EP4630571A1 - Enzymatic detection of non-lactate hydroxy acids - Google Patents

Enzymatic detection of non-lactate hydroxy acids

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Publication number
EP4630571A1
EP4630571A1 EP23817412.2A EP23817412A EP4630571A1 EP 4630571 A1 EP4630571 A1 EP 4630571A1 EP 23817412 A EP23817412 A EP 23817412A EP 4630571 A1 EP4630571 A1 EP 4630571A1
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European Patent Office
Prior art keywords
lactate
enzyme
hydroxy acid
acid
activity
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EP23817412.2A
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German (de)
French (fr)
Inventor
Peter Herzog
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DirectSens GmbH
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DirectSens GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/30—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving catalase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention relates to the field of detection and quantification of analytes, in particular to methods and means for the detection and/or quantification of non-lactate hydroxy acids with enzyme-based methods.
  • Hydroxy acids are suitable analytes for determination of risk factors.
  • the hydroxy acid a-hydroxybutyric acid (2-HBA) or its respective salt a-hydroxybutyrate has been described in several 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 potent predictors for different types of diabetes (Type II, gestational diabetes). From hundreds of compounds, 2-HBA occurred repeatedly as one of the most suitable predictors.
  • Another example of such a suitable analyte for determination of risk factors is the analysis of glycolate in blood for metabolic acidemia (Roberts et al., 2022).
  • 2-HBA is a chiral molecule that occurs in a L- and D-enantiomer and resembles the major metabolite lactate. Due to this similarity with lactate so far only GC/LC-MS has been successfully used to quantify 2-HBA in human serum and urinary samples in clinical studies.
  • US 2019/0107530 A1 discloses a method for assessing the risk of developing occult pancreatic beta cell dysfunction in a patient by measuring the level of a- hydroxybutyrate in a sample of the patient.
  • WO 2017/210097 A1 discloses the detection and determination of analytes such as 2-HBA in samples by mass spectrometry.
  • WO 2015/010042 A2 discloses clinical testing of biomarkers such as a- hydroxybutyrate to predict the likelihood of a subject having impaired glucose tolerance or insulin resistance. Thereby, methods for determination of the biomarkers are e.g. mass spectrometry, NMR, or devices for immunological detection. Maughan et al. (1982) discloses a method for enzymatically determining glucose, lactate, pyruvate, alanine, 3-hydroxybutyrate, and acetoacetate on a 20 pL blood sample.
  • WO 96/39534 A1 discloses oxidoreductases such as lactate dehydrogenase conjugated to a TAG for generating a chemiluminescent signal for use in biosensors and kits.
  • CN 101825625 A discloses a kit for simultaneously determining urinary lactic acid, creatinine, and beta-hydroxybutyric acid in urine.
  • WO 2022/125537 A2 discloses biosensors based on oxidoreductases such as lactate oxidase, lactate dehydrogenase, or 3-hydroxybutyrate dehydrogenase for the determination of lactate and 3-hydroxybutyrate in a sample.
  • oxidoreductases such as lactate oxidase, lactate dehydrogenase, or 3-hydroxybutyrate dehydrogenase for the determination of lactate and 3-hydroxybutyrate in a sample.
  • the inventors of the present invention surprisingly found that by combination of specific and subsequent reaction steps, a specific determination of non-lactate hydroxy acids such as 2-HBA is enabled.
  • a method is provided herein for determining a non-lactate hydroxy acid in complex samples even in the presence of molecules disturbing determination of such a hydroxy acid, i.e., in the presence of lactate.
  • the invention described herein enables a fast and accurate determination of 2-HBA in a sample comprising lactate. Such a method is highly desired for scaled diagnostic purposes.
  • an analyte can be measured in human samples and thus, the methods described herein can be utilized in in-vitro diagnostic (IVD) assay kits and enable determination of abnormal concentration levels of the analyte, which in return allows assessing the risk of various forms of metabolic disorders, such as diabetes.
  • IVD in-vitro diagnostic
  • a method for determining a non- lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
  • the non-lactate hydroxy acid is a non-lactate 2-hydroxy acid.
  • non-lactate 2-hydroxy acid is a compound having the general formula I wherein
  • Ri denotes H or C1-6 alkyl
  • R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • R1 denotes -CH3.
  • R2 denotes phenyl
  • non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
  • the enzyme in ii. is an enzyme having non-lactate hydroxy acid dehydrogenase activity, or an enzyme having non-lactate hydroxy acid oxidase activity. More specifically, the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
  • the enzyme in ii. is FCb2 or LDH.
  • an enzyme having lactate oxidizing activity specifically an enzyme having lactate oxidase activity.
  • the method described herein comprises further adding an agent removing hydrogen peroxide in i., preferably an enzyme having catalase activity is added.
  • determining the non-lactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
  • 2-hydroxybutyric acid is determined in a sample comprising 2- hydroxybutyric acid and lactate, said 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-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
  • the sample is a human sample.
  • an electrode comprising the enzyme having non-lactate hydroxy acid oxidizing activity is used in ii..
  • an enzyme having non-lactate hydroxy acid oxidizing activity specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non-lactate hydroxy acid in a sample in a method described herein.
  • the sample is a human sample.
  • an electrode comprising an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non- lactate hydroxy acid in a sample in a method described herein.
  • the sample is a human sample.
  • kits for determining a non- lactate hydroxy acid in a sample comprising non-lactate hydroxy acid and lactate comprising an enzyme having lactate oxidizing activity and an enzyme having non- lactate hydroxy acid oxidizing activity.
  • non-lactate hydroxy acid is a 2-hydroxy acid.
  • non-lactate hydroxy acid is 2-hydroxybutyric acid.
  • the enzyme having lactate oxidizing activity is lactate oxidase.
  • the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity.
  • the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
  • the enzyme having non-lactate hydroxy acid oxidizing activity is FCb2 or LDH.
  • the enzyme having non-lactate hydroxy acid oxidizing activity is part of an electrode.
  • an electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
  • non-lactate 2-hydroxy acid is a compound having the general formula I wherein
  • Ri denotes H or Ci -ealkyl
  • R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • R1 denotes -CH3.
  • R2 denotes phenyl
  • non-lactate 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-hydroxy-isobutyric acid.
  • the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity.
  • the enzyme having non-lactate 2-hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
  • the enzyme is FCb2 or LDH.
  • Figure 1 Example of an enzymatic assay principle for the determination of 2- HBA in two steps.
  • Figure 2 Technical triplicate reactions for L-lactate removal and 2-HBA measurement in presence and absence of L-lactate interferant and LOx catalyst.
  • amino acids refer to twenty naturally occurring amino acids encoded by sixty-one triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:
  • the “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: Alanine (Ala, A; nonpolar, neutral), Asparagine (Asn, N; polar, neutral), Cysteine (Cys, C; nonpolar, neutral), Glutamine (Gin, Q; polar, neutral), Glycine (Gly, G; nonpolar, neutral), Isoleucine (He, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, 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 (Vai, V; nonpolar, neutral), and Histidine (His, H;
  • the “positively” charged amino acids are: Arginine (Arg, R; polar, positive), and Lysine (Lys, K; polar, positive).
  • the “negatively” charged amino acids are: Aspartic acid (Asp, D; polar, negative), and Glutamic acid (Glu, E; polar, negative).
  • determining refers to detecting and/or quantifying nonlactate hydroxy acid.
  • detecting the non-lactate hydroxy acid refers to the general determination if non-lactate hydroxy acid is present. Detection does not require the exact quantification of non-lactate hydroxy acid but rather provides the user of the method with the information if e.g., non-lactate hydroxy acid is present with a concentration above a certain threshold. These thresholds are to be adapted to the respective application and sample.
  • quantifying refers to the determination of the concentration or amount of non-lactate hydroxy acid.
  • Quantification may refer to the determination of an exact amount of an analyte or may alternatively refer to a semi- quantitative determination of an analyte e.g., if the amount of the analyte in a sample is in a certain range. Such a range may be a concentration range suitable for the respective purpose of the determination of the analyte.
  • the method described herein may be used for the quantification or detection of non-lactate hydroxy acid.
  • the method may be used for determining the amount of non-lactate hydroxy acid or for determining if non-lactate hydroxy acid is present in a certain concentration range. Further, the method described herein may be used for determining if non-lactate hydroxy acid is present above or below a certain threshold.
  • non-lactate hydroxy acid refers to a hydroxy acid different from lactate or to a salt of a hydroxy acid different from lactate.
  • lactate refers to lactic acid or the salt thereof. Specifically, in the context of selectively removing lactate from the sample, and in the context of a non- lactate hydroxy acid, the term “lactate” refers to L-lactic acid or the salt thereof, i.e. , L- lactate.
  • the non-lactate 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-hydroxy- isobutyric acid, D-lactic acid, or the respective salt of any one of the foregoing.
  • the non-lactate hydroxy acid is a non-lactate 2- hydroxy acid.
  • non-lactate 2-hydroxy acid refers to a subgroup of non-lactate hydroxy acids.
  • non-lactate 2-hydroxy acid refers to 2-hydroxy acid different from lactate, or to a salt of a 2-hydroxy acid different from lactate.
  • 2-hydroxy acids are commonly known also as alpha hydroxy acids or a-hydroxy acids.
  • 2-hydroxy acids are a class of chemical compounds that consist of a carboxylic acid with a hydroxyl group substituent on the adjacent (alpha) carbon.
  • the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
  • Ri denotes H or C1-6 alkyl
  • R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • alkyl when used alone or in combination with other groups or atoms, refers to a saturated straight or branched chain consisting solely of 1 to 6 hydrogensubstituted carbon atoms, and includes methyl, ethyl, propyl, isopropyl, n-butyl, 1- methylpropyl, isobutyl, t-butyl, 2,2-dimethylbutyl, 2,2-dimethyl-propyl, n-pentyl, 2- methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like.
  • aryl refers to an aromatic mono- or bicyclic group containing from 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, that may be optionally fused with a fully or partially saturated or unsaturated carbocyclic ring and may optionally be substituted with one or more identical or different substituents, suitably one to three substituents.
  • aryl groups include phenyl, naphthyl, indanyl, and the like.
  • the substituent “-C(O)OH” refers to a carboxylic acid substituent.
  • Ri denotes H or C1-6 alkyl.
  • Ri denotes H or -CHs.
  • Ci-ealkyl is Ci alkyl.
  • R2 denotes H, Ce-8 aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • R2 denotes H.
