Enzymatic detection of non-lactic hydroxy acids

Abstract

The present invention relates to methods and means for the detection and / or quantification of substances, in particular to methods and means for the detection and / or quantification of non-lactic acid hydroxy acids, such as alpha-hydroxybutyric acid, using enzyme-based methods. These methods and means enable the determination of, for example, non-lactic hydroxy acid alpha-hydroxybutyric acid in complex samples comprising lactic acid (e.g., blood samples).

Description

Technical Field

[0001] The present invention relates to the field of detection and quantification of analytes, and in particular to methods and means for detecting and / or quantifying non-lactic acid hydroxy acids using enzyme-based methods. Background Art

[0002] The assessment of various disease risk factors is crucial for disease prevention and early detection. Therefore, the determination of analytes in human samples is becoming increasingly important in in vitro diagnostic kits or tests.

[0003] Hydroxy acids are suitable analytes for risk factor determination.

[0004] For example, the hydroxy acid α-hydroxybutyric acid (2-HBA) or its corresponding salt α-hydroxybutyrate has been described in multiple clinical cohort studies (Alesi et al., 2021; Cobb et al., 2016; Gall et al., 2010; Lu et al., 2021; Wang et al., 2021) and analyzed in serum / plasma to identify reliable and powerful predictors of different types of diabetes (type 2 diabetes, gestational diabetes). Among hundreds of compounds, 2-HBA repeatedly emerged as one of the most suitable predictors.

[0005] Another example of an analyte suitable for risk factor determination is the analysis of glycolic acid in the blood of patients with metabolic acidosis (Roberts et al., 2022).

[0006] 2-HBA is a chiral molecule that exists as L- and D-enantiomers, similar to its primary metabolite, lactate. Due to this similarity to lactate, only GC / LC-MS has been successfully used to quantify 2-HBA in human serum and urine samples in clinical studies to date.

[0007] US2019 / 0107530 A1 discloses a method for assessing a patient's risk of developing occult pancreatic beta cell dysfunction by measuring the level of α-hydroxybutyrate in a patient sample.

[0008] WO 2017 / 210097 A1 discloses the detection and determination of analytes (e.g., 2-HBA) in a sample by mass spectrometry.

[0009] WO 2015 / 010042 A2 discloses clinical detection of biomarkers (e.g., α-hydroxybutyrate) to predict the likelihood of impaired glucose tolerance or insulin resistance in a subject. Thus, the method for determining the biomarker is, for example, mass spectrometry, NMR, or an apparatus for immunoassay.

[0010] Maughan et al. (1982) disclosed a method for the enzymatic determination of glucose, lactate, pyruvate, alanine, 3-hydroxybutyrate, and acetoacetate in a 20 μL blood sample.

[0011] WO 96 / 39534 A1 discloses oxidoreductases (eg lactate dehydrogenase coupled to TAG) for generating chemiluminescent signals for use in biosensors and kits.

[0012] CN 101825625 A discloses a kit for simultaneously determining lactic acid, creatinine and beta-hydroxybutyric acid in urine.

[0013] WO 2022 / 125537 A2 discloses a biosensor based on an oxidoreductase (such as lactate oxidase, lactate dehydrogenase or 3-hydroxybutyrate dehydrogenase) for measuring lactate and 3-hydroxybutyrate in a sample.

[0014] To date, there have been no reports of rapid and accurate measurement of hydroxy acids other than L-lactic acid, as L-lactic acid interferes with the detection of the corresponding non-lactic hydroxy acids. For example, to date, there have been no reports of rapid and accurate measurement of 2-hydroxybutyrate in the presence of L-lactic acid. Hydroxy acids, such as 2-hydroxybutyric acid (2-HBA), have been measured to date using GC / LC-MS.

[0015] However, these methods (eg, GC / LC-MS) are highly complex and time-consuming; therefore, rapid and accurate determination of 2-HBA for large-scale diagnostic purposes is highly desirable.

[0016] Therefore, there is an urgent need in the prior art for methods and means for detecting and / or quantifying non-lactic acid hydroxy acids based on specific, reliable, rapid and simple methods. Summary of the Invention

[0017] The object of the present invention is to provide a method and means for detecting and / or quantifying non-lactic acid hydroxy acids based on a specific, reliable, rapid and simple method.

[0018] The subject matter of the present invention solves the above-mentioned aims.

[0019] The inventors of the present invention have surprisingly found that, by a combination of specific subsequent reaction steps, non-lactic hydroxy acids (e.g., 2-HBA) can be specifically determined. Therefore, a method is provided herein for determining non-lactic hydroxy acids in complex samples, even in the presence of molecules that interfere with the determination of such hydroxy acids (i.e., in the presence of lactic acid). Specifically, the invention described herein is capable of quickly and accurately determining 2-HBA in samples containing lactic acid. This method is highly desirable for large-scale diagnostic purposes. Utilizing the methods described herein, analytes can be measured in human samples, and therefore, the methods described herein can be used in in vitro diagnostic (IVD) assay kits and can determine abnormal concentration levels of analytes, which in turn allows for the assessment of the risk of various types of metabolic disorders (e.g., diabetes).

[0020] According to the present invention, a method for determining non-lactic hydroxy acids in a sample is provided, wherein the sample contains non-lactic hydroxy acids and lactic acid, and the method comprises the following steps:

[0021] i. selectively removing lactic acid from the sample;

[0022] ii. incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity;

[0023] iii. Determining non-lactic hydroxy acids in the sample.

[0024] Specifically, the non-lactic hydroxy acid is a non-lactic 2-hydroxy acid.

[0025] Specifically, the non-lactic 2-hydroxy acid is a compound having the general formula I:

[0026]

[0027] in,

[0028] R1 is H or C 1-6 alkyl,

[0029] R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0030] Specifically, R1 is -CH3.

[0031] Specifically, R2 is phenyl.

[0032] Specifically, the non-lactic acid hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, 2-hydroxyisobutyric acid, D-lactic acid and the corresponding salts of any one of the above.

[0033] Specifically, the enzyme in step ii is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity, or an enzyme having non-lactic acid hydroxy acid oxidase activity.

[0034] More specifically, the enzyme having non-lactate hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0035] Specifically, the enzyme in step ii is FCb2 or LDH.

[0036] Specifically, an enzyme is used in step i.

[0037] In particular, an enzyme having lactate oxidizing activity, in particular an enzyme having lactate oxidase activity, is used in step i.

[0038] Specifically, the method described herein further comprises adding a reagent for removing hydrogen peroxide in step i, preferably adding an enzyme having catalase activity.

[0039] Specifically, the non-lactic hydroxy acid is determined by colorimetry, photometry, fluorescence or electrochemistry.

[0040] Specifically, 2-hydroxybutyric acid is determined in a sample containing 2-hydroxybutyric acid and lactic acid, and the method comprises the following steps:

[0041] a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity;

[0042] b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity;

[0043] c. Determine 2-hydroxybutyric acid in the sample.

[0044] In particular, the sample is a human sample.

[0045] Specifically, an electrode containing an enzyme having non-lactic acid hydroxy acid oxidizing activity is used in step ii.

[0046] According to the present invention, there is also provided an enzyme having non-lactic hydroxy acid oxidation activity, specifically an enzyme having non-lactic hydroxy acid dehydrogenase activity, for use in the method described herein for determining non-lactic hydroxy acids in a sample.

[0047] In particular, the sample is a human sample.

[0048] According to the present invention, there is also provided use of an electrode comprising an enzyme having non-lactic hydroxy acid oxidation activity (particularly an enzyme having non-lactic hydroxy acid dehydrogenase activity) for determining non-lactic hydroxy acids in a sample in the method described herein.

[0049] In particular, the sample is a human sample.

[0050] According to the present invention, there is also provided a kit for determining non-lactic hydroxy acids in a sample containing non-lactic hydroxy acids and lactic acid, the kit comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactic hydroxy acid oxidizing activity.

[0051] In particular, the non-lactic hydroxy acid is a 2-hydroxy acid.

[0052] Specifically, the non-lactic hydroxy acid is 2-hydroxybutyric acid.

[0053] Specifically, the enzyme having lactate oxidation activity is lactate oxidase.

[0054] Specifically, the enzyme having non-lactic acid hydroxy acid oxidizing activity is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity.

[0055] Specifically, the enzyme having non-lactic acid hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0056] Specifically, the enzyme having non-lactic acid hydroxy acid oxidation activity is FCb2 or LDH.

[0057] In particular, the enzyme having non-lactic acid hydroxy acid oxidation activity is part of the electrode.

[0058] According to the present invention, there is also provided an electrode comprising an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity.

[0059] Specifically, the non-lactic 2-hydroxy acid is a compound having the general formula I:

[0060]

[0061] in,

[0062] R1 is H or C 1-6 alkyl,

[0063] R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0064] Specifically, R1 is -CH3.

[0065] Specifically, R2 is phenyl.

[0066] Specifically, the non-lactic acid 2-hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid and 2-hydroxyisobutyric acid.

[0067] Specifically, the enzyme having non-lactic acid 2-hydroxy acid oxidation activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactic acid 2-hydroxy acid oxidase activity.

[0068] Specifically, the enzyme having non-lactate 2-hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0069] Specifically, the enzyme is FCb2 or LDH. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 : Example of the principle of the two-step enzymatic assay for the determination of 2-HBA.

[0071] Figure 2 : Three technical replicates of L-lactic acid removal and 2-HBA determination in the presence and absence of L-lactic acid interferor and LOx catalyst. DETAILED DESCRIPTION

[0072] Unless otherwise indicated or defined, all terms used herein have their usual meaning in the art, which is clear to those skilled in the art. For example, reference may be made to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017); and Berg et al, "Stryer Biochemie", Springer Verlag, 2018.

[0073] The claimed subject matter is specifically directed to artificial products or methods that employ or produce these artificial products, which may be variants of naturally occurring (wild-type) products. Although some sequence identity may exist with naturally occurring structures, it is understood that the materials, methods, and uses of the present invention, for example, specifically directed to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, and modified proteins and enzymes, are "artificial" or synthetic and, therefore, are not considered to be the result of "the laws of nature."

[0074] As used herein, the terms "comprising," "containing," "having," and "including" are used interchangeably and are to be understood as open-ended definitions, permitting the presence of additional members, parts, or elements. "Comprising" is considered the most closed definition, excluding additional elements beyond those constituting the defining features. Therefore, "comprising" is broader and encompasses the definition of "comprising."

[0075] As used herein, the term "about" refers to a value that is the same as a given value or that is + / - 5% different from a given value.

[0076] As used herein and in the claims, singular forms such as "a," "an," and "the" include plural forms unless the context clearly indicates otherwise.

[0077] As used herein, amino acids refer to the twenty naturally occurring amino acids encoded by sixty-one triplet codons. These twenty amino acids can be divided into three categories based on their charge properties: neutral, positive, and negative:

[0078] The "neutral" amino acids and their corresponding three-letter and one-letter codes and polarity are as follows: alanine (Ala, A; nonpolar, neutral), asparagine (Asn, N; polar, neutral), cysteine ​​(Cys, C; nonpolar, neutral), glutamine (Gln, Q; polar, neutral), glycine (Gly, G; nonpolar, neutral), isoleucine (Ile, I; nonpolar, neutral), leucine (Leu, L; nonpolar, neutral), methionine (Me, L; polar, neutral), t, M; nonpolar, neutral), phenylalanine (Phe, F; nonpolar, neutral), proline (Pro, P; nonpolar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; nonpolar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Val, V; nonpolar, neutral), histidine (His, H; polar, 10% positive charge, 90% neutral).

[0079] The "positively charged" amino acids are: arginine (Arg, R; polar, positive charge) and lysine (Lys, K; polar, positive charge).

[0080] The "negatively charged" amino acids are: aspartic acid (Asp, D; polar, negative charge) and glutamic acid (Glu, E; polar, negative charge).

[0081] As used herein, the term "determining" refers to detecting and / or quantifying a non-lactic hydroxy acid. The term "detecting" a non-lactic hydroxy acid refers to the general process of determining whether a non-lactic hydroxy acid is present. Detection does not require precise quantification of the non-lactic hydroxy acid, but rather provides information to the user of the method (e.g., whether the non-lactic hydroxy acid is present in a concentration above a certain threshold value). These threshold values ​​should be adjusted according to the respective application and sample. The term "quantifying" refers to determining the concentration or amount of the non-lactic hydroxy acid. Quantification can refer to determining the exact amount of an analyte, or it can refer to semi-quantitative determination of an analyte, for example, determining whether the amount of an analyte in a sample is within a certain range. Such a range can be a concentration range suitable for determining the analyte for the respective purpose.

[0082] According to one embodiment, the methods described herein can be used to quantify or detect non-lactic hydroxy acids. Depending on the specific application of the method, the method can be used to determine the amount of a non-lactic hydroxy acid or to determine whether a non-lactic hydroxy acid is present within a specific concentration range. Additionally, the methods described herein can be used to determine whether a non-lactic hydroxy acid is present above or below a specific threshold.

[0083] As used herein, the term "non-lactic hydroxy acid" refers to a hydroxy acid other than lactic acid, or a salt of a hydroxy acid other than lactic acid.

[0084] The term "lactic acid" refers to lactic acid or a salt thereof. Specifically, in the context of selectively removing lactic acid from a sample and in the context of non-lactic hydroxy acids, the term "lactic acid" refers to L-lactic acid or a salt thereof (ie, L-lactate).

[0085] According to one embodiment of the present invention, the non-lactic hydroxy acid is 2-hydroxybutyric acid, 3-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, 2-hydroxyisobutyric acid, D-lactic acid or a corresponding salt of any one of the foregoing.

[0086] According to one embodiment, the non-lactic hydroxy acid is a non-lactic 2-hydroxy acid.

