate oxidase A96C of this disclosure were observed at various pH values ​​and / or salinity levels (Figure 7). The data shown in Figure 7 support the fact that the detection capability of the mutant lactate oxidase A96C of this disclosure is not affected or interfered with by the salinity of synthetic sweat.

[0136] In summary, the data from Examples 1 to 3 demonstrate that the lactate oxidase variants of this disclosure have superior lactate detection capabilities, unaffected by the pH and / or salinity of the liquid sample, and achieve broad versatility in lactate detection.

[0137] The above description of embodiments is provided for illustrative purposes only, and those skilled in the art will understand that various modifications can be made. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. As described above, various embodiments of this invention have been described with certain specificity or by reference to one or more individual embodiments, but those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

1. A lactate oxidase mutant having the amino acid sequence of SEQ ID NOs: 2, 3, 5, or 6, A lactate oxidase mutant with lower binding affinity to lactate than wild-type lactate oxidase.

2. A method for detecting and quantifying lactic acid in a liquid sample, (a) A step of contacting the liquid sample with the lactate oxidase variant of claim 1, (b) A step of measuring the electric current generated by the reaction of the lactate oxidase variant of claim 1 with lactate in a liquid sample, and (c) A step of determining the lactic acid concentration in a liquid sample by interpolating or extrapolating the current value measured in step (b) using the current value of a control sample having a known lactic acid concentration. A method that includes this.

3. The method according to claim 2, wherein the liquid sample has a pH value in the range of 4 to 9.

4. The method according to claim 2, wherein the liquid sample has a salinity between 0 and 1000 mM.

5. The method according to claim 2, wherein the liquid sample is sweat.

6. The method according to claim 2, wherein the method can detect lactic acid in a liquid sample in the range of 0 to 300 mM.

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