  • R2 denotes Ce-8 aryl.
  • Ce-8 aryl is Ce aryl.
  • R2 denotes phenyl
  • R2 denotes H, Ce-8 aryl, or C1-14 alkyl optionally substituted by -C(O)OH.
  • R2 denotes H, phenyl, or C1-14 alkyl optionally substituted by -C(O)OH.
  • R2 denotes C1-20 alkyl optionally substituted by - C(O)OH.
  • R2 denotes -CH2-CH3.
  • R2 denotes H, phenyl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • the non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
  • the non-lactate hydroxy acid is a-hydroxybutyric acid (2-hydroxybutyric acid, 2-HBA) or its respective salt i.e., a- hydroxybutyrate.
  • 2-HBA 2-hydroxybutyric acid
  • Synonyms for the abbreviation 2-HBA are: HBA, a-HBA, aHB.
  • 2-HBA is a chiral molecule having the two enantiomers (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid.
  • the sample may be any material for which determining the presence of non-lactate hydroxy acid is relevant or of interest.
  • the sample is a human or animal sample, specifically any one of body fluid, interstitial fluid, blood, blood plasma, blood serum, dermal fluid, urine, tears, sweat, saliva, skin, flesh, tissue, eyeballs, cornea, and gastric fluid.
  • human blood is used as a sample in the methods and means described herein.
  • Blood contains various different non-lactate hydroxy acids and contains also lactate.
  • the sample contains or is suspected to contain a non-lactate hydroxy acid and lactate.
  • the sample contains or is suspected to contain lactate at a concentration in the range of 1 to 2 mM.
  • the sample contains or is suspected to contain at least a 10-fold higher concentration of lactate than 2-HBA.
  • the sample contains or is suspected to contain 2-HBA at a concentration in the range of 0.05 to 0.10 mM.
  • the methods and means described herein enable the determination of a non-lactate hydroxy acid in a sample suspected to contain also lactate e.g., in a blood sample which contains also lactate, by selectively removing lactate prior to the determination of the non-lactate hydroxy acid.
  • a method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate comprising the sequential steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
  • described herein is a method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
  • described herein is a method for determining 2-hydroxybutyric acid in a sample comprising 2-hydroxybutyric acid and lactate, said method comprising the sequential steps of: a.
  • a method for determining 2-hydroxybutyric acid in a sample comprising 2-hydroxybutyric acid and lactate comprising the steps of: a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity prior to step b. and c.; b. incubating the sample with an enzyme having 2-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
  • a method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate comprising the sequential steps of: i. optionally selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
  • a method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate comprising the steps of: i. optionally selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
  • selectively removing lactate from the sample is optional.
  • selectively removing refers to the removal of lactate while not substantially changing the amount or concentration of the non-lactate hydroxy acid in the sample.
  • removing refers to the modification of lactate e.g., to the oxidation of lactate to pyruvate.
  • Non-limiting examples of methods for selectively removing lactate are enzyme reaction, precipitation, emulsion liquid membrane separation techniques, adsorption, extraction, polymerization, and esterification.
  • Non-limiting examples of extracting lactate are extraction with niosomes, microfiltration, and reactive extraction (see Roque L., et al., 2020).
  • polymerization is polymerization using catalysts and heat (see Lunt, James, 1998, and Chafran, Liana S., et al., 2019).
  • esterification is vapor permeation-assisted esterification (see Khunnonkwao, Panwana, et al., 2012).
  • modification of lactate is accompanied by the accumulation of modified lactate in the sample.
  • the modified lactate is not necessarily removed from the sample, but may remain in the sample also during the determination of the non-lactate hydroxy acid.
  • the reaction product pyruvate is not necessarily removed from the reaction mixture and may remain in the sample.
  • the modified lactate may be removed from the sample.
  • pyruvate can remain in the sample, e.g., if a lactate oxidase characterized by a low product inhibition is used.
  • the reaction product pyruvate may be removed from the reaction mixture.
  • enzyme refers to any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reaction(s).
  • an enzyme having an activity is a functionally active molecule such as a functional enzyme.
  • a functional enzyme is specifically characterized by a catalytic centre recognizing the enzyme substrate and catalysing the conversion of the substrate to a conversion product.
  • Enzyme variants are considered functional or functionally active upon determining their enzymatic activity in a standard test system, e.g., wherein the enzymatic activity is at least 50% of the activity of the parent (not modified or wild-type enzyme), or at least any of 60%, 70%, 80%, 90%, 100%, or even more than 100%.
  • Enzyme activity is generally given in units. Thereby, one unit of enzymatic activity is defined as the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per min under the respective conditions of the determination method. For example, one unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of substrate such as e.g., lactate or non-lactate hydroxy acid, per min under the respective conditions of the determination method.
  • the specific activity is given in “ll/mg”, “II mg -1 ” or “II per mg”.
  • Volumetric activity is given in units per volume such as in “U/mL”, “U/ml”, “U per mL”, “U per ml”, “U mL’ 1 ”, or “U ml’ 1 ”.
  • an enzyme is used in step i. for selectively removing lactate, such as enzyme is referred to as herein also as enzyme A.
  • enzyme A is an enzyme having lactate oxidizing activity. In step i, enzyme A does not substantially change the concentration of non-lactate hydroxy acid to be determined in the sample.
  • enzyme A is selected from the group consisting of lactate oxidases, lactate monooxygenases, and lactate dehydrogenases.
  • step ii. an enzyme having non- lactate hydroxy acid oxidizing activity is used.
  • the enzyme having non-lactate hydroxy acid oxidizing activity is herein also referred to as enzyme B.
  • enzyme B has non-lactate hydroxy acid oxidizing activity but may also be able to oxidize lactate. Lactate oxidizing activity of enzyme B does not disturb the determination of the non-lactate hydroxy acid as lactate is removed in step i.
  • enzyme B is selected from the group consisting of non-lactate hydroxy acid dehydrogenases, non-lactate hydroxy acid oxidases, and non-lactate hydroxy acid monooxygenases.
  • enzymes such as LDH, FCb2, LOx, or hydroxy acid oxidase may be used as enzyme B.
  • enzyme A is an enzyme capable of modifying lactate to such an extent that the enzyme B cannot catalyze a reaction with the modified lactate.
  • enzyme A is an enzyme capable of selectively oxidizing lactate but does not substantially oxidize the non-lactate hydroxy acid.
  • enzyme A is an enzyme having lactate oxidizing activity.
  • the term “oxidizing” in the context of an oxidizing agent such as an enzyme having oxidizing activity refers to an agent that oxidizes a substance and gains or “accepts” an electron from said substance.
  • the enzyme has “substance oxidizing activity”.
  • Such a substance may also be referred to as substrate. Therefore, an enzyme having lactate oxidizing activity catalyzes the oxidation of lactate.
  • An enzyme having non-lactate hydroxy acid oxidizing activity catalyzes the oxidation of non-lactate hydroxy acid.
  • the term “capable of oxidizing” may be alternatively used for the term “oxidizing” in the context of an enzyme having oxidizing activity.
  • an enzyme having substance oxidizing activity gains or accepts one or more electrons from the substance.
  • the enzyme itself or a cofactor of the enzyme gets reduced.
  • an enzyme cannot catalyze another oxidation reaction of a substance. Therefore, the enzyme or the cofactor of the enzyme needs to be re-oxidized by transferring the gained electrons to an electron acceptor before another oxidation reaction of a substance can be catalyzed.
  • the enzyme or the cofactor may be re-oxidized by transferring the gained electrons to oxygen, to a molecule with electrochemical activity such as a redox mediator, or to an electrode.
  • the preference or degree of electron transfer of an enzyme to an electron acceptor differs depending on the specific enzyme and on the specific electron acceptor used.
  • an enzyme having substance oxidizing activity is known as “oxidase” if the enzyme uses dioxygen as preferred electron acceptor for the reoxidation of the enzyme. If an enzyme having substance oxidizing activity uses an electron acceptor different than dioxygen as preferred electron acceptor for the reoxidation of the enzyme, then such an enzyme is generally known as a “dehydrogenase”. According to a specific embodiment of the invention, enzyme A may be an enzyme having lactate oxidase activity or alternatively an enzyme having lactate dehydrogenase activity.
  • lactate oxidase activity refers to the activity of an enzyme catalyzing the oxidation of lactate with dioxygen as electron acceptor forming pyruvate and hydrogen peroxide as products. Thereby, two electrons are transferred from lactate to the cofactor of the enzyme e.g., to FAD, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide.
  • dioxygen may be used as electron acceptor for the re-oxidation of the enzyme.
  • enzyme A is a lactate oxidase (LOx).
  • Lactate oxidases belong to the enzyme family of E.C 1.1.3.2. Specifically, various lactate oxidases may be used in the method described herein e.g., lactate oxidase from Aerococcus viridans, Nostoc sp. (PCC7120), Lactobacillus jensenii, Lysinibacillus sphaericus, Chlamydomonas reinhardtii, Alicycliphilus denitrificans, Lacticaseibacillus rhamnosus, Lentilactobacillus hilgardii, Roseobacter sp.
  • lactate oxidase may be a functional variant of any one of the foregoing lactate oxidases having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the respective amino acid sequence of the foregoing lactate oxidases.
  • enzyme A may be a lactate monooxygenase. Therefore, various lactate monooxygenases may be used in the method described herein e.g., lactate 2-monooxygenase from Mycolicibacterium smegmatis.
  • the lactate oxidase is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L-lactate.
  • enzyme A may be an engineered variant of an enzyme.
  • an engineered variant may be an oxidase engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.
  • enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate.
  • an oxidase naturally oxidizing a different molecule than lactate may have been engineered towards using lactate as substrate.
  • the LOx described herein is a functionally active variant of a LOx peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LOx sequence, compared to the respective 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, specifically resulting in one or more amino acid substitutions, additions, or deletions, or the like.
  • the functional variant of the LOx peptide sequence is a full-length LOx peptide sequence comprising point mutations, or it is a fragment of the full-length LOx peptide sequence with retained enzymatic activity.
  • a variant of a LOx sequence is functionally active if it is capable of converting L-lactate to pyruvate or capable of converting a non-lactate hydroxy acid to the respective oxidized non-lactate hydroxy acid.
  • a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence with lactate or non-lactate hydroxy acid.
  • a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence, wherein said enzymatic activity is determined with the following assays.