[0087] As used herein, the term "non-lactic 2-hydroxy acid" refers to a subclass of non-lactic hydroxy acids. Thus, as used herein, the term "non-lactic 2-hydroxy acid" refers to a 2-hydroxy acid other than lactic acid, or a salt of a 2-hydroxy acid other than lactic acid. 2-hydroxy acids are also commonly referred to as alpha-hydroxy acids. 2-hydroxy acids are a class of chemical compounds consisting of a carboxylic acid with a hydroxyl substituent on the adjacent (alpha) carbon.

[0088] According to one embodiment, the non-lactic 2-hydroxy acid is a compound having the general formula I:

[0089]

[0090] in,

[0091] R1 is H or C 1-6 alkyl,

[0092] R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0093] The term "alkyl", when used alone or in combination with other groups or atoms, refers to a saturated straight or branched chain consisting of carbon atoms replaced only by 1 to 6 hydrogen atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, 1-methylpropyl, isobutyl, tert-butyl, 2,2-dimethylbutyl, 2,2-dimethylpropyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl, etc.

[0094] The term "aryl" refers to an aromatic monocyclic or bicyclic group containing 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, which may be optionally fused to a fully or partially saturated or unsaturated carbocyclic ring and may be substituted with one or more identical or different substituents, suitably one to three substituents. Examples of aryl groups include phenyl, naphthyl, indolealkyl, and the like.

[0095] The substituent "-C(O)OH" refers to a carboxylic acid substituent.

[0096] According to one embodiment, R1 is H or C 1-6 alkyl

[0097] According to a particular embodiment, R1 is H or -CH3.

[0098] According to a specific embodiment, C 1-6 The alkyl group is a C1 alkyl group.

[0099] According to one embodiment, R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0100] According to one embodiment, R2 is H.

[0101] According to one embodiment, R2 is C 6-8 Aryl.

[0102] According to a specific embodiment, C 6-8 The aryl group is a C6 aryl group.

[0103] According to one particular embodiment, R2 is phenyl.

[0104] According to a specific embodiment, R2 is H, C 6-8Aryl, or C optionally substituted by -C(O)OH 1-14 alkyl.

[0105] According to a particular embodiment, R2 is H, phenyl, or C optionally substituted with -C(O)OH. 1-14 alkyl.

[0106] According to one embodiment, R2 is C optionally substituted with -C(O)OH 1-20 alkyl.

[0107] According to one particular embodiment, R2 is -CH2-CH3.

[0108] According to one embodiment, R2 is H, phenyl, or C optionally substituted with -C(O)OH. 1-20 alkyl.

[0109] According to one embodiment of the present invention, the non-lactic 2-hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, 2-hydroxyisobutyric acid, D-lactic acid and the corresponding salts of any one of the foregoing.

[0110] According to a specific embodiment of the present invention, the non-lactic hydroxy acid is α-hydroxybutyric acid (2-hydroxybutyric acid, 2-HBA) or its corresponding salt (i.e., α-hydroxybutyrate). 2-HBA's abbreviations and synonyms include: HBA, α-HBA, and aHB.

[0111] According to another embodiment, 2-HBA is a chiral molecule having two enantiomers, (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid.

[0112] According to one embodiment, the sample can be any material that is relevant to or targeted for determining the presence of non-lactic hydroxy acids. Specifically, the sample is a human or animal sample, specifically any one of body fluids, interstitial fluid, blood, plasma, serum, skin fluid, urine, tears, sweat, saliva, skin, meat, tissue, eyeball, cornea, and gastric fluid.

[0113] According to a specific embodiment, the methods and means described herein use human blood as a sample. Blood contains various non-lactic hydroxy acids, as well as lactic acid.

[0114] According to one embodiment of the present invention, the sample contains or is suspected of containing non-lactic hydroxy acids and lactic acid.

[0115] According to a specific embodiment, the sample contains or is suspected of containing lactate at a concentration in the range of 1-2 mM.

[0116] According to a specific embodiment, the sample contains or is suspected of containing lactic acid at a concentration that is at least 10 times higher than 2-HBA.

[0117] According to a specific embodiment, the sample contains or is suspected of containing 2-HBA at a concentration in the range of 0.05-0.10 mM.

[0118] According to the present invention, the methods and means described herein are capable of measuring non-lactic hydroxy acids in samples suspected of also containing lactic acid (eg, blood samples containing lactic acid) by selectively removing lactic acid prior to measuring the non-lactic hydroxy acids.

[0119] According to one embodiment, described herein is a method for determining a non-lactic hydroxy acid in a sample, the sample comprising a non-lactic hydroxy acid and lactic acid, the method comprising the following consecutive steps:

[0120] i. selectively removing lactic acid from the sample;

[0121] ii. incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity;

[0122] iii. Determining non-lactic hydroxy acids in the sample.

[0123] According to one embodiment, described herein is a method for determining a non-lactic hydroxy acid in a sample, the sample comprising a non-lactic hydroxy acid and lactic acid, the method comprising the steps of:

[0124] i. prior to step ii and step iii, selectively removing lactic acid from the sample;

[0125] ii. incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity;

[0126] iii. Determining non-lactic hydroxy acids in the sample.

[0127] According to one embodiment, described herein is a method for determining 2-hydroxybutyrate in a sample, the sample comprising 2-hydroxybutyrate and lactate, the method comprising the following consecutive steps:

[0128] a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity;

[0129] b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity;

[0130] c. Determine 2-hydroxybutyric acid in the sample.

[0131] According to one embodiment, described herein is a method for determining 2-hydroxybutyrate in a sample, the sample comprising 2-hydroxybutyrate and lactate, the method comprising the steps of:

[0132] a. prior to step b and step c, selectively removing lactate from the sample by an enzyme having lactate oxidase activity;

[0133] b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity;

[0134] c. Determine 2-hydroxybutyric acid in the sample.

[0135] According to one embodiment, described herein is a method for determining a non-lactic 2-hydroxy acid in a sample, the sample comprising a non-lactic 2-hydroxy acid and optionally lactic acid, the method comprising the following consecutive steps:

[0136] i. optionally selectively removing lactic acid from the sample;

[0137] ii. incubating the sample with an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity;

[0138] iii. Determination of non-lactic 2-hydroxy acids in the sample.

[0139] According to one embodiment, described herein is a method for determining a non-lactic 2-hydroxy acid in a sample, the sample comprising a non-lactic 2-hydroxy acid and optionally lactic acid, the method comprising the steps of:

[0140] i. prior to step ii and step iii, optionally selectively removing lactic acid from the sample;

[0141] ii. incubating the sample with an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity;

[0142] iii. Determination of non-lactic 2-hydroxy acids in the sample.

[0143] According to one embodiment, selective removal of lactic acid from the sample is optional.

[0144] As used herein, the term "selectively remove" refers to removing lactate without substantially changing the amount or concentration of non-lactic hydroxy acids in the sample. Specifically, as used herein, the term "remove" refers to modifying lactate, such as oxidizing lactate to pyruvate.

[0145] Non-limiting examples of methods for selectively removing lactic acid include enzyme reactions, precipitation, emulsion liquid membrane separation technology, adsorption, extraction, polymerization, and esterification.

[0146] Non-limiting examples of extracting lactic acid are extraction (using vesicles), microfiltration, and reactive extraction (see Roque L. et al., 2020).

[0147] A non-limiting example of polymerization is polymerization using a catalyst and heat (see Lunt, James, 1998, and Chafran, Liana S. et al., 2019).

[0148] A non-limiting example of esterification is steam permeation assisted esterification (see Khunnonkwao, Panwana, et al., 2012).

[0149] According to one embodiment, modification of lactic acid is accompanied by accumulation of the modified lactic acid in the sample. Thus, the modified lactic acid is not necessarily removed from the sample but may remain in the sample during the determination of non-lactic hydroxy acids. For example, if oxidation of lactic acid is accompanied by accumulation of its reaction product, pyruvic acid, the reaction product, pyruvic acid, is not necessarily removed from the reaction mixture but may remain in the sample.

[0150] According to another alternative embodiment, the modified lactate may be removed from the sample.

[0151] According to one embodiment, pyruvate can be retained in the sample, for example, when a lactate oxidase with low product inhibition properties is used. Alternatively, the reaction product pyruvate can be removed from the reaction mixture.

[0152] As used herein, the term "enzyme" refers to any substance consisting entirely or mainly of proteins or polypeptides, which catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.

[0153] As used herein, the term "activity" refers to an enzyme-catalyzed reaction, for example, in the context of enzyme activity. Thus, an enzyme with activity is a molecule with functional activity, for example, a functional enzyme. A functional enzyme is characterized in that it has a catalytic center that can recognize an enzyme substrate and catalyze the conversion of the substrate into a transformation product. When enzyme activity is measured in a standard assay system, an enzyme variant is considered to be functional or to have functional activity, for example, an enzyme activity that is at least 50% of the activity of the parent (unmodified or wild-type enzyme), or at least 60%, 70%, 80%, 90%, 100% or even higher.

[0154] Enzyme activity is usually expressed in units. Here, one unit of enzyme activity is defined as the amount of enzyme that catalyzes the reaction of 1 μmol of substrate per minute under the corresponding assay conditions. For example, one unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of substrate (e.g. lactate or non-lactic hydroxy acid) per minute under the corresponding assay conditions. Specific activity is expressed in "U / mg", "U·mg -1” or “U per mg”. Volume activity is expressed in units per volume, such as “U / mL”, “U / ml”, “U per mL”, “U per ml”, “U·mL -1 ” or “U·ml -1 ”.

[0155] As used herein, if an enzyme is used in step i to selectively remove lactic acid, such enzyme is also referred to herein as enzyme A.

[0156] According to one embodiment of the present invention, the enzyme A is an enzyme having lactate oxidizing activity. In step i, the enzyme A does not substantially change the concentration of non-lactic acid hydroxy acids in the sample to be determined.

[0157] According to a particular embodiment, enzyme A is selected from the group consisting of lactate oxidase, lactate monooxygenase and lactate dehydrogenase.

[0158] According to one embodiment of the present invention, an enzyme having non-lactic acid hydroxy acid oxidation activity is used in step ii. Thus, the enzyme having non-lactic acid hydroxy acid oxidation activity is also referred to as enzyme B herein.

[0159] According to a specific embodiment, enzyme B has non-lactic acid hydroxy acid oxidation activity, but may also be able to oxidize lactic acid. However, since lactic acid has been removed in step i, the lactic acid oxidation activity of enzyme B does not interfere with the determination of non-lactic acid hydroxy acids.

[0160] According to a specific embodiment, enzyme B is selected from the group consisting of non-lactic hydroxy acid dehydrogenase, non-lactic hydroxy acid oxidase and non-lactic hydroxy acid monooxygenase. Thus, enzymes such as LDH, FCb2, LOx or hydroxy acid oxidase can be used as enzyme B.

[0161] In general, for example, whether to use LDH, FCb2, and LOx as enzyme A or enzyme B depends on the substrate specificity of the particular enzyme and the specific non-lactic hydroxy acid to be measured in the sample.

[0162] According to one embodiment of the present invention, enzyme A is an enzyme that can modify lactic acid to the extent that enzyme B cannot catalyze the reaction with the modified lactic acid.

[0163] According to a specific embodiment of the present invention, enzyme A is an enzyme that is capable of selectively oxidizing lactic acid but does not substantially oxidize non-lactic acid hydroxy acids.

[0164] According to one embodiment of the present invention, the enzyme A is an enzyme having lactate oxidizing activity.

[0165] Generally speaking, in the context of an oxidizing agent (e.g., an enzyme with oxidizing activity), the term "oxidizing" refers to an agent that is capable of oxidizing a substance and acquiring or "accepting" electrons from the substance. Thus, the enzyme has "substance oxidizing activity." Such substances may also be referred to as substrates. Thus, an enzyme with lactate oxidizing activity catalyzes the oxidation of lactic acid. An enzyme with non-lactic hydroxy acid oxidizing activity catalyzes the oxidation of non-lactic hydroxy acids. Herein, in the context of an enzyme with oxidizing activity, the term "capable of oxidizing" may be used in place of the term "oxidizing."

[0166] Generally speaking, enzymes with oxidative activity acquire or accept one or more electrons from a substance. This process reduces the enzyme itself or its cofactor. In this reduced state, the enzyme cannot catalyze another oxidation reaction of the substance. Therefore, before catalyzing another oxidation reaction of the substance, the enzyme or its cofactor must be reoxidized by transferring the acquired electrons to an electron acceptor.

[0167] According to one embodiment, the enzyme or enzyme cofactor can be reoxidized by transferring the acquired electrons to oxygen, an electrochemically active molecule (eg, a redox mediator), or an electrode.

[0168] In general, the preference or extent to which an enzyme transfers electrons to an electron acceptor depends on the specific enzyme and the specific electron acceptor used.

[0169] Generally speaking, if the enzyme uses dioxygen as the preferred electron acceptor for enzymatic reoxidation, the enzyme with substance-oxidizing activity is called an "oxidase." If the enzyme uses an electron acceptor other than dioxygen as the preferred electron acceptor for enzymatic reoxidation, the enzyme is usually called a "dehydrogenase."

[0170] According to a particular embodiment of the present invention, enzyme A may be an enzyme having lactate oxidase activity, or alternatively an enzyme having lactate dehydrogenase activity.

[0171] The term "lactate oxidase activity" refers to the activity of an enzyme that catalyzes the oxidation of lactate with dioxygen as an electron acceptor, forming pyruvate and hydrogen peroxide as products. In this process, two electrons are transferred from lactate to an enzyme cofactor (e.g., FAD), and the resulting electrons are subsequently transferred to dioxygen to produce hydrogen peroxide.

[0172] According to a specific embodiment of the present invention, if an enzyme having lactate oxidase activity is used as enzyme A, dioxygen can be used as an electron acceptor for enzyme reoxidation.

[0173] According to one embodiment, enzyme A is lactate oxidase (LOx). Lactate oxidase belongs to the E.C1.1.3.2 enzyme family. In particular, a variety of lactate oxidases can be used in the methods described herein, such as lactate oxidases from Aerococcus viridans, Nostoc sp. (PCC7120), Lactobacillus jensenii, Lysinibacillus sphaericus, Chlamydomonas reinhardtii, Alicycliphilus denitrificans, Lacticaseibacillus rhamnosus, Lentilactobacillus shilgardii, Roseobacter sp. (strain GAI101), Streptococcus iniae, and Pediococcus acidilactici. Specifically, the lactate oxidase can be a functional variant of any of the aforementioned lactate oxidases, which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding amino acid sequence of the aforementioned lactate oxidase.

[0174] According to another embodiment, enzyme A may be lactate monooxygenase. Thus, a variety of lactate monooxygenases may be used in the methods described herein, such as lactate 2-monooxygenase from Mycolicibacterium smegmatis.

[0175] According to a specific embodiment, the lactate oxidase is characterized by its enantioselectivity and specificity for the natural lactate substrate L-lactic acid.

[0176] According to a specific embodiment of the present invention, enzyme A can be an engineered variant of an enzyme. For example, the engineered variant can be an oxidase engineered to use lactate as a substrate instead of 2-HBA or the like (by reducing the activity of the enzyme on 2-HBA).

[0177] According to another alternative embodiment of the present invention, enzyme A may be an engineered variant of an enzyme that is naturally incapable of oxidizing lactate. For example, an oxidase that naturally oxidizes molecules other than lactate may be engineered to use lactate as a substrate.

[0178] In a specific embodiment, the LOx described herein is a functionally active variant of a LOx peptide sequence comprising one or more point mutations in the nucleotide sequence encoding the LOx sequence compared to the corresponding parent LOx sequence. Specifically, it comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations, wherein these mutations result in the substitution, addition, or deletion of one or more amino acids. Specifically, the functional variant of the LOx peptide sequence is a full-length LOx peptide sequence comprising the point mutations, or a fragment of the full-length LOx peptide sequence that retains enzymatic activity. Specifically, according to the use of the Lox as an enzyme A or enzyme B described elsewhere herein, the LOx sequence variant is functionally active if it is capable of converting L-lactic acid to pyruvate, or is capable of converting a non-lactic hydroxy acid to the corresponding oxidized non-lactic hydroxy acid. Specifically, the functionally active variants of the LOx sequence have an enzymatic activity towards lactate or a non-lactic hydroxy acid that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the enzymatic activity of the corresponding parent LOx sequence. Specifically, the functionally active variants of the LOx sequence have at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the enzymatic activity of their corresponding parent LOx sequence as determined using the following assay.

[0179] For example, the lactate oxidase activity of the enzyme can be determined by the Amplex Red assay. Thus, a 5-well plate containing 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM Amplex Red (resorufin: ε560 nm = 54.0 mM) was used. -1 cm -1 Oxidase activity is measured by a peroxidase-coupled reaction with oxygen (O2). Oxygen is present at an ambient concentration of approximately 250 μM (Kadowaki, MAS, et al., 2020). Oxidase activity of enzymes using non-lactic hydroxy acids as substrates can be determined by using the method for measuring lactate oxidase activity and substituting non-lactic hydroxy acids for lactate.

[0180] As used herein, the term "lactate dehydrogenase activity" refers to the activity of an enzyme that catalyzes the oxidation of lactate, with a molecule other than dioxygen serving as an electron acceptor to form an oxidized lactate molecule (e.g., pyruvate) and a corresponding reduced electron acceptor as products. For example, such reactions are catalyzed by lactate dehydrogenase (LDH) or flavocytochrome b2 (FCb2).

[0181] According to a specific embodiment of the present invention, if an enzyme having lactate dehydrogenase activity is used as enzyme A, a molecule other than dioxygen is used as the electron acceptor for enzyme reoxidation. In a more specific embodiment, a system for regenerating spent electron acceptors can be implemented in the methods described herein.

[0182] According to a specific embodiment, the regeneration system of the electron acceptor may include an enzymatic, chemical, electrochemical, homogeneous catalytic, photocatalytic, or heterogeneous catalytic regeneration system.

[0183] As used herein, the term "lactate dehydrogenase" is abbreviated as LDH. Generally, LDH is an enzyme that catalyzes the oxidation of lactate to pyruvate. During this process, two electrons are transferred from lactate to a cofactor of LDH (e.g., FMN or FAD). The resulting electrons are then transferred to a suitable electron acceptor (e.g., DCIP). Therefore, LDH is generally unresponsive or nearly unresponsive to dioxygen as an electron acceptor.

[0184] According to one embodiment, LDH is used in the methods described herein as enzyme A. Specifically, a variety of LDHs can be used in the methods described herein, including but not limited to NAD+-dependent LDHs, such as LDHs from Sus scrofa, Homo sapiens, Mus musculus, Rattus norvegicus, Lactobacillus casei, Geobacillus stearothermophilus, Lactiplantibacillus pentosus, Deinococcus radiodurans, Thermus caldophilus, Thermotoga maritima, Bacillus subtilis, and Thermus thermophilus; or FAD- or FMN-dependent LDHs, such as LDHs from Pediococcus acidilactici. Specifically, the LDH can be a functional variant of any of the aforementioned LDHs, which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding amino acid sequence of the aforementioned LDH.

[0185] According to a specific embodiment of the present invention, enzyme A can be an engineered variant of an enzyme. For example, an engineered variant of a dehydrogenase (e.g., lactate dehydrogenase) can be engineered to use lactate as a substrate while not using 2-HBA or the like as a substrate (by reducing the activity of the enzyme on 2-HBA).

[0186] According to another alternative embodiment of the present invention, enzyme A can be an engineered variant of an enzyme that is naturally incapable of oxidizing lactate. For example, a dehydrogenase that naturally oxidizes a molecule other than lactate can be engineered to use lactate as a substrate, and thus, the engineered variant can also be an enzyme having lactate dehydrogenase activity.

[0187] According to a particular embodiment, the LDH used as enzyme A is characterized by its enantioselectivity and specificity for the natural lactate substrate L-lactic acid.

[0188] In a specific embodiment, the LDH described herein is a functionally active variant of an LDH peptide sequence comprising one or more point mutations in the nucleotide sequence encoding the LDH sequence compared to the corresponding parent LDH sequence. Specifically, it comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 point mutations, specifically, these mutations result in substitution, addition or deletion of one or more amino acids. Specifically, the functionally active variant of the LDH peptide sequence is a full-length LDH peptide sequence comprising a point mutation, or a fragment of the full-length LDH peptide sequence that retains enzymatic activity. Specifically, according to the application of LDH as enzyme A or enzyme B as described elsewhere herein, if the LDH sequence variant is capable of converting L-lactic acid into pyruvic acid, or is capable of converting a non-lactic hydroxy acid into the corresponding oxidized non-lactic hydroxy acid, then the variant has functional activity. Specifically, the functionally active variants of the LDH sequence have an enzymatic activity towards lactic acid or a non-lactic hydroxy acid of at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the enzymatic activity of the corresponding parent LDH sequence. Specifically, the functionally active variants of the LDH sequence have at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the enzymatic activity of the corresponding parent LDH sequence as determined using the following assay.

[0189] The enzyme activity of lactate dehydrogenase or LDH and its variants can be determined by the DCIP test, which is performed by detecting the extinction coefficient of 2,6-dichlorophenol indophenol sodium salt hydrate (DCIP) at 520 nm or 600 nm at 30 ° C (molar extinction coefficient ε520 nm = 6.8 mM). -1 cm -1 ; Molar extinction coefficient ε600nm=8.98mM -1 cm -1) is used to assess enzyme activity, for example, by referring to previously described methods (WJ Bao, SN et al. (1993); Krondorfer, I. et al. (2014); Harreither, W. et al. (2011)). The assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCl, and 3 mM KCl and contains 10 mM lactate and 120 μM DCIP (as an electron acceptor). One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of lactate per minute under the assay conditions. Since each lactate molecule acquires two electrons and transfers them to a single DCIP molecule, the stoichiometric ratio of the reaction between lactate and DCIP is 1:1. If the activity of other substrates is to be detected, lactate can be replaced with other compounds. If the activity of a non-lactic hydroxy acid dehydrogenase is to be detected, lactate is replaced with a specific non-lactic hydroxy acid. For example, if the 2-HBA dehydrogenase activity of the enzyme is to be measured, lactate is replaced with 2-HBA.

[0190] The enzymatic activity of LDH variants can also be determined by measuring 500 μM 1,4-benzoquinone (1,4-BQ) (molar extinction coefficient ε290 nm = 2.24 mM -1 cm -1 ) or 160 μM ferrocene hexafluorophosphate (FcPF6) (molar extinction coefficient ε300nm=4.3mM -1 cm -1 ) is assessed by a colorimetric decrease in the amount of ferrocene hexafluorophosphate (FFP). The assay mixture is prepared identically to the DCIP assay, but contains 500 μM 1,4-benzoquinone or 160 μM ferrocene hexafluorophosphate (FFP) instead of DCIP. Because each lactate molecule acquires two electrons and transfers them to a single 1,4-benzoquinone molecule, the stoichiometric ratio of the reaction between lactate and 1,4-benzoquinone is 1:1. Conversely, the stoichiometric ratio of the reaction between lactate and ferrocene hexafluorophosphate is 1:2, because each lactate molecule acquires two electrons and transfers them to two FFP molecules. To test activity on other substrates, lactate can be replaced with other compounds (Brugger, D. et al., 2014; Sygmund, C. et al., 2011).

[0191] The enzymatic activity of LDH and its variants can also be determined using oxygen as the electron acceptor, in which case the oxidase activity of LDH is specifically measured. If the ability of LDH to transfer electrons to oxygen is very low, its oxidase activity may not be detected. As an example of a suitable method, the Amplex Red test can be used. This method uses a 5-well plate containing 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM Amplex Red (resorufin: ε560nm = 54.0 mM -1 cm -1) to measure oxidase activity using a peroxidase-coupled reaction. Oxygen is present at an ambient concentration of approximately 250 μM (Kadowaki, MAS, et al. (2020)).

[0192] According to a specific embodiment, the enzyme having lactate oxidation activity (e.g., lactate oxidase activity or lactate dehydrogenase activity) used as enzyme A in the methods described herein does not substantially change the concentration or amount of non-lactic hydroxy acids. For example, the enzyme having lactate oxidase or dehydrogenase activity used as enzyme A does not have or substantially does not have 2-HBA oxidase activity or 2-HBA dehydrogenase activity.

[0193] According to a particular embodiment, the specific activity of enzyme A towards non-lactic hydroxy acids is lower than 10%, 5%, 4%, 3%, 2%, 1% relative to its specific activity towards lactic acid. Specifically, the specific activity of enzyme A towards non-lactic hydroxy acids relative to its specific activity towards lactic acid is 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.

[0194] According to a specific embodiment, the sample is treated with enzyme A for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more.

[0195] According to a specific embodiment, the sample is treated with enzyme A at room temperature.

[0196] According to a specific embodiment, the sample is treated with enzyme A at 10, 15, 20, 25, 30, 35 or 40°C.

[0197] According to a specific embodiment, the final concentration of enzyme A added to the sample is at least 0.01 mg / mL. Specifically, the final concentration of enzyme A added to the sample is 0.01 to 0.1 mg / mL or higher.

[0198] According to one embodiment, the duration of the selective removal of lactic acid (pretreatment step, step i) depends on the specific activity of the enzyme towards lactic acid and the final concentration of the enzyme in the sample. Thus, the duration of the pretreatment step can be adjusted depending on the specific enzyme and the specific concentration of the enzyme in the sample.

[0199] According to one embodiment, enzyme A and optionally catalase may be inactivated after selective removal of lactate and before incubation with enzyme B. For example, the sample may be heated to 100° C. for 5, 10, 15 or 20 minutes to inactivate the enzymes.

[0200] According to one embodiment, the determination of non-lactic hydroxy acids described herein can be calibrated by a predetermined correction factor. Such a predetermined correction factor may be derived from a side reaction of enzyme A with a specific non-lactic hydroxy acid.

[0201] According to one embodiment, enzyme A does not substantially change the concentration of non-lactic hydroxy acids in the sample. Thus, the extent to which the concentration of non-lactic hydroxy acids substantially changes can be predetermined and a correction factor for the non-lactic hydroxy acid determination can be determined.

[0202] According to one embodiment of the present invention, hydrogen peroxide may be produced as a byproduct during the step of selectively removing lactic acid from the sample. Therefore, a reagent capable of removing hydrogen peroxide may be added to the method of the present invention. This reagent may be added during step i (i.e., the step of selectively removing lactic acid from the sample) of the method of the present invention. Alternatively, this reagent may be added after step i but before step ii, or simultaneously with step ii.

[0203] According to a specific embodiment, the agent capable of removing hydrogen peroxide is an enzyme having catalase activity. Catalase activity refers to the activity of an enzyme that catalyzes the decomposition of hydrogen peroxide into water and molecular oxygen. Non-limiting examples of such enzymes are catalases, such as catalases from Aspergillus niger, bovine liver, human erythrocytes, and other known sources.

[0204] The catalase activity of the enzyme or the enzymatic activity of catalase and its variants can be measured spectrophotometrically (continuous spectrophotometric rate reduction assay at 240 nm). During this process, the rate of disappearance of H2O2 is tracked by observing the rate of decrease in absorbance at 240 nm. One unit of catalase decomposes 1.0 μmol of H2O2 per minute at 25°C and pH 7.0, while the concentration of H2O2 decreases from 10.3 mM to 9.2 mM. Beers, RF Jr, & Sizer, IW (1952) described a spectrophotometric method for determining the decomposition of hydrogen peroxide by catalase.

[0205] According to a specific embodiment, catalase is added to the sample in step i. Thus, catalase can decompose the hydrogen peroxide produced by lactate oxidase into water and molecular oxygen. The generated molecular oxygen can again serve as an electron acceptor for lactate oxidase.

[0206] According to one embodiment, catalase is used to enhance the processing and / or detection reaction of lactate oxidase.