  • the oxidase activity of an enzyme with non-lactate hydroxy acid as substrate can be determined using the method for determining lactate oxidase activity and using the non-lactate hydroxy acid instead of lactate.
  • lactate dehydrogenase activity refers to the activity of an enzyme catalyzing the oxidation of lactate with an electron acceptor different from dioxygen forming an oxidized lactate molecule e.g., pyruvate, and the respective reduced electron acceptor as products. Such a reaction is e.g., performed by a lactate dehydrogenase (LDH) or by a flavocytochrome b2 (FCb2).
  • LDH lactate dehydrogenase
  • FCb2 flavocytochrome b2
  • an enzyme having lactate dehydrogenase activity is used as enzyme A
  • a molecule different from dioxygen is used as electron acceptor for the re-oxidation of the enzyme.
  • a system for regeneration of the used electron acceptor may be implemented in the method described herein.
  • a system for regeneration of an electron acceptor may comprise an enzymatic, chemical, electrochemical, homogeneous catalytic, photocatalytic, or heterogeneous catalytic regeneration system.
  • lactate dehydrogenase is abbreviated herein as LDH.
  • LDH lactate dehydrogenase
  • a LDH is an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN or FAD, and the so gained electrons are subsequently transported towards a suitable electron acceptor like DCIP.
  • the LDH is commonly irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
  • a LDH is used in the method described herein as enzyme A.
  • various LDHs may be used in the method described herein such as but not limited to NAD+ dependent LDH e.g., from Sus scrofa, Homo sapiens, Mus musculus, Rattus norvegicus, Lactobacillus easel, Geobacillus stearothermophilus, Lactiplantibacillus pentosus, Deinococcus radiodurans, Thermus caldophilus, Thermotoga maritima, Bacillus subtilis, and Thermus thermophilus; or FAD- or FMN- dependent LDH e.g., from Pediococcus acidilactici.
  • the LDH may be a functional variant of any one of the foregoing LDHs having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the respective amino acid sequence of the foregoing LDHs.
  • enzyme A may be an engineered variant of an enzyme.
  • an engineered variant of a dehydrogenase e.g., a lactate dehydrogenase, engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.
  • enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate.
  • a dehydrogenase naturally oxidizing a different molecule than lactate may be engineered towards using lactate as substrate and thus, an engineered variant may also be an enzyme having lactate dehydrogenase activity.
  • the LDH used as enzyme A is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L- lactate.
  • the LDH described herein is a functionally active variant of a LDH peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LDH sequence, compared to the respective 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 resulting in one or more amino acid substitutions, additions or deletions, or the like.
  • the functional variant of the LDH peptide sequence is a full-length LDH peptide sequence comprising point mutations, or it is a fragment of the full-length LDH peptide sequence with retained enzymatic activity.
  • a variant of a LDH sequence is functionally active, if it is capable of converting L-lactate to pyruvate or capable of converting a non-lactate hydroxy acid to the respective oxidized non-lactate hydroxy acid.
  • a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence with lactate or non-lactate hydroxy acid.
  • a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence, wherein said enzymatic activity is determined with the following assays.
  • DCIP 2,6-dichlorophenol-indophenol sodium salt hydrate
  • the assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCI, 3 mM KCI and contains 10 mM lactate and 120 pM DCIP, which acts as an electron acceptor.
  • One unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of lactate per min under the assay conditions.
  • the reaction stoichiometry of lactate: DCIP is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single DCIP molecule. For the detection of activity with other substrates, lactate can be exchanged for other compounds.
  • the lactate is exchanged for this specific non-lactate hydroxy acid.
  • the lactate is exchanged for 2-HBA.
  • the assay mixture is formulated as is described for the DCIP assay but contains 500 pM 1 ,4-benzoquinone or 160 pM ferrocenium hexafluorophosphate instead of DCIP.
  • the reaction stoichiometry of lactate: 1 ,4-benzoquinone is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single molecule of 1 ,4-benzoquinone.
  • the reaction stoichiometry of lactate : ferrocenium hexafluorophosphate is 1 : 2, since two electrons are gained per lactate molecule and transferred individually to two molecules ferrocenium hexafluorophosphate.
  • lactate can be exchanged for other compounds (Brugger, D, et al. (2014), Sygmund, C. et al. (2011)).
  • the determination of the enzymatic activity of a LDH or a variant thereof can be also determined for oxygen as electron acceptor. Thereby, specifically the oxidase activity of a LDH is measured. The oxidase activity might not be detectable if the capability of the LDH to transfer electrons to oxygen is very low.
  • the enzyme having lactate oxidizing activity such as e.g., lactate oxidase activity or lactate dehydrogenase activity, used as enzyme A in the method described herein does not substantially alter the concentration or amount of the non-lactate hydroxy acid.
  • the enzyme having lactate oxidase or dehydrogenase activity used as enzyme A does not have or does substantially not have 2-HBA oxidase or 2-HBA dehydrogenase activity.
  • enzyme A has a specific activity with the non-lactate hydroxy acid of less than 10, 5, 4, 3, 2, 1 % relative to the specific activity of said enzyme with lactate.
  • enzyme A has a specific activity with the non- lactate hydroxy acid of 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 % relative to the specific activity of said enzyme with lactate.
  • the sample is treated 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 even longer with the enzyme A.
  • the sample is treated at room temperature with the enzyme A.
  • the sample is treated at 10, 15, 20, 25, 30, 35, or 40 °C with the enzyme A.
  • the enzyme A is added to a final concentration in the sample of at least 0.01 mg/mL. Specifically, the enzyme A is added to a final concentration in the sample of 0.01 to 0.1 mg/mL or even higher.
  • the duration of selectively removing lactate depends on the specific activity of the enzyme with lactate and on the final concentration of the enzyme in the sample.
  • the duration of the pretreatment step may be adapted depending on the specific enzyme and on the specific concentration of enzyme in the sample.
  • the enzyme A and optionally the catalase may be inactivated after selectively removing lactate and prior to incubation with the enzyme B.
  • the sample may be heated up to 100 °C for 5, 10, 15, or 20 minutes for inactivating the enzyme(s).
  • the determination of the non-lactate hydroxy acid as described herein may be corrected by a pre-determined factor.
  • a pre-determined factor may be derived from the side-reactivity of enzyme A with the specific non-lactate hydroxy acid.
  • enzyme A does not substantially change the concentration of non-lactate hydroxy acid in the sample.
  • the degree of this substantially change of the concentration of non-lactate hydroxy acid may be predetermined and a correction factor for the determination of non-lactate hydroxy acid may be determined.
  • hydrogen peroxide may be produced as a by-product in the step of selectively removing lactate from the sample. Therefore, an agent capable of removing hydrogen peroxide may be added in the method described herein. Such an agent may be added in step i. of the method described herein i.e., in the step of selectively removing lactate from the sample. Alternatively, such an agent may be added after step i. but before step ii., or simultaneously to step ii.
  • the agent capable of removing hydrogen peroxide is an enzyme having catalase activity.
  • Catalase activity refers to the activity of an enzyme catalyzing the reaction of decomposing hydrogen peroxide to water and molecular oxygen.
  • Non-limiting examples of such enzymes are catalases, e.g., catalase from Aspergillus niger, bovine liver, human erythrocytes, and any other known catalase.
  • the catalase activity of an enzyme or the enzymatic activity of a catalase or a variant thereof can be determined spectrophotometrically by continuous spectrophotometric rate reduction determination at 240 nm. Thereby, the rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm.
  • One unit of catalase will decompose 1 .0 pmole of H2O2 per minute at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM.
  • a spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase is described by Beers, R. F. Jr, & Sizer, I. W. (1952).
  • catalase is added to the sample in step i.. Thereby, catalase decomposes the hydrogen peroxide produced by the lactate oxidase to water and molecular oxygen.
  • the produced molecular oxygen may again serve as electron acceptor for the lactate oxidase.
  • catalase is used to boost treatment and/or detection reactions with lactate oxidase.
  • enzyme B is an enzyme having non-lactate hydroxy acid oxidizing activity.
  • Enzyme B may be an enzyme having nonlactate hydroxy acid dehydrogenase activity or an enzyme having non-lactate hydroxy acid oxidase activity.
  • non-lactate hydroxy acid oxidizing activity refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid.
  • hydroxy acid oxidizing activity may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
  • non-lactate hydroxy acid dehydrogenase activity refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid with an electron acceptor different from dioxygen and thereby forming an oxidized hydroxy acid and the respective reduced electron acceptor as products.
  • Such enzymes having dehydrogenase activity are commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
  • hydroxy acid dehydrogenase activity may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
  • the enzyme having hydroxy acid dehydrogenase activity or hydroxy acid oxidase activity may also have the respective lactate dehydrogenase or lactate oxidase activity. Since the lactate present in the sample is selectively removed from the sample in step i., there is no lactate present anymore in step ii. and thus, the enzymatic reaction of step ii. is not disturbed by lactate irrespective if the used enzyme has the theoretical capability of oxidizing lactate.
  • the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-HBA dehydrogenase activity.
  • an enzyme having 2-HBA dehydrogenase activity for example, flavocytochrome b2 (FCb2) and lactate dehydrogenase (LDH) are enzyme having 2-HBA dehydrogenase activity.
  • the enzyme having non-lactate hydroxy acid dehydrogenase activity is flavocytochrome b2 (FCb2) or lactate dehydrogenase (LDH).
  • such an enzyme having hydroxy acid dehydrogenase activity may also be a functionally active variant of e.g., FCb2 or LDH.
  • lactate dehydrogenase is described elsewhere herein as an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
  • LDH non-lactate hydroxy acid
  • lactate dehydrogenase an enzyme catalyzing the oxidation of the non-lactate hydroxy acid e.g., 2-HBA, to its corresponding oxidized form whereby two electrons are transferred from the non-lactate hydroxy acid to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
  • a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
  • lactate dehydrogenases are known and described elsewhere herein.
  • the lactate dehydrogenase may be FAD-dependent or NAD+-dependent.
  • a FAD-dependent lactate dehydrogenase is the lactate dehydrogenase from Pediococcus acidilactici which was previously described as lactate oxidase (see Ashok, Y., et al., 2020).
  • a LDH is used in the method described herein as enzyme B.
  • a LDH is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.
  • FCb2 is used in the method described herein as enzyme B.
  • a FCb2 is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.
  • FCb2 refers to a L-lactate-cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b2, FCb2, L-lactate cytochrome c oxidoreductase).