[0207] According to one embodiment of the present invention, enzyme B is an enzyme having non-lactic acid hydroxy acid oxidizing activity. Enzyme B can be an enzyme having non-lactic acid hydroxy acid dehydrogenase activity, or an enzyme having non-lactic acid hydroxy acid oxidase activity.

[0208] As used herein, the term "non-lactic acid hydroxy acid oxidation activity" refers to the activity of an enzyme that catalyzes the oxidation of non-lactic acid hydroxy acids. Alternatively, the term "hydroxy acid oxidation activity" may also be used, as the enzyme may also catalyze lactate oxidation as described elsewhere herein.

[0209] As used herein, the term "non-lactic hydroxy acid dehydrogenase activity" refers to an enzyme activity that catalyzes the oxidation of a non-lactic hydroxy acid, wherein a molecule other than dioxygen serves as an electron acceptor, thereby forming an oxidized hydroxy acid and a corresponding reduced electron acceptor as products. Such enzymes having dehydrogenase activity are typically unresponsive or non-responsive to accepting dioxygen as an electron acceptor. Alternatively, the term "hydroxy acid dehydrogenase activity" may also be used, as such enzymes may also catalyze the oxidation of lactate as described elsewhere herein.

[0210] Generally speaking, an enzyme having hydroxy acid dehydrogenase activity or hydroxy acid oxidase activity may also have corresponding lactate dehydrogenase activity or lactate oxidase activity. Since lactic acid present in the sample is selectively removed in step i, lactic acid is no longer present in step ii. Therefore, regardless of whether the enzyme used has the theoretical ability to oxidize lactic acid, the enzymatic reaction in step ii will not be interfered with by lactic acid.

[0211] According to one embodiment of the present invention, the enzyme having non-lactic acid hydroxy acid dehydrogenase activity is an enzyme having 2-HBA dehydrogenase activity. For example, flavocytochrome b2 (FCb2) and lactate dehydrogenase (LDH) are enzymes having 2-HBA dehydrogenase activity.

[0212] According to one embodiment, the enzyme having non-lactate hydroxyacid dehydrogenase activity is flavocytochrome b2 (FCb2) or lactate dehydrogenase (LDH).

[0213] According to another embodiment of the present invention, the enzyme having hydroxyacid dehydrogenase activity may also be a functionally active variant, such as a functionally active variant of FCb2 or LDH.

[0214] The term "lactate dehydrogenase" (LDH) is described elsewhere herein as an enzyme that catalyzes the oxidation of lactate to pyruvate, during which two electrons are transferred from lactate to a cofactor of LDH (e.g., FMN), and the resulting electrons are subsequently transferred to a suitable electron acceptor (e.g., DCIP), and is generally unresponsive or barely reactive to dioxygen as an electron acceptor. However, in the methods described herein, if the term "LDH" or "lactate dehydrogenase" is used in the context of enzyme B, it refers to an enzyme that catalyzes the oxidation of a non-lactic hydroxy acid (e.g., 2-HBA) to its corresponding oxidized form, during which two electrons are transferred from the non-lactic hydroxy acid to a cofactor of LDH (e.g., FMN), and the resulting electrons are subsequently transferred to a suitable electron acceptor (e.g., DCIP), and is generally unresponsive or barely reactive to dioxygen as an electron acceptor.

[0215] According to one embodiment, several different lactate dehydrogenases are known and described elsewhere herein. Thus, the lactate dehydrogenase can be FAD-dependent or NAD+-dependent. For example, a FAD-dependent lactate dehydrogenase is the lactate dehydrogenase from Pediococcus acidilactici, which was previously described as a lactate oxidase (see Ashok, Y. et al., 2020).

[0216] According to one embodiment of the present invention, LDH is used as enzyme B in the method of the present invention.

[0217] According to a specific embodiment, in the method according to the present invention, LDH is used as enzyme B, which is capable of oxidizing 2-HBA.

[0218] According to another alternative embodiment of the present invention, FCb2 is used as enzyme B in the method according to the present invention.

[0219] According to a specific embodiment, in the method according to the present invention, FCb2 is used as enzyme B, which is capable of oxidizing 2-HBA.

[0220] The term "FCb2" refers to L-lactate-cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b2, FCb2, L-lactate cytochrome c oxidoreductase). Generally, FCb2 catalyzes electron transfer from L-lactate to cytochrome c in yeast mitochondria. In yeast, L-lactate is converted to pyruvate by L-lactate cytochrome c oxidoreductase (EC 1.1.2.3), which is referred to herein as "flavocytochrome b2" or "FCb2." Native yeast flavocytochrome b2 (FCb2) has two functional domains connected by a "hinge" linker (57 kDa monomer). FCb2 from Saccharomyces cerevisiae is the best-studied representative and has been crystallized (PDB 1FCB). However, in the methods described herein, if the term "FCb2" or its synonyms are used in the context of describing an enzyme having the ability to oxidize a non-lactic hydroxy acid to be determined, these terms refer to an enzyme that catalyzes the oxidation of a non-lactic hydroxy acid (e.g., 2-HBA) to its corresponding oxidized form, wherein FCb2 catalyzes the transfer of electrons from the non-lactic hydroxy acid to cytochrome c.

[0221] According to a specific embodiment, the FCb2 described herein may comprise a sequence based on the mature form of FCb2 naturally present in the intermembrane space of yeast mitochondria, comprising a cytochrome b2 domain, a flavin domain, a hinge region connecting the cytochrome b2 domain and the flavin domain, and a tail region located at the C-terminus. The mature FCb2 peptide sequence is the sequence of the FCb2 peptide naturally present in yeast mitochondria (specifically, in the mitochondrial intermembrane space).

[0222] According to a specific embodiment, the FCb2 described herein comprises an FCb2 peptide sequence comprising at least a yeast heme domain and a yeast flavin domain.

[0223] In one embodiment, the FCb2 described herein is a functionally active variant of the FCb2 peptide sequence found in the intermembrane space of yeast mitochondria, comprising one or more point mutations in the nucleotide sequence encoding the FCb2 sequence compared to the corresponding native mature FCb2 sequence. Specifically, it comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations, each of which results in the replacement, addition, or deletion of one or more amino acids. Specifically, a functional variant of the FCb2 peptide sequence is a full-length mature FCb2 peptide sequence comprising a point mutation, or a fragment of the full-length mature FCb2 peptide sequence that retains enzymatic activity. Specifically, a variant of the FCb2 sequence is functionally active if, using the methods of the present invention, it is capable of converting a non-lactic hydroxy acid to be assayed into its corresponding oxidized form. Specifically, when a non-lactic hydroxy acid is used as a substrate, the enzymatic activity of the functionally active variant of the FCb2 sequence is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even higher than the enzymatic activity of the corresponding wild-type FCb2 sequence. Specifically, when the enzymatic activity is determined using the CytC assay and a corresponding non-lactic hydroxy acid (e.g., 2-HBA) as a substrate, the functionally active variant of the FCb2 sequence has at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or even higher than the enzymatic activity of the corresponding wild-type FCb2 sequence.

[0224] The enzymatic activity of the flavocytochrome b2 variant can be readily determined by assays known in the art, such as colorimetric reduction assays for cytochrome c, ferrocyanide, or 2,6-dichloroindophenol (DCIP). Specifically, the FCb2 described herein is determined by the CytC assay described by Diêp Lê et al. (2009), using the corresponding non-lactic hydroxy acid or lactic acid as a substrate.

[0225] According to a specific embodiment, FCb2 may comprise the amino acid sequence of FCb2 from the source: S. cerevisiae, W. anomalus, K. marxianus, O. parapolymorpha, Candida glabrata, Kluyveromyces lactis, Lachancea thermotolerans, Saccharomycodes ludwigii, Naumovozyma castelli, Zygosaccharomyces bailii, Zygosaccharomyces parabalii, Lachancea mirantina, Tetrapis poraphaffii, Saccharomyces eubayanus), Saccharomyces kudriavzevii, Saccharomyces paradoxus, Vanderwaltozyma polyspora, Lachancea dasiensis, Wickerhamomyces ciferri, Kluyveromyces dobzhanskii, Kazachstania naganishii, Zygosaccharomyces mellis, Kazachstania saulgeensis, Candida boidinii, Lachancea fermentati, Zygosaccharomyces rouxii rouxii), Cyberlindnera fabianii, Cyberlindnera jadinii, Kazachstania africana, Lachancea quebecensis, Kuraishiacapsulata), Torulaspora delbrueckii, Komogatella pastoris, Komagatella phaffii, Lachancea nothofagi, or Naumovomyces dairenensis. Specifically, FCb2 may be a functional variant of any of the above-mentioned FCb2s, comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above-mentioned FCb2 amino acid sequences.

[0226] Specifically, the recombinant FCb2 described herein comprises a peptide sequence of FCb2 derived from Saccharomyces cerevisiae, Kluyveromyces marxianus, Wickerhamomyces anomalus, Naumovozyma castelli, or Cyberlindnera fabianii.

[0227] The amino acid sequence of a polypeptide derived from an organism can be easily obtained from a publicly available database, for example, from the database provided by the National Center for Biotechnology Information (NCBI).

[0228] According to another alternative embodiment, enzyme B may also be an enzyme having non-lactate hydroxy acid oxidase activity.

[0229] As used herein, the term "non-lactic hydroxy acid oxidase activity" refers to an enzyme activity that catalyzes the oxidation of a non-lactic hydroxy acid with dioxygen as an electron acceptor to form the corresponding oxidized non-lactic hydroxy acid and hydrogen peroxide as products. In this process, two electrons are transferred from the non-lactic hydroxy acid to a cofactor of the enzyme (e.g., FAD cofactor), and the electrons gained are subsequently transferred to dioxygen to produce hydrogen peroxide. Alternatively, the term "hydroxy acid oxidase activity" may be used, as this enzyme may also catalyze the oxidation of lactate as described elsewhere herein.

[0230] According to a particular embodiment of the present invention, the enzyme having non-lactate hydroxyacid oxidase activity may be selected from enzyme class EC: 1.1.3.15.

[0231] According to a specific embodiment, the enzyme having non-lactic acid hydroxyacid oxidase activity can be a hydroxyacid oxidase from mouse (Mus musculus), rat (Rattus norvegicus), Homo sapiens (Homo sapiens), Arabidopsis thaliana (Arabidopsis thaliana), or any other known enzyme having hydroxyacid oxidase activity.

[0232] According to specific embodiments of the present invention, the enzymes used in the methods described herein can be active in acidic, neutral, or alkaline pH ranges. Specifically, the enzymes of the present invention can be used at a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or the pH of any body fluid (e.g., sweat or blood). Typically, the pH of blood is between 7.35 and 7.45.

[0233] According to a specific embodiment of the present invention, in the methods described herein, a lactate dehydrogenase-based detection system (flavocytochrome b2, Fcb2) is used in combination with lactate oxidase (LOx)-dependent lactate removal. Specifically, these enzymes either have a high turnover rate for the target analyte (2-HBA) or have high specificity for interfering substances (e.g., lactate).

[0234] According to one embodiment, described herein is the use of oxygen-dependent enzymes in combination with oxygen-independent enzymes to achieve undisturbed detection or undisturbed colorimetric reactions. For example, the use of LOx to remove lactate does not affect the colorimetric reaction of FCb2.

[0235] According to another embodiment, the methods described herein may include combining an engineered lactate oxidase variant specific for L-lactic acid with an engineered flavocytochrome b2 variant having improved reactivity to 2-HBA. In this process, the lactate oxidase variant is used in a pretreatment step to specifically oxidize L-lactic acid. The improved FCb2 variant is used to oxidize 2-HBA while simultaneously developing the reagent, thereby converting the 2-HBA concentration into a quantifiable signal (e.g., absorbance, fluorescence).

[0236] As used herein, the term "incubation" refers to contacting a sample with an enzyme to allow the enzyme to react with the non-lactic hydroxy acid. In this process, for example, after adding the enzyme, the incubation can be performed directly in the sample. Alternatively, before adding the enzyme, an aliquot is taken from the sample and mixed with a suitable liquid (e.g., a buffer). Depending on the specific assay method, this suitable liquid may also contain other compounds, such as compounds or electron acceptors required for colorimetric detection.

[0237] According to one embodiment of the present invention, the step of incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity can be performed sequentially or simultaneously with the step of determining the non-lactic acid hydroxy acid in the sample.

[0238] According to one embodiment of the present invention, the enzyme having non-lactic hydroxy acid oxidizing activity can be any enzyme capable of oxidizing a non-lactic hydroxy acid and transferring the resulting electrons to a suitable electron acceptor. This electron acceptor can be any suitable molecule that can be used to measure the electron transfer reaction using a colorimetric, photometric, fluorometric, or electrochemical method. In this process, the signal obtained by detecting the electron transfer reaction is used to determine the non-lactic hydroxy acid in the sample.

[0239] According to an alternative embodiment, if an enzyme with hydroxy acid oxidase activity is used, the electron acceptor may be dioxygen. In this case, hydrogen peroxide is produced by electron transfer from the non-lactic hydroxy acid to dioxygen. The hydrogen peroxide can also be detected by a suitable method, such as colorimetry, photometry, fluorescence, bioelectrochemistry, or electrochemistry, possibly using an enzyme-based assay. In this process, the presence or amount of the non-lactic hydroxy acid can be calculated by detecting hydrogen peroxide.

[0240] According to another alternative embodiment of the present invention, the electron acceptor for the non-lactic hydroxy acid oxidation reaction can also be a polypeptide. This polypeptide is naturally associated with an enzyme capable of oxidizing non-lactic hydroxy acids, or is artificially linked to the enzyme. For example, the enzyme FCb2 contains a heme domain that accepts electrons from the cofactor of the FCb2 catalytically active domain (i.e., the flavin domain) and is capable of oxidizing non-lactic hydroxy acids. In addition, the polypeptide used as an electron acceptor can transfer the obtained electrons to a terminal electron acceptor. Examples of such terminal electron acceptors are molecules such as cytochrome c, ferrocyanide, or 2,6-dichloroindophenol (DCIP), which can be used in colorimetric and photometric detection methods. Another example of a terminal electron acceptor is an electrode surface. The presence or amount of non-lactic hydroxy acids in a sample can be derived from the signal obtained by a specific detection method.