  • FCb2 catalyzes the electron transfer from L-lactate to cytochrome c in yeast mitochondria.
  • yeast L-lactate is converted to pyruvate by L-lactate cytochrome c- oxidoreductase (EC 1.1.2.3), which is herein referred to as “Flavocytochrome b2” or "FCb2".
  • FCb2 Native yeast flavocytochrome b2
  • FCb2 Native yeast flavocytochrome b2
  • FCb2 has two functional domains that are connected via a “hinge” linker (57 kDa monomer).
  • the FCb2 from S. cerevisiae is the best studied representative and has been crystallized (PDB 1 FCB).
  • FCb2 or a synonym thereof is used in the context of an enzyme having the capability of oxidizing the non-lactate hydroxy acid to be determined with the method described herein, these terms refer to an enzyme catalyzing the oxidation of the non- lactate hydroxy acid e.g., 2-HBA, to its corresponding oxidized form whereby the FCb2 catalyzes the electron transfer from non-lactate hydroxy acid to cytochrome c.
  • the FCb2 described herein may comprise a sequence based on the mature form of FCb2 naturally found in the yeast mitochondrial intermembrane space, which comprises a cytochrome b2 domain, a flavin domain, a hinge region connecting the cytochrome b2 domain and the flavin domain and a tail region at its C-terminus.
  • a mature FCb2 peptide sequence is the sequence of an FCb2 peptide as it is naturally found in the yeast mitochondrion, specifically in the mitochondrial intermembrane space.
  • the FCb2 described herein comprises a FCb2 peptide sequence comprising at least a yeast heme domain and a yeast flavin domain.
  • the FCb2 described herein is a functionally active variant of a FCb2 peptide sequence found in the yeast mitochondrial intermembrane space and comprises one or more point mutations in the nucleotide sequence encoding the FCb2 sequence, compared to the respective 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, specifically resulting in one or more amino acid substitutions, additions or deletions, or the like.
  • the functional variant of the FCb2 peptide sequence is a full-length mature FCb2 peptide sequence comprising point mutations, or it is a fragment of the full-length mature FCb2 peptide sequence with retained enzymatic activity.
  • a variant of a FCb2 sequence is functionally active if it is capable of converting the non-lactate hydroxy acid to be determined with the method to the corresponding oxidized form.
  • a functionally active variant of a FCb2 sequence has at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence with the non-lactate hydroxy acid as substrate.
  • a functionally active variant of a FCb2 sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence, wherein said enzymatic activity is determined with the CytC assay and the respective non-lactate hydroxy acid, e.g., 2-HBA as substrate.
  • the enzymatic activity of a flavocytochrome b2 variant can be readily determined by assays known in the art, such as assays determining the colorimetric reduction of cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP).
  • assays known in the art, such as assays determining the colorimetric reduction of cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP).
  • FCb2 described herein is determined by the CytC assay described by Diep Le et al. (2009) using the respective non-lactate hydroxy acid or alternatively lactate as substrate.
  • the FCb2 may comprise the amino acid sequence of a FCb2 from S. cerevisiae, W. anomalus, K. marxianus, O. parapolymorpha, Candida glabrata, Kluyveromyces lactis, Lachancea thermotolerans, Saccharomycodes ludwigii, Naumovozyma castelli, Zygosaccharomyces bailii, Zygosaccharomyces parabalii, Lachancea mirantina, Tetrapisispora phaffii, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces paradoxus, Vanderwaltozyma polyspora, Lachancea dasiensis, Wickerhamomyces ciferri, Kluyveromyces dobzhanskii, Kazachstania naganishii, Zygosaccharomyces mellis, Kazachstan
  • the FCb2 may be a functional variant of any one of the foregoing and comprise an amino acid sequence having 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% with the amino acid sequence of an FCb2 of any one of the foregoing.
  • the recombinant FCb2 described herein comprises a peptide sequence derived from the FCb2 of Saccharomyces cerevisiae, Kluyveromyces marxianus, Wickerhamomyces anomalus, Naumovozyma castelli or Cyberlindera fabianii.
  • Amino acid sequences of polypeptides derived from organisms may be readily derived from publicly available databases such as e.g., from databases provided by the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • enzyme B may also be an enzyme having non-lactate hydroxy acid oxidase activity.
  • non-lactate hydroxy acid oxidase activity refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid with dioxygen as electron acceptor forming the respective oxidized non-lactate hydroxy acid and hydrogen peroxide as products. Thereby, two electrons are transferred from the non-lactate hydroxy acid to the cofactor of the enzyme e.g., to a FAD cofactor, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide.
  • the term “hydroxy acid oxidase activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
  • an enzyme having non- lactate hydroxy acid oxidase activity may be an enzyme selected from the enzyme class EC: 1.1.3.15.
  • an enzyme having non-lactate hydroxy acid oxidase activity may be hydroxy acid oxidase from Mus musculus, Rattus norvegicus, Homo sapiens, Arabidopsis thaliana, or any other known enzyme having hydroxy acid oxidase activity.
  • the enzymes used in the method described herein may be active at acidic, neutral, or alkaline pH ranges.
  • the enzymes of the invention may 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 at any pH of a body fluid e.g., of sweat or blood.
  • blood has a pH between 7.35 and 7.45.
  • a lactate dehydrogenase-based detection system flavocytochrome b2, Fcb2
  • LOx lactate oxidase
  • the enzymes are characterized either by high turnover rates with the analyte of interest (2- HBA) or high specificity to the otherwise interfering substances such as lactate.
  • the combination of an oxygen-dependent enzyme and an oxygen-independent enzyme for unaffected detection or unaffected colorimetric reaction is described herein.
  • the colorimetric reaction with FCb2 is unaffected by lactate removal with LOx.
  • the method described herein may comprise the combination of an engineered lactate oxidase variant with specificity for L-lactate and an engineered flavocytochrome b2 variant with improved reactivity towards 2-HBA.
  • the lactate oxidase variant is used in a pre-treatment step to specifically oxidize L-lactate.
  • the improved FCb2 variant is used to oxidize 2-HBA and colorize a reagent concomitantly to translate 2-HBA concentration into a quantifiable signal (e.g., absorbance, fluorescence).
  • incubating refers to contacting the sample with an enzyme to allow the enzyme to react with the non-lactate hydroxy acid. Thereby, incubating may be performed directly in the sample e.g., after adding the enzyme. Alternatively, an aliquot of the sample may be taken from the sample and mixed with a suitable liquid e.g., a buffer, prior to adding the enzyme. Depending on the specific determination method, such a suitable liquid may also comprise other compounds such as e.g., a compound necessary for the colorimetric detection or an electron acceptor.
  • the step of incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity may be sequentially or simultaneously performed with the step of determining the non-lactate hydroxy acid in the sample.
  • the enzyme having non-lactate hydroxy acid oxidizing activity may be any enzyme capable of oxidizing the non-lactate hydroxy acid and transferring the so gained electrons to a suitable electron acceptor.
  • an electron acceptor may be any suitable molecule which can be used for the colorimetric, photometric, fluorimetric, or electrochemical detection of the electron transfer reaction.
  • the signal obtained from detecting the electron transfer reaction is used for the determination of the non-lactate hydroxy acid in the sample.
  • an enzyme having hydroxy acid oxidase activity such an electron acceptor may also be dioxygen. In this case, hydrogen peroxide is produced through the electron transfer from non-lactate hydroxy acid to dioxygen.
  • Hydrogen peroxide can also be detected by suitable methods such as e.g., by colorimetric, photometric, fluorimetric, bioelectrochemical, electrochemical methods, possibly using enzyme based assays. Thereby, the presence or amount of non-lactate hydroxy acid can be calculated from the detection of hydrogen peroxide.
  • the electron acceptor used in the oxidation reaction of the non-lactate hydroxy acid may also be a polypeptide.
  • a polypeptide may be naturally attached to the enzyme capable of oxidizing the non-lactate hydroxy acid or synthetically attached to said enzyme.
  • the enzyme FCb2 comprises a heme domain which accepts electrons from the cofactor of the catalytically active domain of the FCb2 i.e. , flavin domain, capable of oxidizing the non-lactate hydroxy acid.
  • a polypeptide used as electron acceptor may transfer the so gained electrons to a terminal electron acceptor.
  • terminal electron acceptor examples include molecules such as cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP) which can be used in colorimetric and photometric detection methods.
  • cytochrome c ferricyanide
  • DCIP 2,6-dichloroindophenol
  • Another example of such a terminal electron acceptor is an electrode surface. The presence or amount of non-lactate hydroxy acid in the sample can be derived from a signal obtained by the specific detection method.
  • determining the non-lactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
  • colorimetrically refers to the application of a colorimetric analysis for the determination of the presence or the amount of non-lactate hydroxy acid in the sample.
  • concentration of a chemical element or chemical compound in a solution is determined with the aid of a color reagent.
  • the color reagent may be visually detected or detected with suitable equipment e.g., a colorimeter.
  • enzymatic analysis as described herein, the color reaction is preceded by a reaction catalyzed by an enzyme such as the enzyme capable of oxidizing a non- lactate hydroxy acid.
  • a general example of such a colorimetric determination is the detection of a colored complex formed by a peroxidase from hydrogen peroxide and ABTS.
  • photometrically refers to the usage of a photometer or a spectrophotometer for enzymatically determining a substance in a sample, or alternatively also for determining the enzyme activity, by following the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution.
  • a colorimetric detection may be coupled or performed by a photometer.
  • fluorimetrically refers to the detection of fluorescence by determining the difference in the fluorescence spectra of substrate from product to measure the enzyme reaction or determine the substrate or product concentration.
  • the substrate and product may be different from the direct substrates and products of enzymes described herein e.g., different from non-lactate hydroxy acid and oxidized non-lactate hydroxy acid.
  • the Amplex red assay which is elsewhere described herein is an example of an assay based on a fluorimetric detection method.
  • electrochemically refers to the usage of an electrochemical biosensor based on the measurement of biological binding eventdependent changes in conductance, resistance, or capacitance of the biosensor surface.
  • one of the electrodes is immobilized with a biological recognition molecule.
  • the binding of the analyte to the biological recognition molecule triggers a change in the electrical properties due to oxidation and reduction reactions taking place as a result of biological interaction activity, thus providing the sensor signal.
  • Electrochemical biosensors rely mostly on enzyme-catalyzed reactions to produce current/potential difference which is then detected.
  • the electron acceptor used in the oxidation reaction of the non-lactate hydroxy acid for the determination of the amount of non-lactate hydroxy acid in the sample may also be an electrode equipped with an enzyme capable of oxidizing the non-lactate hydroxy acid.
  • Such an electrode may be part of a biosensor.
  • detecting the oxidation of non- lactate hydroxy acid by the enzyme capable of oxidizing the non-lactate hydroxy acid may be carried out by a sensor, specifically a bio-electrochemical sensor, configured to detect and/or quantify non-lactate hydroxy acid in a sample via (bio)electrochemical redox reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of the analyte non-lactate hydroxy acid.
  • Electrochemical biosensors can be impedimetric, potentiometric, or amperometric. In an amperometric biosensor, a biochemical signal is transduced into a quantifiable amperometric signal.
  • amperometric biosensors are commonly divided into three main generations depending on the electron transfer method used for the measurement of the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediators, and cofactors).
  • First-generation biosensors measure the concentration of analytes and/or products of enzymatic reactions that diffuse to the transducer surface and generate an electrical response. They are also called mediatorless amperometric biosensors.
  • oxidases are used in first-generation biosensors. Oxidases need molecular oxygen as a second substrate so the oxidase-based biosensors are oxygen dependent.
  • Second-generation biosensors require an electron mediator for the transfer of electrons obtained from enzymatic reactions to the transducer surface and thereby generate an electrical response.
  • direct electron transfer is enabled between the redox-active biomolecule i.e., the enzyme, and the electrode surface.
  • an electrode comprising an enzyme capable of oxidizing the non-lactate hydroxy acid is contacted with the sample.
  • This contact between electrode and sample can be performed by any approach which brings the electrode and the sample in contact in order that the enzyme is allowed to react with the non-lactate hydroxy acid or with the sample suspected to contain non-lactate hydroxy acid.
  • the term “electrode” refers to any suitable surface for accepting electrons from the enzyme via mediatorless, mediated, or direct electron transfer.
  • the electrode is of a material capable of accepting electrons.
  • the electrode may be of any material suitable or modified with any material to adsorb or immobilize the enzyme capable of oxidizing the non-lactate hydroxy acid.
  • Non-limiting examples of such a material are platinum, gold, boron doped diamond and carbons such as graphite, pyrolytic graphite and glassy carbon where all of them can additionally be modified with carbon nanotubes (single or multi-walled), carbon fibers, nanoparticles e.g. gold nanoparticles or promoters as e.g., thiols.
  • the electrode may be also of any material to increase the specific surface are of the electrode. According to one embodiment of the invention, the electrode may be used as single electrode or as a stack of electrodes of e.g., 2, 3, 4, 5, or more electrodes.
  • the electrode provided herein is a working electrode.
  • the electrode comprising the enzyme capable of oxidizing the non-lactate hydroxy acid of the invention enables the detection and/or quantification of non-lactate hydroxy acid based on mediatorless, mediated, or direct electron transfer.
  • Mediatorless electron transfer typically employs the transfer of electrons from hydrogen peroxide, produced through the enzymatic reaction, to the electrode.
  • Mediated electron transfer in biosensors typically employs a two-step procedure in which the enzyme takes part in a first redox reaction with the substrate and is in turn re-oxidized by a redox mediator. Finally, the redox mediator is oxidized by the electrode.
  • Redox mediators are artificial electron transferring agents that can readily participate in the redox reaction with the biological component and thus help in rapid electron transfer to the electrode.
  • a "redox mediator” is an electron-transfer agent for carrying electrons between an analyte, an analyte-reduced or analyte-oxidized enzyme, and an electrode, either directly, or via one or more additional electron-transfer agents.
  • a redox mediator that includes a polymeric backbone may also be referred to as a redox polymer.
  • said redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.
  • the oxidation of non-lactate hydroxy acid is performed in the presence of a redox mediator.
  • the method of the invention can also be performed in the presence of more than one redox mediator e.g., in the presence of two or more different redox mediators.
  • the redox mediator may be present on the electrode, in an enzyme composition comprising the enzyme and the redox mediator, or may be present in the sample.
  • the redox mediator may be any molecule or material able to carry electrons between the enzyme capable of oxidizing the non-lactate hydroxy acid and electrode.
  • said redox mediator is selected from the group consisting of any one of organic redox mediators, soluble redox mediators, insoluble redox mediators, redox polymers, transition metal complexes, polymeric transition metal complexes, wired redox mediators, sandwich compounds, and derivatives of these redox mediators.
  • Polymeric transition metal complexes comprise a polymeric backbone, spacers, and transition metal complexes.
  • redox polymers are polymers comprising redox species.
  • redox species used in redox polymers are osmium (Os), ruthenium (Ru), iron (Fe), cobalt (Co), or any transition metal.
  • Non-limiting examples of polymers used for redox polymers are poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).
  • An example of a redox polymer is Os-containing poly(vinylpyridine).
  • the redox mediator may be an osmium transition metal complex with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2'- bipyridine, 1 ,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole, or derivatives thereof.
  • the redox mediator may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof.
  • an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2' -bipyridine, 1 ,10- phenanthroline, or derivatives thereof, the two ligands not necessarily being the same.
  • Some derivatives of 2,2' -bipyridine for complexation with the osmium cation include but are not limited to 4,4'-dimethyl-2,2'- bipyridine and mono-, di-, and polyalkoxy-2, 2'-bipyri dines, including 4,4'-dimethoxy-2,2'- bipyridine.
  • Derivatives of 1 , 10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl- 1 , 10- phenanthroline and mono, di-, and polyalkoxy-1 , 10- phenanthrolines, such as 4,7- dimethoxy- 1 ,10-phenanthroline.
  • Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1 -vinyl imidazole) and poly(4-vinyl pyridine).
  • Suitable copolymer substituents of poly(1 -vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1 -vinyl imidazole).
  • An example of a redox polymer is also derived from poly(l-vinylimidazole) or a copolymer of (1 -vinyl imidazole) bound to a metal ion selected from the group consisting of Os.sup.3+/2+, Ru.sup.3+/2+, and Fe.sup.3+/2+.
  • transition metal refers to an element whose atom has a partially filled d sub-shell, or which can give rise to cations with an incomplete d sub-shell. Thereby, transition metals are elements in the d-block of the periodic table and also lanthanides and actinides.
  • Non-limiting examples of transition metal complexes include complexes comprising 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 ferricyanide, ruthenium hexamine, metalloporphyrins such as heme b or heme c. In these complexes, the transition metal is coordinatively bound to one or more ligands, which are typically mono-, di-, tri-, or tetradentate.
  • 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.
  • 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.
  • a sandwich compound is a chemical compound featuring a metal bound by haptic covalent bonds to two arene ligands.
  • the arenes have the formula C n Hn, substituted derivatives (for example C n (CH3)n) and heterocyclic derivatives (for example BC n Hn+i).
  • a special class of sandwich complexes are metallocenes.
  • a metallocene contains a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure, i.e. , the two cyclopentadienyl anions are on parallel planes with equal bond lengths and strengths.
  • Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1 ,1'-dimethyl ferrocene [DMF], and ferrocene monocarboxylic acid.
  • Organic redox compounds are organic molecules capable to act as a redox mediator.
  • organic redox mediators are organic molecules such as quinones, compounds having a quinoid structure such as benzoquinones or phenanthroline quinones, phenazine such as 1 -methoxyphenazine methosulfate, tetracyanoquinodimethane (TCNQ), N,N,N', N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivative of these molecules.
  • TCNQ tetracyanoquinodimethane
  • TMPD N,N,N', N'-tetramethyl-p-phenylenediamine
  • DCIP tetrathiafulvalene
  • a direct electron transfer biosensor may be used for the determination of non-lactate hydroxy acids, wherein the biosensor comprises an electrode comprising an enzyme capable of oxidizing the non-lactate hydroxy acid and said enzyme is able to transfer the electrons gained from the oxidation of the non-lactate hydroxy acid directly to the electrode surface.
  • an enzyme capable of oxidizing the non-lactate hydroxy acid enabling direct electron transfer is FCb2.
  • the enzyme capable of oxidizing the non-lactate hydroxy acid is immobilized on the electrode by adsorption, physical entrapment in a polymer, complex formation, preferably via an additional complexing linker, covalent binding, in particular cross-linking, or ionic binding and/or the immobilized enzyme can be cross-linked, in particular by bifunctional agents, to increase stability or activity.
  • Cross-linking agents are e.g., dialdehydes such as glutaraldehyde.
  • the electrode of the invention is part of a biosensor.
  • a specific use of the electrodes of the invention is in the provision of a biosensor, more specifically a first, second, or third-generation non-lactate hydroxy acid biosensor using mediatorless, mediated, or direct electron transfer properties to detect non-lactate hydroxy acid and/or to measure the non-lactate hydroxy acid concentration.
  • the biosensor may be suitable for use at acidic, neutral, or alkaline pH.
  • the biosensor may be suitable for use at room temperature or at body temperature.
  • the biosensor may be suitable for the detection and/or quantification at 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.
  • the biosensor may have one or more electrodes comprising the enzyme capable of oxidizing the non-lactate hydroxy acid.
  • the non-lactate hydroxy acid biosensor includes: a working electrode comprising a conductive material, wherein the enzyme capable of oxidizing the non-lactate hydroxy acid is in proximity to the conductive material.
  • One or more other electrodes may be included such as one or more counter electrodes, one or more reference electrodes and/or one or more counter/reference electrodes.
  • the biosensor may depend on the use for which the biosensor is intended and the conditions under which it will operate.
  • the biosensor may be a single use biosensor for the detection of non-lactate hydroxy acid.
  • the biosensor may be a biosensor strip.
  • a kit for determining, e.g., detecting and/or quantifying, non-lactate hydroxy acid in a sample comprising an enzyme capable of selectively removing lactate from the sample and an enzyme capable of oxidizing the non-lactate hydroxy acid.
  • a kit for determining 2-HBA in a sample.
  • the kit may further comprise an agent for selectively removing hydrogen peroxide, specifically an enzyme, specifically a catalase.
  • the enzyme capable of oxidizing the non-lactate hydroxy acid may be part of an electrode.
  • the electrode may be part of a biosensor.
  • the kit described herein further comprises an instruction manual.
  • the kit may also comprise auxiliary substances, like buffers, molecules necessary for detection e.g., electron acceptors, and containers such as a sample holding means, and/or non-lactate hydroxy acid standards.