[0241] According to one embodiment of the present invention, the non-lactic hydroxy acid is determined by a colorimetric, photometric, fluorometric or electrochemical method.

[0242] The term "colorimetry" as used herein refers to the use of colorimetric analysis to determine the presence or amount of non-lactic hydroxy acids in a sample. In a colorimetric analysis, the concentration of a chemical element or chemical compound in a solution is determined by using a colorimetric reagent. The colorimetric reagent can be detected by visual observation or using a suitable device (e.g., a colorimeter). In the enzymatic assays described herein, the color development reaction is preceded by a reaction catalyzed by an enzyme (e.g., an enzyme capable of oxidizing non-lactic hydroxy acids). A common example of such a colorimetric assay is detection using a colored complex formed by hydrogen peroxide and ABTS with a peroxidase.

[0243] As used herein, the term "photometry" refers to the use of a photometer or spectrophotometer to track the progress of an enzymatic reaction by measuring changes in the intensity of light absorbed or scattered by the reaction solution, thereby enzymatically determining a substance in a sample, or can also be used to determine enzyme activity. Colorimetric detection can be coupled or performed using a photometer.

[0244] As used herein, the term "fluorescence" refers to a fluorescence assay that detects an enzyme reaction or determines the concentration of a substrate or product by measuring the difference in the fluorescence spectra of the substrate and product. Thus, the substrate and product may differ from the direct substrate and product of the enzyme described herein, for example, from a non-lactic hydroxy acid and an oxidized non-lactic hydroxy acid. The Amplex red assay described elsewhere herein is an example of an assay based on fluorescence detection methods.

[0245] The term "electrochemical" as used herein refers to the use of electrochemical biosensors, which are based on measuring changes in conductivity, resistance, or capacitance at the biosensor surface in response to a biological binding event. In such electrochemical biosensors, a biorecognition molecule is immobilized on one of the electrodes. When an analyte binds to the biorecognition molecule, a redox reaction triggered by the biological interaction triggers a change in the electrical properties, providing a sensor signal. Electrochemical biosensors primarily rely on enzyme-catalyzed reactions to generate a current or potential difference, which is then detected.

[0246] According to one embodiment of the present invention, the electron acceptor used in the non-lactic hydroxy acid oxidation reaction for determining the amount of non-lactic hydroxy acid in a sample can also be an electrode equipped with an enzyme capable of oxidizing the non-lactic hydroxy acid. Such an electrode can be part of a biosensor.

[0247] According to one embodiment of the present invention, the oxidation of non-lactic hydroxy acids by enzymes capable of oxidizing non-lactic hydroxy acids can be detected by a sensor (particularly a bioelectrochemical sensor) configured to detect and / or quantify non-lactic hydroxy acids in a sample by (bio) electrochemical redox reactions. These reactions can generally be converted into electrical signals that can be correlated with the amount or concentration of the analyte non-lactic hydroxy acid.

[0248] Electrochemical biosensors can be impedance-based, potentiometric, or amperometric. In amperometric biosensors, the biochemical signal is converted into a quantifiable amperometric signal.

[0249] As described by Rocchitta G. et al. (2016), amperometric biosensors are generally divided into three generations based on the electron transfer method used to measure the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediator, and cofactor). First-generation biosensors measure the concentration of analytes and / or enzymatic reaction products that diffuse to the transducer surface and generate an electrical response. They are also called mediator-free amperometric biosensors. Typically, oxidases are used in first-generation biosensors. Oxidase-based biosensors require molecular oxygen as a second substrate, making them oxygen-dependent. Second-generation biosensors require an electron mediator to transfer electrons obtained in the enzymatic reaction to the transducer surface, thereby generating an electrical response. In third-generation biosensors, direct electron transfer is achieved between redox-active biomolecules (i.e., enzymes) and the electrode surface.

[0250] According to one embodiment of the present invention, to electrochemically determine non-lactic hydroxy acids, an electrode containing an enzyme capable of oxidizing the non-lactic hydroxy acid is contacted with a sample. This contact between the electrode and the sample can be achieved by any means that allows the electrode and the sample to be contacted, allowing the enzyme to react with the non-lactic hydroxy acid or a sample suspected of containing the non-lactic hydroxy acid.

[0251] The term "electrode" refers to any suitable surface that can accept electrons from enzymes without a medium, with a medium or in a direct electron transfer mode. Therefore, an electrode is a material that can accept electrons. In addition, the electrode can be made of any suitable material, or modified with any material to adsorb or fix the enzyme that can oxidize non-lactic acid hydroxy acids. Non-limiting examples of this material include platinum, gold, boron-doped diamond and carbon (such as graphite, pyrolytic graphite and glassy carbon), and they can also be additionally modified with carbon nanotubes (single-walled or multi-walled), carbon fibers, nanoparticles (such as gold nanoparticles) or promoters (such as mercaptans). The electrode can also be made of any material that can increase the electrode specific surface area.

[0252] According to one embodiment of the present invention, the electrode can be used as a single electrode, or as an electrode stack, for example consisting of 2, 3, 4, 5 or more electrodes.

[0253] According to one embodiment of the present invention, the electrode provided herein is a working electrode.

[0254] According to one embodiment, the electrode comprising an enzyme capable of oxidizing non-lactic hydroxy acids of the present invention can detect and / or quantify non-lactic hydroxy acids based on a mediator-free, mediator-based, or direct electron transfer approach.

[0255] Mediator-free electron transfer typically utilizes hydrogen peroxide generated by an enzymatic reaction to transfer electrons to an electrode.

[0256] Mediated electron transfer in biosensors typically follows a two-step process, in which the enzyme first undergoes a redox reaction with the substrate, which is then reoxidized by the redox mediator. Finally, the redox mediator is oxidized by the electrode.

[0257] Redox mediators are artificial electron transfer agents that readily undergo redox reactions with biological components, thereby facilitating rapid electron transfer to an electrode. A "redox mediator" is an electron transfer agent that transfers electrons between an analyte, an analyte-reducing enzyme, or an analyte-oxidizing enzyme, and an electrode, either directly or through one or more additional electron transfer agents. Redox mediators comprising a polymer backbone may also be referred to as redox polymers.

[0258] In particular, the redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.

[0259] According to one embodiment of the present invention, the oxidation of the non-lactic hydroxy acid is carried out in the presence of a redox mediator. The method of the present invention can also be carried out in the presence of multiple redox mediators, for example, in the presence of two or more different redox mediators. In this process, the redox mediator can be present on the electrode, in an enzyme composition comprising an enzyme and a redox mediator, or in the sample.

[0260] According to one embodiment, the redox mediator can be any molecule or material capable of transferring electrons between the enzyme capable of oxidizing the non-lactic hydroxy acid and the electrode. Specifically, the redox mediator is selected from the group consisting of any one of the following: an organic redox mediator, a soluble redox mediator, an insoluble redox mediator, a redox polymer, a transition metal complex, a polymer transition metal complex, a wired redox mediator, a sandwich compound, and derivatives of these redox mediators.

[0261] The polymer transition metal complex comprises a polymer backbone, a spacer group and a transition metal complex.

[0262] Specifically, redox polymers are polymers comprising a redox species. Non-limiting examples of redox species for redox polymers include osmium (Os), ruthenium (Ru), iron (Fe), cobalt (Co) or any transition metal. Non-limiting examples of polymers for redox polymers include poly(vinyl pyridine), polythiophene, polyaniline, polypyrrole or polyacetylene. An example of a redox polymer is poly(vinyl pyridine) containing osmium.

[0263] According to a specific embodiment, when the redox mediator comprises osmium, the redox mediator can be an osmium transition metal complex having one or more ligands, each ligand having a nitrogen-containing heterocycle (e.g., 2,2'-bipyridine, 1,10-phenanthroline, 1-methyl-2-pyridylbiimidazole, or a derivative thereof. The redox mediator can also have one or more ligands covalently bound to a polymer, each ligand having at least one nitrogen-containing heterocycle (e.g., pyridine, imidazole, or a derivative thereof). An example of an electron transfer agent includes: (a) a polymer or copolymer having pyridine or imidazole functional groups; and (b) an osmium cation complexed with two ligands, each ligand containing 2,2'-bipyridine, 1,10-phenanthroline, or a derivative thereof, the two ligands not necessarily being the same. Some derivatives of 2,2'-bipyridine for complexing with the osmium cation include, but are not limited to, 4,4'-dimethylpyridinium. alkyl-2,2'-bipyridine and mono-, di- and poly-alkoxy-2,2'-bipyridine, including 4,4'-dimethoxy-2,2'-bipyridine. Derivatives of 1,10-phenanthroline complexed with osmium cations include, but are not limited to, 4,7-dimethyl-1,10-phenanthroline and mono-, di- and poly-alkoxy-1,10-phenanthroline, such as 4,7-dimethoxy-1,10-phenanthroline. Polymers complexed with osmium cations include, but are not limited to, polymers and copolymers of poly(1-vinylimidazole) and poly(4-vinylpyridine). Suitable copolymer substituents for poly(1-vinylimidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinylimidazole, for example, an electron transfer agent complexed with a polymer or copolymer of osmium and poly(1-vinylimidazole). An example of a redox polymer is also derived from poly(1-vinylimidazole) or a polymer selected from the group consisting of Os 3+ / 2+ 、Ru 3+ / 2+ and Fe 3+ / 2+ The composition comprises a metal ion-bound (1-vinylimidazole) copolymer.

[0264] The term "transition metal" refers to an element whose atoms have a partially filled d subshell or are capable of producing cations with incomplete d subshells. Thus, transition metals are elements in the d block of the periodic table, which also includes the lanthanides and actinides.

[0265] Non-limiting examples of transition metal complexes include complexes containing titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium and platinum. Specific examples of transition metal complexes are ferrocyanide, hexaamine ruthenium, metalloporphyrins (e.g., heme b or heme c). In these complexes, the transition metal is coordinated with one or more ligands, which are typically monodentate, bidentate, tridentate or quadridentate ligands.

[0266] Non-limiting examples of transition metal complexes include complexes comprising lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0267] Non-limiting examples of transition metal complexes include complexes comprising actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, and lawrencium.

[0268] A sandwich compound is a chemical compound characterized by a metal bonded to two aromatic ligands via a thixotropic covalent bond. Aromatics have the molecular formula C n H n , substituted derivatives (e.g. C n (CH3) n ) and heterocyclic derivatives (such as BC n H n+1 A special class of sandwich complexes are metallocenes. Metallocenes consist of a transition metal and two cyclopentadienyl ligands coordinated in a sandwich configuration, meaning the two cyclopentadienyl anions lie in parallel planes with equal bond lengths and strengths. Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1,1'-dimethylferrocene [DMF], and ferrocene monocarboxylic acid.

[0269] Organic redox compounds are organic molecules that can act as redox mediators. Non-limiting examples of organic redox mediators are organic molecules (e.g., quinones), compounds having quinone structures (e.g., benzoquinone or phenanthroline quinone), phenazines (e.g., 1-methoxyphenazine methylsulfate), tetracyanoquinodimethane (TCNQ), N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivatives of these molecules.

[0270] According to one embodiment, a direct electron transfer biosensor can be used to measure non-lactic hydroxy acids, wherein the biosensor comprises an electrode comprising an enzyme capable of oxidizing the non-lactic hydroxy acid, and the enzyme is capable of directly transferring electrons obtained from the oxidation of the non-lactic hydroxy acid to the electrode surface. An example of such an enzyme capable of oxidizing the non-lactic hydroxy acid and achieving direct electron transfer is FCb2.

[0271] According to one embodiment of the present invention, the enzyme capable of oxidizing non-lactic hydroxy acids is immobilized on the electrode by adsorption, physical embedding in a polymer, complex formation (preferably through an additional complexing linker), covalent binding (particularly cross-linking) or ionic binding, and / or the immobilized enzyme can be cross-linked (particularly through a bifunctional reagent) to improve stability or activity. The cross-linking agent is, for example, a dialdehyde (e.g., glutaraldehyde).

[0272] According to one embodiment of the present invention, the electrode of the present invention is part of a biosensor. Thus, a particular use of the electrode of the present invention is to provide a biosensor, more specifically, a first, second, or third generation non-lactic hydroxy acid biosensor that uses mediatorless, mediator-based, or direct electron transfer properties to detect non-lactic hydroxy acids and / or measure their concentration. The biosensor can be used at acidic, neutral, or alkaline pH. The biosensor can be used at room temperature or body temperature. Specifically, the biosensor can be used for detection and / or quantification at temperatures of 4°C, 10°C, 15°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or higher.

[0273] According to another embodiment, a biosensor may have one or more electrodes comprising an enzyme capable of oxidizing a non-lactic hydroxy acid. In further embodiments, a non-lactic hydroxy acid biosensor includes a working electrode comprising a conductive material, wherein the enzyme capable of oxidizing a non-lactic hydroxy acid is proximate to the conductive material. One or more additional electrodes may also be included, such as one or more counter electrodes, one or more reference electrodes, and / or one or more counter / reference electrodes.

[0274] The specific configuration of the biosensor may depend on the intended use of the biosensor and the conditions under which it operates.

[0275] In one embodiment of the present invention, the biosensor may be a disposable biosensor for detecting non-lactic acid hydroxy acids. Thus, the biosensor may be a biosensor strip.

[0276] According to another embodiment of the present invention, a kit for determining (e.g., detecting and / or quantifying) non-lactic acid hydroxy acids in a sample is provided, the kit comprising an enzyme capable of selectively removing lactic acid from the sample and an enzyme capable of oxidizing the non-lactic acid hydroxy acids. These enzymes are described elsewhere herein.

[0277] According to one embodiment, a kit for measuring 2-HBA in a sample is provided.

[0278] According to one embodiment, the kit may further comprise a reagent for selectively removing hydrogen peroxide, in particular an enzyme, in particular catalase.

[0279] According to one embodiment, in the kit described herein, the enzyme capable of oxidizing a non-lactic acid hydroxy acid may be part of an electrode. In particular, the electrode may be part of a biosensor.

[0280] According to one embodiment, the kit described herein further comprises instructions for use.

[0281] According to one embodiment, the kit may also include auxiliary substances, such as buffers, molecules necessary for detection (e.g., electron acceptors), and containers (e.g., sample holders) and / or non-lactic hydroxy acid standards. Non-lactic hydroxy acid standards can be used to calibrate the assay. The kit may also include a signal reader (particularly an electrochemical signal reader, such as a potentiostat), a computer-readable storage device with calibration and / or measurement calculation software.

[0282] According to one embodiment of the present invention, enzyme as herein described can be recombinantly expressed by methods well known in the art. For example, standard methods for cloning, transforming and recombinant production can be used to express enzyme as herein described in suitable host organisms (e.g., Escherichia coli or Pichia pastoris).

[0283] As used herein, the terms "increase in activity", "improvement in activity" or similar terms may refer to a detectable increase in enzyme activity. As used herein, "increase in activity" or "improvement in activity" may mean that the modified enzyme (variant) exhibits a higher activity than a reference enzyme of the same type (e.g., an enzyme without specific modification). As another example, the modified enzyme may comprise a sequence change in a polypeptide or nucleotide sequence encoding the enzyme. For example, the activity of a modified or engineered enzyme may be higher than the activity of a non-engineered enzyme of the same type (e.g., a wild-type enzyme), for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more. The activity of a particular protein or enzyme in a recombinant or engineered cell may be higher than the activity of the same type of protein or enzyme in a parental cell (e.g., non-engineered cell), for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more. The increase in enzyme or protein activity in a cell can be verified by any method known in the art. Similarly, the terms "activity reduction," "activity decline," or similar terms used herein may refer to a detectable reduction in enzyme activity.

[0284] The increase or decrease in activity may also be specific to a specific target substrate. For example, the activity of an enzyme used herein may decrease when a non-lactic acid hydroxy acid is used as a substrate, but may remain essentially unchanged or even increase when lactic acid is used as a substrate.

[0285] The term "functional variant" or "functionally active variant" also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, allelic variants (also referred to as homologs) are alternative forms of nucleic acids or peptides, characterized by substitutions, deletions or additions of one or more nucleotides or amino acids, and these changes do not substantially alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may comprise substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, or a combination of these variations. Specifically, substitutions, deletions and / or additions may be conservative modifications. Specifically, substitutions, deletions and / or additions do not reduce the specific activity of the enzyme. Specifically, the functionally active variants of the enzyme capable of oxidizing a non-lactic hydroxy acid as described herein have a specific enzyme activity of at least 1 U / mg for the non-lactic hydroxy acid as determined by the corresponding tests described herein.

[0286] Specifically, the functional variants described herein comprise no more than or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid replacements, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not reduce the specific activity of the enzyme. Specifically, the functionally active variants described herein comprise at most 15, preferably at most 10 or 5 amino acid replacements, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not reduce the specific activity of the enzyme.

[0287] In particular, the functionally active variants described herein comprise at least 40%, 50%, 60%, 70%, 80% or 90%, or even more, of the enzymatic activity of the corresponding wild-type enzyme.

[0288] Functional variants can be obtained by making sequence changes to the polypeptide or nucleotide sequence, for example, by one or more point mutations, wherein the sequence changes retain or improve the characteristics of the enzyme, such as its stability or activity. Such sequence changes can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions. Conservative substitutions are substitutions that occur within a family of amino acids with related side chains and chemical properties. Examples of such families are amino acids with basic side chains, amino acids with acidic side chains, amino acids with non-polar aliphatic side chains, amino acids with non-polar aromatic side chains, amino acids with uncharged polar side chains, amino acids with small side chains, amino acids with large side chains, etc.

[0289] Specifically, point mutations can be understood as engineering of a polynucleotide that results in the expressed amino acid sequence differing from the non-engineered amino acid sequence by substitution, exchange, deletion or insertion of one or more single (non-consecutive) or pairs of amino acids.

[0290] According to a specific embodiment, the enzyme described herein may comprise one or more tag sequences, specifically N-terminal tag sequences. Specifically, such tag sequences are located at the C-terminus of the N-terminal methionine of the enzyme described herein. Such tag sequences may comprise more than 2, 4, 5, 6 or 10 amino acids, and any number of amino acids up to 20 or 50 or more amino acids. Specifically, the tag sequences used herein may be any tag sequences known to those skilled in the art. Specifically, the tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.

[0291] Affinity tags are amino acid sequences that can be used for example for protein purification, and they are attached to proteins. These affinity tags have high affinity to the appropriate ligands of solid supports (e.g., chromatographic resins) or directly to the resin. By selectively binding the protein with the affinity tag to a specific resin, it is possible to purify the protein very effectively with only one chromatography step. According to a specific embodiment, the affinity tag sequence used herein is selected from histidine (His) tags, specifically polyhistidine tags, polyarginine tags, FLAG tags, Strep tags, streptavidin binding peptide (SBP) tags, calmodulin binding peptide (CBP) tags, S tags, HA tags, c-Myc tags and SUMO tags, or any other tags known to be useful for effectively purifying proteins fused thereto. Preferably, the tag is a His tag, which comprises one or more H, specifically hexahistidine tags. Specifically, proteins comprising polyhistidine or hexahistidine tags (His tags) can be captured and purified using chromatography, for example, by immobilized metal affinity chromatography (IMAC).

[0292] The solubility enhancing tag can be fused to the N-terminus of the enzyme described herein. Compared to protein expression without the tag, the solubility enhancing tag can increase the titer of soluble protein when expressed in a host cell (e.g., in the cytoplasm of Pichia pastoris). According to another specific embodiment, the solubility enhancing tag sequence used herein is selected from calmodulin binding peptide (CBP), polyarginine (poly Arg), polylysine (poly Lys), protein D tag (dTAG), the Z domain of Staphylococcus aureus protein A, and thioredoxin, or any other known tag that can improve the solubility of the protein fused thereto during, for example, host cell expression. In particular, the solubility-enhancing tag is a T7 tag, preferably selected from the group consisting of T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7B, T7Bl, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7BlO, T7Bl 1, T7B12, T7B13 and T7C.

[0293] According to another specific embodiment, the monitoring tag sequence used in this article is m-Cherry, green fluorescent protein (GFP) or fibrous actin (f-Actin), or any other tag for detecting or quantifying the recombinant enzyme by a simple in situ, online or offline detector (such as UV, IR, Raman, fluorescence, etc. detector) in the production steps (including fermentation, separation and purification).

[0294] As used herein, the term "sequence identity" is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and is described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homolog, or ortholog compared to a parent nucleotide or amino acid sequence indicates the degree of identity between two or more sequences. Two or more amino acid sequences may have identical or conserved amino acid residues at corresponding positions to a certain extent, up to 100%. Two or more nucleotide sequences may have identical or conserved base pairs at corresponding positions to a certain extent, up to 100%.

[0295] Sequence similarity searching is an efficient and reliable strategy for identifying homologs with high (e.g., at least 50%) sequence identity. For example, commonly used sequence similarity searching tools include BLAST, FASTA, and HMMER.

[0296] Sequence similarity searches can identify these homologous proteins or polynucleotides by detecting excessively high similarities and statistically significant similarities that reflect common ancestry. Homologs may include orthologs, which are understood herein to be identical proteins in different organisms, such as variants of such proteins in different organisms or species.

[0297] To determine the percent complementarity of two complementary sequences, one of the sequences is converted to its complement and then the percent complementarity is calculated as the percent identity between the first sequence and the second converted sequence using the algorithm described above.

[0298] For the amino acid sequences, homologs and orthologs described herein, "percent identity (%)" is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the specified polypeptide sequence, after aligning the sequences and introducing gaps (if necessary), and not considering any conservative substitutions as part of the sequence identity. One skilled in the art can determine appropriate parameters for the alignment, including any algorithm required to achieve the highest scoring alignment over the full length of the sequences being compared. In determining the percentage of sequence identity, arithmetic decimal places may appear, which is not possible for complete nucleotides or amino acids. In such cases, the percentage should be rounded up to the whole nucleotide or amino acid.

[0299] For the purposes described herein, sequence identity between two amino acid sequences is determined using standard methods, for example using the NCBI BLAST program version 2.2.29 (January 6, 2014) or online using the multiple sequence alignment tool EMBL-EBI Luxtal Omega (Sievers, F. et al. (2011)).

[0300] "Percent identity (%)" for nucleotide sequences (e.g., nucleic acid molecules or portions thereof, particularly encoding DNA sequences) is defined as the percentage of nucleotides in the candidate DNA sequence that are identical to the nucleotides in the DNA sequence, after aligning the sequences and introducing gaps (if necessary), and not considering any conservative substitutions as part of the sequence identity. Comparisons for determining percent identity of nucleotide sequences can be accomplished in various ways within the skill in the art, for example, using publicly available computer software. Appropriate parameters for the alignment, including any algorithm needed to achieve the highest scoring alignment over the full length of the compared sequences, can be determined by one skilled in the art.

[0301] Any suitable sequence alignment algorithm can be used to determine optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on MAFFT (Multiple Sequence Alignment using Fast Fourier Transforms), algorithms based on the Burrows-Wheeler transform (e.g., the Burrows Wheeler aligner), Clustal W, Clustal X, BLAT, Novoalign (provided by Novocraft Technologies, available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0302] In a structural alignment, the largest set of corresponding amino acid residue pairs that produce a good structural match when the structures are superimposed (i.e., overlapped) is identified. Thus, the positions of the protein backbone C-alpha atoms and / or the positions of secondary structural elements are taken into account in the alignment. Tools for performing structural alignments are available, for example, the Protein Data Bank provides a tool for pairwise structural alignments. In particular, structural superposition is also a tool for determining the positions of corresponding amino acids in different enzymes. The molecular graphics system PyMOL ( Company) and use the "align" command to perform structural superposition.

[0303] The present invention further provides the following:

[0304] 1. A method for determining a non-lactic 2-hydroxy acid in a sample, the sample comprising a non-lactic 2-hydroxy acid and optionally lactic acid, the method comprising the following steps:

[0305] i. optionally selectively removing lactic acid from the sample;

[0306] ii. incubating the sample with an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity;

[0307] iii. Determination of non-lactic 2-hydroxy acids in the sample.

[0308] 2. The method according to item 1, wherein the non-lactic 2-hydroxy acid is a compound having the general formula I

[0309]

[0310] in

[0311] R1 is H or C 1-6 alkyl,

[0312] R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0313] 3. The method according to item 2, wherein R1 is -CH3.

[0314] 4. The method according to item 2, wherein R2 is phenyl.

[0315] 5. The method according to any one of items 1 to 4, wherein the non-lactic 2-hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, 2-hydroxyisobutyric acid, D-lactic acid, and the corresponding salts of any one of the foregoing.

[0316] 6. The method according to any one of items 1 to 5, wherein in step ii, the enzyme is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity or an enzyme having non-lactate 2-hydroxy acid oxidase activity.

[0317] 7. The method according to item 6, wherein the enzyme having non-lactate 2-hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0318] 8. The method according to any one of items 1 to 7, wherein in step ii, the enzyme is FCb2 or LDH.

[0319] 9. The method according to any one of items 1 to 8, wherein an enzyme is used in step i.

[0320] 10. The method according to any one of items 1 to 9, wherein an enzyme having lactate oxidizing activity is used in step i.

[0321] 11. The method according to item 10, wherein an enzyme having lactate oxidase activity is used.

[0322] 12. The method according to any one of items 1 to 11, further comprising adding a reagent for removing hydrogen peroxide in step i.

[0323] 13. The method according to item 12, wherein an enzyme having catalase activity is added.

[0324] 14. The method according to any one of items 1 to 13, wherein the non-lactic 2-hydroxy acid is determined by a colorimetric, photometric, fluorometric or electrochemical method.

[0325] 15. The method according to any one of items 1 to 14, wherein 2-hydroxybutyric acid is determined in a sample comprising 2-hydroxybutyric acid and optionally lactic acid, the method comprising the steps of:

[0326] a. optionally selectively removing lactate from the sample by an enzyme having lactate oxidase activity;

[0327] b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity;

[0328] c. Determine 2-hydroxybutyric acid in the sample.

[0329] 16. The method according to any one of items 1 to 15, wherein the sample is a human sample.

[0330] 17. The method according to any one of items 1 to 16, wherein an electrode comprising an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is used in step ii.

[0331] 18. The method of item 17, wherein the electrode is part of a biosensor.

[0332] 19. Use of an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity for determining non-lactic acid 2-hydroxy acid in a sample in the method according to any one of items 1 to 18.

[0333] 20. The use according to item 19, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity.

[0334] 21. Use of an electrode comprising an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity for measuring non-lactic acid 2-hydroxy acid in a sample.

[0335] 22. The use according to item 21, for use in a method according to any one of items 1 to 18.

[0336] 23. The use according to item 21 or 22, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity.

[0337] 24. A kit for measuring non-lactic acid 2-hydroxy acid in a sample, the sample comprising non-lactic acid 2-hydroxy acid and lactic acid, the kit comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity.

[0338] 25. The kit according to item 24, wherein the non-lactic 2-hydroxy acid is 2-hydroxybutyric acid.

[0339] 26. The kit according to item 24 or 25, wherein the enzyme having lactate oxidizing activity is lactate oxidase.

[0340] 27. The kit according to any one of items 24 to 26, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity.

[0341] 28. The kit according to item 27, wherein the enzyme having non-lactate 2-hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0342] 29. The kit according to any one of items 24 to 28, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is FCb2 or LDH.

[0343] 30. The kit according to any one of items 24 to 29, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidation activity is part of an electrode.

[0344] 31. An electrode comprising an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity.