  • auxiliary substances like buffers, molecules necessary for detection e.g., electron acceptors, and containers such as a sample holding means, and/or non-lactate hydroxy acid standards.
  • Non- lactate hydroxy acid standards may be used to calibrate the assay.
  • the kit may also comprise a reader for a signal, especially an electrochemical signal such as a potentiostat, a computer readable memory device with software for calibration and/or measurement calculations.
  • the enzymes described herein may be recombinantly expressed by methods commonly known in the art.
  • the enzymes described herein may be expressed using standard methods for cloning, transformation, and recombinant production in suitable host organisms e.g., in Escherichia coli or in Pichia pastoris.
  • the terms “increase in activity”, “increased activity”, or the like used herein may refer to a detectable increase in activity of an enzyme.
  • the terms “increase in activity”, or “increased activity” used herein may mean that a modified enzyme (variant) shows higher activity than a comparable enzyme of the same type, like an enzyme that does not have the particular modification.
  • the modified enzyme may comprise sequence alterations in the polypeptide or the nucleotide sequence encoding the enzyme.
  • activity of a modified or engineered enzyme may be higher than activity of a non-engineered enzyme of the same type, for example, a wild-type enzyme 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 a protein or enzyme of the same type in a parent cell, for example, a non-engineered cell 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.
  • Increased activity of an enzyme or protein in a cell may be verified by any methods known in the art.
  • the term “decrease in activity”, “decreased activity”, or the like used herein may refer to a detectable decrease in activity of an enzyme.
  • an increase or decrease in activity may also target only a specific substrate of interest.
  • an enzyme used herein may have a decreased activity with nonlactate hydroxy acid as a substrate but a substantially non-altered or even increased activity with lactate.
  • a functional variant or “functionally active variant” also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
  • an allelic variant, or also referred to as homologue is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide.
  • a functional variant may comprise a substitution, deletion and/or addition 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 thereof.
  • substitutions, deletions and/or additions may be conservative modifications. Specifically, substitutions, deletions and/or additions do not decrease the enzyme’s specific activity.
  • a functionally active variant of the enzyme capable of oxidizing non-lactate hydroxy acid as described herein comprises specific enzymatic activity towards a non-lactate hydroxy acid of at least 1 ll/rng, as determined by the respective assay as described herein.
  • a functional variant as described herein comprises no more than or up to 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 substitutions, deletions and/or additions.
  • these modifications may be conservative modifications.
  • these modifications do not decrease the enzyme’s specific activity.
  • a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5, amino acid substitutions, deletions and/or additions.
  • these modifications may be conservative modifications.
  • these modifications do not decrease the enzyme’s specific activity.
  • a functionally active variant described herein comprises at least 40, 50, 60, 70, 80 or 90% or even more of the enzymatic activity of the respective wild type enzyme.
  • Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence e.g., by one or more point mutations, wherein the sequence alterations retain or improve a feature of the enzyme, such as its stability or activity for example.
  • sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions.
  • Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.
  • a point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the nonengineered amino acid sequence in the substitution, or exchange, deletion, or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.
  • the enzymes described herein may comprise one or more tag sequences, specifically N-terminal tag sequences.
  • tag sequence is C-terminal of the N-terminal methionine of the enzymes described herein.
  • tag sequence may comprise any number of amino acids of more than 2, 4, 5, 6 or 10 amino acids and up to 20 or 50 or more amino acids.
  • tag sequences used herein may be any tag sequence known to the person skilled in the art.
  • tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.
  • Affinity tags are amino acid sequences that can be used for example for the purification of proteins where they are attached to. These affinity tags have high affinity to appropriate ligands of a solid support, like chromatography resins or directly to the resins. By selectively binding of the protein having the affinity tag to the particular resin the protein can be purified highly effective by only one chromatography step.
  • affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, poly-arginine tag, FLAG tag, Strep tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP) tag, S-tag, HA tag, c-Myc tag, and SUMO tag, or any other tag known to be useful for the efficient purification of a protein it is fused to.
  • the tag is a His tag comprising one or more H, specifically a hexahistidine tag.
  • proteins comprising a poly-, or hexa-histidine tag (His-tag) can be captured and purified using chromatography, e.g. by Immobilized Metal Affinity Chromatography (IMAC).
  • IMAC Immobilized Metal Affinity Chromatography
  • Solubility enhancement tags can be fused N-terminal to the enzymes described herein. Solubility enhancement tags can increase the titer of the soluble protein when expressed in a host cell, e.g. in the cytosol of P. pastoris, compared to expression of the proteins without the tag.
  • solubility enhancement tag sequences used herein are selected from calmodulin-binding peptide (CBP), poly Arg, poly Lys, protein D tag (dTAG), Z domain of Staphylococcal protein A, and thioredoxin or any other tag known to improve the solubility of the protein it is fused to e.g. during expression in a host cell.
  • the solubility enhancement tag is a T7 tag, preferably selected from the group consisting of T7A, T7A1 , T7A2, T7A3, T7A4, T7A5, T7B, T7B1 , T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11 , T7B12, T7B13, and T7C.
  • the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin or any other tag useful for detection or quantification of the recombinant enzyme during production steps including fermentation, isolation and purification by simple in-situ, inline, online or atline detectors, like UV, IR, Raman, fluorescence and the like.
  • sequence identity is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity.
  • sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences.
  • Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%.
  • Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.
  • Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.
  • Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry.
  • Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.
  • one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.
  • Percent (%) identity with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity.
  • Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared.
  • percentages determined for sequence identities it is possible that arithmetical decimal places may result which are not possible with regard to full nucleotides or amino acids. In this case, the percentages shall be rounded up to whole nucleotides or amino acids.
  • sequence identity between two amino acid sequences is determined using standard methods, e.g. using the NCBI BLAST program version 2.2.29 (Jan-06-2014) or online using the multiple sequence alignment tool EMBL-EBI Clustal Omega (Sievers, F. et al. (2011)).
  • Percent (%) identity with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, MAFFT based algorithms: multiple alignment using fast fourier transform, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g., the Burrows Wheeler Aligner
  • Clustal W Clustal X
  • BLAT Novoalign
  • ELAND Illumina, San Diego, CA
  • SOAP available at soap.genomies.org.cn
  • Maq available at maq.sourceforge.
  • a structure alignment In a structure alignment the maximal set of corresponding pairs of amino acid residues that gives a good structural match when the structures are overlaid, i.e., superposed, is identified. Thereby, the positions of the protein’s backbone C-alpha atoms and/or location of secondary structural elements are considered in this alignment.
  • Tools for performing a structure alignment are available, e.g., the protein data bank provides a tool for pairwise structure alignment.
  • structure superposition is also a tool for determining corresponding amino acid positions in different enzymes. Structure superposition can be performed using the Molecular Graphics System PyMOL, (Schrodinger) using the command “align”.
  • the invention further provides the following ITEMS:
  • a method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate comprising the steps of: i. optionally selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
  • Ri denotes H or C1-6 alkyl
  • R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
  • kits for determining a non-lactate 2-hydroxy acid in a sample comprising non-lactate 2-hydroxy acid and lactate comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
  • kit of any one of items 24 to 26, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity.
  • kit of any one of items 24 to 28, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is FCb2 or LDH.
  • kit of any one of items 24 to 29, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is part of an electrode.
  • An electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
  • Ri denotes H or C i-ealkyl
  • R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
  • non-lactate 2- hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2- hydroxy valeric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, and 2-hydroxy-isobutyric acid.
  • enzyme having non- lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity.
  • a method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate comprising the steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
  • non-lactate hydroxy acid is 2- hydroxybutyric acid, 3-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2- hydroxyoctanoic acid, 2-hydroxypalmitic acid, or glycolic acid.
  • an electrode comprising an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non-lactate hydroxy acid in a sample in a method of any one of items 40 to 47.
  • kits for determining a non-lactate hydroxy acid, specifically 2- hydroxybutyric acid, in a sample comprising non-lactate hydroxy acid and lactate comprising an enzyme having lactate oxidizing activity and an enzyme having non- lactate hydroxy acid oxidizing activity.
  • kit of item 50 wherein the enzyme having lactate oxidizing activity is lactate oxidase.
  • the kit of item 50 or 51 , wherein the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity, preferably having 2-hydroxybutyric acid dehydrogenase activity.
  • kit of any one of items 50 to 52, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is FCb2 or LDH.
  • kit of any one of items 50 to 53, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is part of an electrode.
  • Example 1 Enzymatic assay principle for the determination of 2-HBA in two steps
  • lactate oxidase is used as an example of an enzyme for the removal of L-lactate.
  • Flavocytochrome b2 is used as an example of an enzyme for the detection of the non-lactate hydroxy acid.
  • 2-HBA is used as an example for the non-lactate hydroxy acid.
  • a general scheme of the reaction is shown in Fig. 1.
  • a specific detection of 2-HBA is performed in samples, where interfering L-lactate can be removed using a dedicated enzymatic pre-treatment step with lactate oxidase (LOx).
  • LOx lactate oxidase
  • an enzyme is used for removal which uses oxygen as electron acceptor, H2O2 accumulates from O2 reduction simultaneous to the oxidation of L-lactate, and can be partly recovered (half-stoichiometrically) employing an enzymatic conversion with catalase: 2 H2O2 2 H2O + O2.
  • This regeneration using catalase may aid in maintaining a steady oxygen concentration for the LOx reaction.
  • the 2-HBA analyte can be detected without interference using an enzyme like FCb2.
  • L-lactate is present at >10-fold higher concentrations (1-2 mM) than the 2-HBA analyte (0.05-0.10 mM).
  • This example provides the general proof of principle that the method described herein can be used for the determination of a non-lactate hydroxy acid in samples comprising L-lactate. Furthermore, results of spiking the non-lactate hydroxy acid in physiological samples is shown.
  • Multititer plate 96-well (Greiner, Polystyrole, Merck # M2936)
  • Reagent 1 is used for the pretreatment step.
  • Reagent 1 Aerococcus viridans
  • Reagent 2 Candida glabrata FCb2 at 50 pg/mL and 80 pM equine Cytochrome C (Sigma-Aldrich, #C2506) in 11 mM PBS, pH 7.4
  • the determination of 2-HBA concentrations is performed at room temperature (22 °C), including the 5 min pre-treatment reaction and the 3 min measurement using a photometric plate reader.