[0345] 32. The electrode according to item 31, wherein the non-lactic 2-hydroxy acid is a compound having the general formula I

[0346]

[0347] in

[0348] R1 is H or C 1-6 alkyl,

[0349] R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

[0350] 33. The electrode according to item 32, wherein R1 is -CH3.

[0351] 34. The electrode according to item 32, wherein R2 is phenyl.

[0352] 35. The electrode according to any one of items 31 to 34, wherein the non-lactic 2-hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid and 2-hydroxyisobutyric acid.

[0353] 36. The electrode according to any one of items 31 to 35, wherein the enzyme having non-lactic acid 2-hydroxy acid oxidation activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity or an enzyme having non-lactic acid 2-hydroxy acid oxidase activity.

[0354] 37. The electrode according to item 36, wherein the enzyme having non-lactate 2-hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0355] 38. The electrode according to any one of items 31 to 37, wherein the enzyme is FCb2 or LDH.

[0356] 39. The electrode according to any one of items 31 to 38, wherein the electrode is part of a biosensor.

[0357] 40. A method for determining non-lactic hydroxy acids in a sample, the sample comprising non-lactic hydroxy acids and lactic acid, the method comprising the steps of:

[0358] i. selectively removing lactic acid from the sample;

[0359] ii. incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity;

[0360] iii. Determining non-lactic hydroxy acids in the sample.

[0361] 41. The method of item 40, wherein the non-lactic hydroxy acid is 2-hydroxybutyric acid, 3-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, or glycolic acid.

[0362] 42. The method according to item 40 or 41, wherein the enzyme in step ii is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity, or an enzyme having non-lactic acid hydroxy acid oxidase activity.

[0363] 43. The method according to item 42, wherein the enzyme having non-lactate hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity, preferably FCb2 or LDH.

[0364] 44. The method according to any one of items 40 to 43, wherein an enzyme having lactate oxidizing activity, in particular an enzyme having lactate oxidase activity, is used in step i.

[0365] 45. The method according to any one of items 40 to 44, further comprising adding a reagent for removing hydrogen peroxide in step i, preferably adding an enzyme having catalase activity.

[0366] 46. ​​The method according to any one of items 40 to 45, wherein the non-lactic hydroxy acid is determined by a colorimetric, photometric, fluorometric or electrochemical method.

[0367] 47. The method according to any one of items 40 to 46, wherein 2-hydroxybutyrate is determined in a sample comprising 2-hydroxybutyrate and lactic acid, the method comprising the steps of:

[0368] a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity;

[0369] b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity;

[0370] c. Determine 2-hydroxybutyric acid in the sample.

[0371] 48. Use of an enzyme having non-lactic hydroxy acid oxidizing activity, in particular an enzyme having non-lactic hydroxy acid dehydrogenase activity, for determining non-lactic hydroxy acids in a sample in the method according to any one of items 40 to 47.

[0372] 49. Use of an electrode comprising an enzyme having non-lactic hydroxy acid oxidation activity, in particular an enzyme having non-lactic hydroxy acid dehydrogenase activity, in the method according to any one of items 40 to 47 for determining non-lactic hydroxy acids in a sample.

[0373] 50. A kit for determining non-lactic hydroxy acid (particularly 2-hydroxybutyric acid) in a sample, wherein the sample contains non-lactic hydroxy acid and lactic acid, and the kit contains an enzyme having lactate oxidation activity and an enzyme having non-lactic hydroxy acid oxidation activity.

[0374] 51. The kit according to item 50, wherein the enzyme having lactate oxidizing activity is lactate oxidase.

[0375] 52. The kit according to item 50 or 51, wherein the enzyme having non-lactic acid hydroxy acid oxidizing activity is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity, preferably an enzyme having 2-hydroxybutyrate dehydrogenase activity.

[0376] 53. The kit according to any one of items 50 to 52, wherein the enzyme having non-lactic acid hydroxy acid oxidation activity is FCb2 or LDH.

[0377] 54. The kit according to any one of items 50 to 53, wherein the enzyme having non-lactic acid hydroxy acid oxidation activity is part of an electrode.

[0378] The embodiments described herein are intended to illustrate the present invention and are not intended to limit the present invention. Various modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the present invention.

[0379] Example

[0380] Example 1: Principle of the two-step enzymatic assay for 2-HBA determination

[0381] In this example, lactate oxidase (LOx) is used as an example of an enzyme for removing L-lactic acid, flavocytochrome b2 is used as an example of an enzyme for detecting non-lactic hydroxy acids, and 2-HBA is used as an example of a non-lactic hydroxy acid. The overall scheme of the reaction is as follows: Figure 1 shown.

[0382] According to this example, an assay is employed for the specific detection of 2-HBA in a sample, employing a specialized enzymatic pretreatment step using lactate oxidase (LOx) to remove the interfering L-lactic acid. Therefore, since the removal enzyme in this example uses oxygen as an electron acceptor, simultaneous oxidation of L-lactic acid and oxygen reduction results in H2O2 accumulation, which can be partially recovered (semi-stoichiometrically) using catalase's enzymatic conversion: 2H2O2 → 2H2O + O2. This regeneration using catalase helps maintain a stable oxygen concentration during the LOx reaction. Following the removal step, the 2-HBA analyte can be detected without interference using an enzyme such as FCb2.

[0383] Generally, in physiologically relevant samples, the concentration of L-lactic acid (1-2 mM) is more than 10-fold higher than that of the 2-HBA analyte (0.05-0.10 mM).

[0384] This example provides a general proof of principle that the methods described herein can be used to determine non-lactic hydroxy acids in samples containing L-lactic acid. In addition, the results of spiking non-lactic hydroxy acids in physiological samples are shown.

[0385] Material

[0386] Enzymes:

[0387] The amino acid sequence of the enzyme used for removal or detection in this embodiment is as follows:

[0388] AvLOx-amino acid sequence (SEQ ID NO: 1):

[0389] MGSSHHHHHHNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY

[0390] CangFCb2-amino acid sequence (SEQ ID NO: 2):

[0391] MHHHHHHHHDAKFDSSKPKISPSEVIKHNTPEDCWVVIDGYVYDLTNFIALHPGGPDIIKTNAGKDVTAIFDPIHPPDAIEKYIKPEQHVGPLDGKLDAEYICPPYAPGETPDDIARKAALRARLPPLSIMNLYDFEYLASQILSKQAWAYYSSASDDEVSYRENHNAYHRIFFNPKVLVDVSKVDTSTEMLGHKVDVPFYVTATALCKLGNPKEGEKDIARGCGQGPNKTPQMISTLASCSVDEIVNAAPSKDQVIWYQLYVNSDRKITENLIKHVEDLGVKAIFVTVDAPSLGSREKDKKVKFNNTMSGPKSMKKSDVGESEGAAQTLSKFIDPSLSWQDIKILRKKTKLPIVIKGVQRVQDVVKAAEIGCNGVVLSNHGGRQLDFARAPIEVLAETMPVLKEKKLDKNFEVFVDGGVRRGTDVIKALCLGASGVGLGRPFLYANSCYGKDGVQKAIDLLKTEIEMNMRLLGVTSIKDMNPELLDLSSSLHGRTVNVPKDSLYVNVYNKPELAEFLDASD

[0392] Corynebacterium glutamicum catalase (CgCat), Sigma #02071

[0393] Chemical reagents:

[0394] Horse cytochrome C, Sigma #C2506

[0395] Sodium L-lactate, ≥99.0% (NT), Sigma #71718

[0396] Sodium 2-hydroxybutyrate >97%, racemate, Sigma #220116 (CAS 5094-24-6, 126.09 g / mol)

[0397] Phosphate buffered saline (PBS), pH 7.4, Sigma #P3813

[0398] Human plasma (SIGMA#P9523): lyophilized, 4% citrate

[0399] Human serum H3667-20 mL (Sigma, source SLCL8404), heat inactivated: frozen

[0400] 96-well multiwell plate (Greiner, polystyrene, Merck #M2936)

[0401] instrument:

[0402] Photometer (TECAN Infinite M Nanoplate Reader); used for measurement at 550 nm.

[0403] Proof of principle

[0404] method:

[0405] Reagent 1 was used for the pretreatment step. Reagent 1: 0.1 mg / mL lactate oxidase from Aerococcus viridans and 1 μL / mL catalase from Corynebacterium glutamicum dissolved in 11 mM PBS, pH 7.4.

[0406] Reagent 2 was used for the measurement step. Reagent 2: 50 μg / mL Candida glabrata FCb2 and 80 μM equine cytochrome C (Sigma-Aldrich, #C2506), dissolved in 11 mM PBS, pH 7.4.

[0407] Determination of 2-HBA concentration was performed at room temperature (22° C.) and included a 5-minute pretreatment reaction and a 3-minute measurement using a photometric plate reader.

[0408] At reaction time 0, 10 μL of sample (serum, plasma, synthetic control) was transferred to a well of a 96-well plate. The pretreatment reaction was started by adding 90 μL of reagent 1. After 5 minutes, 100 μL of reagent 2 was added and the photometric measurement was started immediately. The reaction (absorbance change) was followed at 550 nm in a photometer for 3 minutes. For this measurement setup, the increase in optical absorbance at 550 nm per minute (ΔAbs550 min) was calculated. -1 ) can be calculated as enzyme activity (U / mL or U / mg) or apparent 2-HBA concentration according to the following formulas (these formulas are only applicable to the above technical settings):

[0409] Formula 1 - Calculate volumetric activity (U / mL) based on the change in optical absorbance at 550 nm:

[0410] Volume activity [U mL -1 ]=ΔAbs min -1 *0.98*df

[0411] Formula 2 - Calculation of 2-HBA concentration based on the change in optical absorbance at 550 nm (this calculation applies only to the given technical setup):

[0412]

[0413] The following applies to the formula shown:

[0414] 0.98: Enzyme factor that combines the dilution factor, extinction coefficient, and pathlength.

[0415] df: Sample dilution factor (equal to 0.05 when 10 μL of sample is added to 200 μL).

[0416] The samples contained different combinations of 5 mM L-lactic acid, 0.1 mM 2-HBA, and 0.1 mg / mL LOx, but in all experiments, a 0.1% (v / v) catalase solution was included. A 5-minute pretreatment reaction was performed at 22°C before measuring the 2-HBA concentration of the samples (which is dependent on a 3-minute cytochrome C (CytC) assay using FCb2). The increase in absorbance at 550 nm caused by CytC reduction was converted into enzyme activity (U / mL) that was dependent on substrate concentration.

[0417] result

[0418] The proof of principle results are as follows Figure 2 shown.

[0419] (1) Samples containing 2-HBA (analyte) and L-lactic acid (interferent) without the addition of LOx will produce high activity (CytC) in subsequent measurements using CangFCb2 due to the presence of high concentrations of L-lactic acid.

[0420] (2) In the presence of LOx, samples containing the 2-HBA analyte and the L-lactic acid interferor yield low activity because the L-lactic acid is oxidized by LOx and therefore cannot be accepted by FCb2 during the colorimetric measurement. The remaining activity comes solely from 2-HBA.

[0421] (3) In contrast, when L-lactic acid was present in the sample but 2-HBA was absent, the FCb2 reaction did not produce a detectable signal.

[0422] (4) When L-lactic acid was absent, an enzyme activity level similar to that of (2) was achieved.

[0423] These results provide proof of principle for the approach described herein, in particular as the following were confirmed:

[0424] Lactic acid removal: regardless of the presence of L-lactic acid; (2) compared to (4): the remaining signals were comparable, indicating that only the 2-HBA fraction produced a signal after treatment.

[0425] This is due to LOx. In the experiment (1) in which Lox was absent, a significantly higher signal was shown.

[0426] When 2-HBA was not present in the sample mixture, no signal remained after LOx treatment, indicating that L-lactic acid was completely removed.

[0427] Determination of the analytical slope of 2-HBA using the FCb2 assay

[0428] method

[0429] 2-HBA was diluted in PBS to give the following μM concentrations: 6.25; 12.5; 25; 50; 100; 200; 300; 400.

[0430] Six technical replicates of 10 μL of each of these samples were measured using the CytC / Fcb2 assay protocol described above, using CangFCb2. The protocol was modified to use 90 μL of PBS instead of Reagent 1, but 100 μL of Reagent 2 containing CangFCb2 was used.

[0431] Based on the calculated FCb2 activity, the data points were used to perform a linear regression to obtain the slope, intercept, and goodness of fit (R 2 ).

[0432] result

[0433] In tests using pure 2-HBA and the FCb2 / CytC assay, a clear linear analytical range was achieved over a sample concentration range of 6.25 to 400 μM. In this measurement setup, no interfering substances were present, and the LOx-catalyzed L-lactic acid removal step was omitted.

[0434] Table 1 below gives the numerical data for the analytical range measurements:

[0435] Table 1: Numerical data of the analyzed range measurements

[0436]

[0437]

[0438] These numerical data were plotted and the following linear calibration function was determined:

[0439] y=1E-04x+0.0029

[0440] R 2 =0.9994

[0441] These results highlight the wide linear range and low standard error of 2-HBA measurements in the CangFCb2 setup.

[0442] Plasma spike with 2-HBA

[0443] method:

[0444] The spiked samples were prepared using the human plasma and serum samples described above and a synthetic standard solution containing 5 mM L-lactic acid and 0.1 mM 2-HBA.

[0445] · Take 10 μL of the spiked sample and perform 6 technical replicates as in the proof of principle experiment, add 90 μL of Reagent 1 for depletion, and then use 100 μL of Reagent 2 containing CangFCb2 for 2-HBA measurement.

[0446] Using the analytical slope parameter obtained from the above 2-HBA assay, the mean value of FCb2 activity was calculated and converted into apparent 2-HBA concentration, which was then compared with the 2-HBA concentration spiked into the samples.

[0447] Based on the calculated FCb2 activity, the data points were used to perform a linear regression to obtain the slope, intercept, and goodness of fit (R 2 ), where the slope, the apparent 2-HBA concentration compared to the spiked 2-HBA concentration, reflects the “recovery”.