  • Equation 2 calculation of 2-HBA concentration from changes in optical absorbance at 550 nm (this calculation only applies for the given technical setup):
  • Samples contained different combinations of 5 mM L-lactate, 0.1 mM 2-HBA, 0.1 mg/mL LOx but contained catalase solution at 0.1 % (v/v) in all assays. Pre-treatment reactions were carried out for 5 minutes at 22 °C prior to sample measurement of 2-HBA concentrations, relying on a 3-minute Cytochrome C (CytC) assay with FCb2. The increase in absorbance at 550 nm originating from CytC reduction translated to a substrate concentration dependent enzyme activity (Ll/mL). Results:
  • Lactate removal Whether L-lactate is present or not; (2) vs. (4): the residual signals are comparable, indicating that the 2-HBA fraction alone leads to the signals after treatment.
  • FCb2 activities were averaged and translated to apparent 2-HBA concentrations, which were the opposed to spiked 2-HBA concentrations in the sample
  • KmFCb2 is used as an exemplary enzyme for the electrochemical detection of 2-HBA.
  • Biosensor electrodes were prepared as is described in Geiss et al. (2021) with the adaptation of using 2 pL of 10 mg/mL engineered KmFCb2 in 100 mM Phosphate buffer. Curing was done at 22 °C for 2 h under dry ambience.
  • the electrode measurement was carried out in triplicates utilizing a potentiostat in a chronoamperometric measuring mode at an applied potential of 0.2 V vs a pseudo-Ag/AgCI reference electrode at 22°C. Electrodes were mounted horizontally, and samples were added/removed step wise to the sensing area to yield increased substrate concentrations over time. The change of currents was measured over time. • Data evaluation: Baseline subtracted currents were evaluated 10 s after each sample addition and plotted versus the substrate concentration. Non-linear regression fitting to Michaelis-Menten equation were calculated.
  • the analyte can be detected using the FCb2 enzyme without the need of a soluble electron acceptor when the FCb2 enzyme is contacted on an electrode.
  • the enzyme delivers proportional catalytic currents that can be measured using an electronic device, such as a potentiostat.
  • This example shows the determination of different non-lactate hydroxy acids using three different FCb2 enzymes.
  • WaFCb2 - amino acid sequence SEQ ID NO: 4
  • FCb2 enzyme activities were determined photometrically following the general principles of the Cytochrome C assay as described herein e.g., in example 1. Therefore, 300 mM stock solutions were prepared in 50 mM PPB, pH 6.5 for each substrate.
  • Table 5 gives the relative enzyme activity with respect to L-lactate for each substrate of CangFCb2, KmFCb2, and WaFCb2. Table 5: Overview over relative substrate-dependent activities of FCb2s
  • Example 4 shows the determination of different non-lactate 2-hydroxy acids using the invention described herein.
  • Example 4 was performed as described in Example 3.
  • the following Table 6 shows the relative substrate-dependent activities.
  • a-Hydroxybutyric acid is a selective metabolite biomarker of impaired glucose tolerance. Diabetes Care, 39(6), 988-995.

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Abstract

The present invention relates to methods and means for detecting and/or quantifying of analytes, in particular to methods and means for the detection and/or quantification of non-lactate hydroxy acids such as α-hydroxybutyric acid using enzyme- based methods. These methods and means enable the determination of e.g., the non-lactate hydroxy acid α-hydroxybutyric acid in complex samples comprising lactate such as in blood samples.

Description

ENZYMATIC DETECTION OF NON-LACTATE HYDROXY ACIDS
FIELD OF THE INVENTION
The present invention relates to the field of detection and quantification of analytes, in particular to methods and means for the detection and/or quantification of non-lactate hydroxy acids with enzyme-based methods.
BACKGROUND OF THE INVENTION
Assessment of risk factors for various diseases is crucial for prevention of diseases and early detection of diseases. Thereby, the determination of analytes in samples provided by humans is gaining importance in in-vitro diagnostic kits or assays.
Hydroxy acids are suitable analytes for determination of risk factors.
For example, the hydroxy acid a-hydroxybutyric acid (2-HBA) or its respective salt a-hydroxybutyrate has been described in several 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 potent predictors for different types of diabetes (Type II, gestational diabetes). From hundreds of compounds, 2-HBA occurred repeatedly as one of the most suitable predictors.
Another example of such a suitable analyte for determination of risk factors is the analysis of glycolate in blood for metabolic acidemia (Roberts et al., 2022).
2-HBA is a chiral molecule that occurs in a L- and D-enantiomer and resembles the major metabolite lactate. Due to this similarity with lactate so far only GC/LC-MS has been successfully used to quantify 2-HBA in human serum and urinary samples in clinical studies.
US 2019/0107530 A1 discloses a method for assessing the risk of developing occult pancreatic beta cell dysfunction in a patient by measuring the level of a- hydroxybutyrate in a sample of the patient.
WO 2017/210097 A1 discloses the detection and determination of analytes such as 2-HBA in samples by mass spectrometry.
WO 2015/010042 A2 discloses clinical testing of biomarkers such as a- hydroxybutyrate to predict the likelihood of a subject having impaired glucose tolerance or insulin resistance. Thereby, methods for determination of the biomarkers are e.g. mass spectrometry, NMR, or devices for immunological detection. Maughan et al. (1982) discloses a method for enzymatically determining glucose, lactate, pyruvate, alanine, 3-hydroxybutyrate, and acetoacetate on a 20 pL blood sample.
WO 96/39534 A1 discloses oxidoreductases such as lactate dehydrogenase conjugated to a TAG for generating a chemiluminescent signal for use in biosensors and kits.
CN 101825625 A discloses a kit for simultaneously determining urinary lactic acid, creatinine, and beta-hydroxybutyric acid in urine.
WO 2022/125537 A2 discloses biosensors based on oxidoreductases such as lactate oxidase, lactate dehydrogenase, or 3-hydroxybutyrate dehydrogenase for the determination of lactate and 3-hydroxybutyrate in a sample.
So far, fast and accurate measurement of hydroxy acids different than L-lactate has not been reported if L-lactate is disturbing the detection of the respective non-lactate hydroxy acid. For example, fast and accurate measurement of 2-hydroxybutyrate in the presence of L-lactate has not been reported so far. So far, the determination of hydroxy acids such as 2-hydroxybutyrate (2-HBA) is performed by GC/LC-MS.
However, methods such as GC/LC-MS are highly sophisticated and time consuming; hence, fast and accurate measurement of 2-HBA is highly desired for scaled diagnostic purpose.
Thus, there is an urgent need in the art for methods and means enabling non- lactate hydroxy acid detection and/or quantification based on specific, reliable, fast, and simple methods.
SUMMARY OF THE INVENTION
It is the objective of the present invention to provide methods and means for enabling non-lactate hydroxy acid detection and/or quantification based on specific, reliable, fast, and simple methods.
The objective is solved by the subject matter of the present invention.
The inventors of the present invention surprisingly found that by combination of specific and subsequent reaction steps, a specific determination of non-lactate hydroxy acids such as 2-HBA is enabled. Thereby, a method is provided herein for determining a non-lactate hydroxy acid in complex samples even in the presence of molecules disturbing determination of such a hydroxy acid, i.e., in the presence of lactate. Specifically, the invention described herein enables a fast and accurate determination of 2-HBA in a sample comprising lactate. Such a method is highly desired for scaled diagnostic purposes. Using the methods described herein, an analyte can be measured in human samples and thus, the methods described herein can be utilized in in-vitro diagnostic (IVD) assay kits and enable determination of abnormal concentration levels of the analyte, which in return allows assessing the risk of various forms of metabolic disorders, such as diabetes.
According to the invention there is provided a method for determining a non- lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
Specifically, the non-lactate hydroxy acid is a non-lactate 2-hydroxy acid.
Specifically, the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C1-6 alkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
Specifically, R1 denotes -CH3.
Specifically, R2 denotes phenyl.
Specifically, the non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
Specifically, the enzyme in ii. is an enzyme having non-lactate hydroxy acid dehydrogenase activity, or an enzyme having non-lactate hydroxy acid oxidase activity. More specifically, the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
Specifically, the enzyme in ii. is FCb2 or LDH.
Specifically, in i. an enzyme is used.
Specifically, in i. an enzyme having lactate oxidizing activity is used, specifically an enzyme having lactate oxidase activity.
Specifically, the method described herein comprises further adding an agent removing hydrogen peroxide in i., preferably an enzyme having catalase activity is added.
Specifically, determining the non-lactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
Specifically, 2-hydroxybutyric acid is determined in a sample comprising 2- hydroxybutyric acid and lactate, said 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-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
Specifically, the sample is a human sample.
Specifically, an electrode comprising the enzyme having non-lactate hydroxy acid oxidizing activity is used in ii..
According to the invention there is further provided the use of an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non-lactate hydroxy acid in a sample in a method described herein.
Specifically, the sample is a human sample.
According to the invention there is further provided a use of an electrode comprising an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non- lactate hydroxy acid in a sample in a method described herein.
Specifically, the sample is a human sample.
According to the invention there is further provided a kit for determining a non- lactate hydroxy acid in a sample comprising non-lactate hydroxy acid and lactate, said kit comprising an enzyme having lactate oxidizing activity and an enzyme having non- lactate hydroxy acid oxidizing activity.
Specifically, the non-lactate hydroxy acid is a 2-hydroxy acid.
Specifically, the non-lactate hydroxy acid is 2-hydroxybutyric acid.
Specifically, the enzyme having lactate oxidizing activity is lactate oxidase.
Specifically, the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity.
Specifically, the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
Specifically, the enzyme having non-lactate hydroxy acid oxidizing activity is FCb2 or LDH.
Specifically, the enzyme having non-lactate hydroxy acid oxidizing activity is part of an electrode.
According to the invention there is further provided an electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
Specifically, the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or Ci -ealkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
Specifically, R1 denotes -CH3.
Specifically, R2 denotes phenyl.
Specifically, the non-lactate 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-hydroxy-isobutyric acid.
Specifically, the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity. Specifically, the enzyme having non-lactate 2-hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
Specifically, the enzyme is FCb2 or LDH.
FIGURES
Figure 1 : Example of an enzymatic assay principle for the determination of 2- HBA in two steps.
Figure 2: Technical triplicate reactions for L-lactate removal and 2-HBA measurement in presence and absence of L-lactate interferant and LOx catalyst.
DETAILED DESCRIPTION
Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, 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.
The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wildtype) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods, and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells and modified proteins and enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.
The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.
The term “about” as used herein refers to the same value or a value differing by +/-5 % of the given value.