[0448] result:

[0449] Enzyme activity was measured from a series of 2-HBA standards (with known 2-HBA concentrations) in buffers, and the kinetic slope was determined, i.e., at a specific 2-HBA concentration point, the kinetic slope varied linearly with increasing concentration. These data points were used to calculate a linear regression characterized by an intercept (d) and slope (x) value based on y = kx + d, which is called the analytical calibration.

[0450] After spiking commercially available human plasma and serum with known amounts of 2-HBA and an unknown concentration of the physiological L-lactate interferor, the apparent concentration of the 2-HBA analyte in serum and plasma was calculated using calibration curve parameters determined in buffer. The correlation between spiked and measured 2-HBA concentrations in this experiment is visualized here and represents the concentrations used in the experiment. Sample concentrations were diluted 20-fold.

[0451] The results are shown in Tables 2 and 3 below:

[0452] Table 2: Numerical data for plasma spike experiments

[0453]

[0454] Table 3: Numerical data for serum spike experiments

[0455]

[0456] A calibration curve for 2-HBA was recorded in buffer for the extended range of 0-40 μM of the analyte in the assay (1:20, 0-800 μM in samples). The signal was converted to concentration using the slope and intercept and compared to the spike concentration.

[0457] • Serum and plasma were spiked with 2-HBA at concentrations ranging from 0 to 10 μM (as tested).

[0458] • The recovery ranges for plasma and serum were similar (approximately 94%).

[0459] • Linear (detectable) range: 0.625-10 μM 2-HBA in the assay, which translates to 12.5-200 μM in the sample.

[0460] Example 2: Electrochemical Detection of Non-Lactic Hydroxy Acids

[0461] In this example, the electrochemical detection of 2-HBA is described, using KmFCb2 as an exemplary enzyme for the electrochemical detection of 2-HBA.

[0462] Material:

[0463] Enzymes:

[0464] KmFCb2-amino acid sequence (SEQ ID NO: 3):

[0465] MHHHHHHHHATKEELNKPKVSPLEVAKHSSPDDCWVVIDGFVYNLTEFISAHPGGPAIIENNAGKDVTAIFGPIHAPDVIEKYIAPENRIGPLDGKMPDDLICAPLTPGETPEDVARKEELRQNMPDLDS LVNIYDFEFLASQILTKQAWSYYSSAADDEVTHRENHAAYHRIFFKPRILVNVKEVDTSTTMLGEKVGVPFYVSATALCKLGNPKEGEKDIARGCGESDVKPIQMISTLASCSLQEIVEAAPSKDQIQWF QLYVNSDRKITEELIKNVEKLGLKAIFVTVDAPSLGNREKDAKVKFTNKDSSAKAMEKSNVKESKGASRALSTFIDPALCWDDIVTLKSKTKLPIVIKGVQCVEDVLKAAEIGAAGVVLSNHGGRQLDFS RAPIEVLAETMPILKEKKLDDKIEIFIDGGVRRGTDILKALCLGAKGVGLGRPFLYANSCYGKEGVKKAIELLKDELEMSMRLLGVTSIDQLSEKYLDLSTLHGRTVSVPRDNLYNGVYVPHEPTDFKEN

[0466] Chemical reagents:

[0467] Sodium 2-hydroxybutyrate >97%, racemate, Sigma #220116 (CAS 5094-24-6, molecular weight 126.09 g / mol)

[0468] Phosphate buffered saline (PBS), pH 7.4, Sigma #P3813

[0469] DropSens carbon screen-printed electrode DRP-C110

[0470] instrument:

[0471] Potentiostat: Palmsens EMstat Blue

[0472] method:

[0473] • Dilute 2-HBA in PBS to give the following mM concentrations: 1.0, 5.0, 10.

[0474] Biosensor electrodes were prepared as described by Geiss et al. (2021) with the following modifications: 2 μL of 10 mg / mL engineered KmFCb2 in 100 mM phosphate buffer was used. Curing was carried out at 22°C for 2 hours under dry conditions.

[0475] Electrode measurements were performed at 22°C using a potentiostat in chronoamperometry mode with three replicates, applying a potential of 0.2 V relative to a pseudo-Ag / AgCl reference electrode. With the electrode mounted horizontally, sample was gradually added to and removed from the sensing area to allow the substrate concentration to increase over time. The current was measured over time.

[0476] Data evaluation: Baseline-subtracted current was evaluated 10 seconds after each sample addition and plotted against substrate concentration. Nonlinear regression fitting was performed using the Michaelis-Menten equation.

[0477] result:

[0478] When the FCb2 enzyme is in contact with an electrode, it can detect analytes without the need for a soluble electron acceptor. Depending on the concentration of the substrate in the assay, the enzyme delivers a catalytic current proportional to the concentration of the substrate, which can be measured using an electronic device such as a potentiostat.

[0479] Table 4 below shows the results of this example.

[0480] Table 4: Catalytic current of electrochemical KmFCb2 measurements of 2-HBA

[0481] 2-HBA Substrate (mM) Current (μA) 0 0.09±0.07 1 2.15±0.03 5 10.71±0.35 10 14.98±0.32 <![CDATA[R 2 ]]> 0.995

[0482] In experiments with electrochemical contact of KmFCb2, 2-HBA could be detected in the relevant range. In this measurement setup, no interfering substances were present, and the L-lactic acid removal step catalyzed by LOx was omitted, as the general demonstration of L-lactic acid removal and its influence on the general 2-HBA determination had already been shown in Example 1.

[0483] These results highlight electrochemical measurement as a viable approach for detecting 2-HBA from samples using FCb2, which may be particularly important for biosensor applications.

[0484] Example 3: Detection of different non-lactic hydroxy acids

[0485] This example demonstrates the detection of different non-lactic hydroxy acids using three different FCb2 enzymes.

[0486] The enzymes used were as follows:

[0487] KmFCb2 and CangFCb2 as described above.

[0488] WaFCb2 - amino acid sequence (SEQ ID NO: 4):

[0489] MHHHHHHHHDVPHWKDIELTPEIVSQHNKKDDLWVVLNGQVYDLTDFLPNHPGGQKIIIRYAGKDATKIFVPIHPPDTIEKFIPPEKHLGPLVGEFEQEEEELSDEEIDRLERIERKPPLSQMINLH DFETIARQILPPPALAYYCSAADDEVTLRENHNAYHRIFFNPKILIDVKDVDISTEFFGEKTSAPFYISATALAKLGHPEGEVAIAKGAGREDVVQMISTLASCSFDEIADARIPGQQQWYQLYVNA DRSITEKAVRHAEERGMKGLFITVDAPSLGRREKDMKMKFEADSDVQGDDEDIDRSQGASRALSSFIDPSLSWKDIAFIKSITKMPIVIKGVQRKEDVLLAAEHGLQGVVLSNHGGRQLDYTRAPVE VLAEVMPILKERGLDQKIDIFVDGGVRRGTDVLKALCLGAKGVGLGRPFLYAMSSYGDKGVTKAIQLLKDEIEMNMRLLGVNKIEELTPELLDTRSIHNRAVPVAKDYLYEQNYQRMSGAEFRPGIED

[0490] The activity of the FCb2 enzyme was determined photometrically according to the general principles of the cytochrome C assay described herein (e.g., in Example 1). Therefore, a 300 mM stock solution was prepared for each substrate in 50 mM potassium phosphate buffer (PPB) at pH 6.5.

[0491] The substrates used were as follows:

[0492] D-lactic acid (#CAS:920-49-0)

[0493] Sodium glycolate (#CAS:2836-32-0)

[0494] (S)-2-Hydroxybutyric acid (α-HB, L(S)-enantiomer) (#CAS:3347-90-8)

[0495] 2-Hydroxybutyric acid (α-HB, racemic) (#CAS:5094-24-6)

[0496] (S)-2-Hydroxyvaleric acid (#CAS:41014-93-1)

[0497] (S)-(+)-Mandelic acid (#CAS:17199-29-0)

[0498] 2-Hydroxyoctanoic acid*(#CAS:617-73-2)

[0499] (C+) Sodium L-lactate (#CAS:867-56-1)

[0500] * Sonicate and heat to 40°C to dissolve

[0501] Table 5 shows the relative enzyme activities of CangFCb2, KmFCb2, and WaFCb2 for each substrate relative to L-lactic acid.

[0502] Table 5: Summary of relative substrate-dependent activities of FCb2

[0503]

[0504] Example 4: Determination of different non-lactic 2-hydroxy acids

[0505] This example illustrates the assay of various non-lactic 2-hydroxy acids using the invention described herein. Example 4 was performed as described in Example 3. Table 6 below shows the relative substrate dependent activities.

[0506] Table 6

[0507]

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Claims

1. A method for determining non-lactic hydroxy acids in a sample, wherein the sample contains non-lactic hydroxy acids and lactic acid, the method comprising the following steps: i. selectively removing lactic acid from the sample; ii. incubating the sample with an enzyme having non-lactic acid hydroxy acid oxidizing activity; iii. Determining non-lactic hydroxy acids in the sample.

2. The method of claim 1, wherein the non-lactic hydroxy acid is a non-lactic 2-hydroxy acid.

3. The method of claim 2, wherein the non-lactic 2-hydroxy acid is a compound having the general formula I in R1 is H or C 1-6 alkyl, R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

4. The method according to claim 3, wherein R1 is -CH3.

5. The method according to claim 3, wherein R2 is phenyl.

6. The method according to any one of claims 1 to 5, wherein the non-lactic hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, 2-hydroxyisobutyric acid, D-lactic acid, and the corresponding salts of any one of the foregoing. 7 . The method according to claim 1 , wherein the enzyme in step ii. is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity, or an enzyme having non-lactic acid hydroxy acid oxidase activity. The method according to claim 7 , wherein the enzyme having non-lactate hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

9. The method according to any one of claims 1 to 8, wherein the enzyme in step ii. is FCb2 or LDH.

10. The process according to any one of claims 1 to 9, wherein an enzyme is used in step i.

11. The method according to any one of claims 1 to 10, wherein an enzyme having lactate oxidizing activity, in particular an enzyme having lactate oxidase activity, is used in step i.

12. The method according to any one of claims 1 to 11, wherein a reagent for removing hydrogen peroxide is further added in step i.

13. The method according to claim 12, wherein an enzyme having catalase activity is added.

14. The method according to any one of claims 1 to 13, wherein the non-lactic hydroxy acid is determined by a colorimetric, photometric, fluorometric or electrochemical method.

15. The method according to any one of claims 1 to 14, wherein 2-hydroxybutyrate is determined in a sample comprising 2-hydroxybutyrate and lactic acid, the method comprising the steps of: a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity; b. incubating the sample with an enzyme having 2-hydroxybutyrate dehydrogenase activity; c. Determine 2-hydroxybutyric acid in the sample.

16. The method according to any one of claims 1 to 15, wherein the sample is a human sample. 17 . The method according to claim 1 , wherein an electrode comprising an enzyme having non-lactic acid hydroxy acid oxidizing activity is used in step ii.

18. Use of an enzyme having non-lactic hydroxy acid oxidizing activity for determining non-lactic hydroxy acids in a sample in the method according to any one of claims 1 to 17. 19 . The use according to claim 18 , wherein the enzyme having non-lactic acid hydroxy acid oxidizing activity is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity.

20. Use of an electrode comprising an enzyme having non-lactic hydroxy acid oxidizing activity for determining non-lactic hydroxy acids in a sample in the method according to any one of claims 1 to 17. 21 . The use according to claim 20 , wherein the enzyme having non-lactic acid hydroxy acid oxidizing activity is an enzyme having non-lactic acid hydroxy acid dehydrogenase activity.

22. The use according to any one of claims 18 to 21, wherein the sample is a human sample.

23. A kit for measuring non-lactic hydroxy acids in a sample, the sample comprising non-lactic hydroxy acids and lactic acid, the kit comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactic hydroxy acid oxidizing activity.

24. The kit of claim 23, wherein the non-lactic hydroxy acid is a 2-hydroxy acid.

25. The kit of claim 23 or 24, wherein the non-lactic hydroxy acid is 2-hydroxybutyric acid. 26 . The kit according to claim 23 , wherein the enzyme having lactate oxidizing activity is lactate oxidase. 27 . The kit according to any one of claims 23 to 26 , wherein the enzyme having non-lactic hydroxy acid oxidizing activity is an enzyme having non-lactic hydroxy acid dehydrogenase activity. The kit according to claim 27 , wherein the enzyme having non-lactate hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

29. The kit according to any one of claims 23 to 28, wherein the enzyme having non-lactic acid hydroxy acid oxidation activity is FCb2 or LDH.

30. The kit according to any one of claims 23 to 29, wherein the enzyme having non-lactic acid hydroxy acid oxidizing activity is part of an electrode.

31. An electrode comprising an enzyme having non-lactic acid 2-hydroxy acid oxidizing activity.

32. The electrode of claim 31 , wherein the non-lactic 2-hydroxy acid is a compound having the general formula I in R1 is H or C 1-6 alkyl, R2 is H, C 6-8 Aryl or C optionally substituted by -C(O)OH 1-20 alkyl.

33. The electrode of claim 32, wherein R1 is -CH3.

34. The electrode according to claim 32, wherein R2 is phenyl.

35. The electrode of any one of claims 31 to 34, wherein the non-lactic 2-hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, and 2-hydroxyisobutyric acid. 36 . The electrode according to claim 31 , wherein the enzyme having non-lactic acid 2-hydroxy acid oxidizing activity is an enzyme having non-lactic acid 2-hydroxy acid dehydrogenase activity or an enzyme having non-lactic acid 2-hydroxy acid oxidase activity.

37. The electrode according to claim 36, wherein the enzyme having non-lactate 2-hydroxyacid dehydrogenase activity is an enzyme having 2-hydroxybutyrate dehydrogenase activity.

38. The electrode according to any one of claims 31 to 37, wherein the enzyme is FCb2 or LDH.

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