As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise. As used herein, amino acids refer to twenty naturally occurring amino acids encoded by sixty-one triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:
The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: Alanine (Ala, A; nonpolar, neutral), Asparagine (Asn, N; polar, neutral), Cysteine (Cys, C; nonpolar, neutral), Glutamine (Gin, Q; polar, neutral), Glycine (Gly, G; nonpolar, neutral), Isoleucine (He, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, 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 (Vai, V; nonpolar, neutral), and Histidine (His, H; polar, positive (10%) neutral (90%)).
The “positively” charged amino acids are: Arginine (Arg, R; polar, positive), and Lysine (Lys, K; polar, positive).
The “negatively” charged amino acids are: Aspartic acid (Asp, D; polar, negative), and Glutamic acid (Glu, E; polar, negative).
The term “determining” as used herein refers to detecting and/or quantifying nonlactate hydroxy acid. The term “detecting” the non-lactate hydroxy acid refers to the general determination if non-lactate hydroxy acid is present. Detection does not require the exact quantification of non-lactate hydroxy acid but rather provides the user of the method with the information if e.g., non-lactate hydroxy acid is present with a concentration above a certain threshold. These thresholds are to be adapted to the respective application and sample. The term “quantifying” refers to the determination of the concentration or amount of non-lactate hydroxy acid. Quantification may refer to the determination of an exact amount of an analyte or may alternatively refer to a semi- quantitative determination of an analyte e.g., if the amount of the analyte in a sample is in a certain range. Such a range may be a concentration range suitable for the respective purpose of the determination of the analyte.
According to one embodiment, the method described herein may be used for the quantification or detection of non-lactate hydroxy acid. Depending on the specific type of application of the method, the method may be used for determining the amount of non-lactate hydroxy acid or for determining if non-lactate hydroxy acid is present in a certain concentration range. Further, the method described herein may be used for determining if non-lactate hydroxy acid is present above or below a certain threshold. The term non-lactate hydroxy acid as used herein refers to a hydroxy acid different from lactate or to a salt of a hydroxy acid different from lactate.
The term “lactate” refers to lactic acid or the salt thereof. Specifically, in the context of selectively removing lactate from the sample, and in the context of a non- lactate hydroxy acid, the term “lactate” refers to L-lactic acid or the salt thereof, i.e. , L- lactate.
According to one embodiment of the invention, the non-lactate 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-hydroxy- isobutyric acid, D-lactic acid, or the respective salt of any one of the foregoing.
According to one embodiment, the non-lactate hydroxy acid is a non-lactate 2- hydroxy acid.
The term “non-lactate 2-hydroxy acid” as used herein refers to a subgroup of non-lactate hydroxy acids. Thereby, the term “non-lactate 2-hydroxy acid” as used herein refers to 2-hydroxy acid different from lactate, or to a salt of a 2-hydroxy acid different from lactate. 2-hydroxy acids are commonly known also as alpha hydroxy acids or a-hydroxy acids. 2-hydroxy acids are a class of chemical compounds that consist of a carboxylic acid with a hydroxyl group substituent on the adjacent (alpha) carbon.
According to one embodiment, the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C1-6 alkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
The term “alkyl”, when used alone or in combination with other groups or atoms, refers to a saturated straight or branched chain consisting solely of 1 to 6 hydrogensubstituted carbon atoms, and includes methyl, ethyl, propyl, isopropyl, n-butyl, 1- methylpropyl, isobutyl, t-butyl, 2,2-dimethylbutyl, 2,2-dimethyl-propyl, n-pentyl, 2- methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like. The term “aryl” refers to an aromatic mono- or bicyclic group containing from 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, that may be optionally fused with a fully or partially saturated or unsaturated carbocyclic ring and may optionally be substituted with one or more identical or different substituents, suitably one to three substituents. Examples of aryl groups include phenyl, naphthyl, indanyl, and the like.
The substituent “-C(O)OH” refers to a carboxylic acid substituent.
According to one embodiment, Ri denotes H or C1-6 alkyl.
According to a specific embodiment, Ri denotes H or -CHs.
According to a specific embodiment, Ci-ealkyl is Ci alkyl.
According to one embodiment, R2 denotes H, Ce-8 aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
According to one embodiment, R2 denotes H.
According to one embodiment, R2 denotes Ce-8 aryl.
According to a specific embodiment, Ce-8 aryl is Ce aryl.
According to a specific embodiment, R2 denotes phenyl.
According to a specific embodiment, R2 denotes H, Ce-8 aryl, or C1-14 alkyl optionally substituted by -C(O)OH.
According to a specific embodiment, R2 denotes H, phenyl, or C1-14 alkyl optionally substituted by -C(O)OH.
According to one embodiment, R2 denotes C1-20 alkyl optionally substituted by - C(O)OH.
According to a specific embodiment, R2 denotes -CH2-CH3.
According to one embodiment, R2 denotes H, phenyl, or C1-20 alkyl optionally substituted by -C(O)OH.
According to one embodiment of the invention, the non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
According to a specific embodiment of the invention, the non-lactate hydroxy acid is a-hydroxybutyric acid (2-hydroxybutyric acid, 2-HBA) or its respective salt i.e., a- hydroxybutyrate. Synonyms for the abbreviation 2-HBA are: HBA, a-HBA, aHB.
According to another specific embodiment, 2-HBA is a chiral molecule having the two enantiomers (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid. According to one embodiment, the sample may be any material for which determining the presence of non-lactate hydroxy acid is relevant or of interest. In particular, the sample is a human or animal sample, specifically any one of body fluid, interstitial fluid, blood, blood plasma, blood serum, dermal fluid, urine, tears, sweat, saliva, skin, flesh, tissue, eyeballs, cornea, and gastric fluid.
According to a specific embodiment, human blood is used as a sample in the methods and means described herein. Blood contains various different non-lactate hydroxy acids and contains also lactate.
According to one embodiment of the invention, the sample contains or is suspected to contain a non-lactate hydroxy acid and lactate.
According to a specific embodiment, the sample contains or is suspected to contain lactate at a concentration in the range of 1 to 2 mM.
According to a specific embodiment, the sample contains or is suspected to contain at least a 10-fold higher concentration of lactate than 2-HBA.
According to a specific embodiment, the sample contains or is suspected to contain 2-HBA at a concentration in the range of 0.05 to 0.10 mM.
According to the invention, the methods and means described herein enable the determination of a non-lactate hydroxy acid in a sample suspected to contain also lactate e.g., in a blood sample which contains also lactate, by selectively removing lactate prior to the determination of the non-lactate hydroxy acid.
According to one embodiment, described herein is a method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the sequential steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
According to one embodiment, described herein is a method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample. According to a specific embodiment, described herein is a method for determining 2-hydroxybutyric acid in a sample comprising 2-hydroxybutyric acid and lactate, said method comprising the sequential 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-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
According to a specific embodiment, described herein is a method for determining 2-hydroxybutyric acid in a sample comprising 2-hydroxybutyric acid and lactate, said method comprising the steps of: a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity prior to step b. and c.; b. incubating the sample with an enzyme having 2-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
According to one embodiment, described herein is a method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate, said method comprising the sequential steps of: i. optionally selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
According to one embodiment, described herein is a method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate, said method comprising the steps of: i. optionally selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
According to one embodiment, selectively removing lactate from the sample is optional. The term “selectively removing” as used herein refers to the removal of lactate while not substantially changing the amount or concentration of the non-lactate hydroxy acid in the sample. Specifically, the term “removing” as used herein refers to the modification of lactate e.g., to the oxidation of lactate to pyruvate.
Non-limiting examples of methods for selectively removing lactate are enzyme reaction, precipitation, emulsion liquid membrane separation techniques, adsorption, extraction, polymerization, and esterification.
Non-limiting examples of extracting lactate are extraction with niosomes, microfiltration, and reactive extraction (see Roque L., et al., 2020).
A non-limiting example of polymerization is polymerization using catalysts and heat (see Lunt, James, 1998, and Chafran, Liana S., et al., 2019).
A non-limiting example of esterification is vapor permeation-assisted esterification (see Khunnonkwao, Panwana, et al., 2012).
According to a specific embodiment, modification of lactate is accompanied by the accumulation of modified lactate in the sample. Thereby, the modified lactate is not necessarily removed from the sample, but may remain in the sample also during the determination of the non-lactate hydroxy acid. For example, if oxidation of lactate is accompanied by the accumulation of its reaction product pyruvate, the reaction product pyruvate is not necessarily removed from the reaction mixture and may remain in the sample.
According to an alternative embodiment, the modified lactate may be removed from the sample.
According to a specific embodiment, pyruvate can remain in the sample, e.g., if a lactate oxidase characterized by a low product inhibition is used. Alternatively, the reaction product pyruvate may be removed from the reaction mixture.
The term "enzyme" as used herein refers to any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reaction(s).
The term "activity” as used herein e.g., in the context of an enzyme activity, shall refer to the catalyzed reaction of the enzyme. Thereby, an enzyme having an activity is a functionally active molecule such as a functional enzyme. A functional enzyme is specifically characterized by a catalytic centre recognizing the enzyme substrate and catalysing the conversion of the substrate to a conversion product. Enzyme variants are considered functional or functionally active upon determining their enzymatic activity in a standard test system, e.g., wherein the enzymatic activity is at least 50% of the activity of the parent (not modified or wild-type enzyme), or at least any of 60%, 70%, 80%, 90%, 100%, or even more than 100%.
Enzyme activity is generally given in units. Thereby, one unit of enzymatic activity is defined as the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per min under the respective conditions of the determination method. For example, one unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of substrate such as e.g., lactate or non-lactate hydroxy acid, per min under the respective conditions of the determination method. The specific activity is given in “ll/mg”, “II mg-1” or “II per mg”. Volumetric activity is given in units per volume such as in “U/mL”, “U/ml”, “U per mL”, “U per ml”, “U mL’1”, or “U ml’1”.
As used herein and if an enzyme is used in step i. for selectively removing lactate, such as enzyme is referred to as herein also as enzyme A.
According to one embodiment of the invention, enzyme A is an enzyme having lactate oxidizing activity. In step i, enzyme A does not substantially change the concentration of non-lactate hydroxy acid to be determined in the sample.
According to a specific embodiment, enzyme A is selected from the group consisting of lactate oxidases, lactate monooxygenases, and lactate dehydrogenases.
According to one embodiment of the invention, in step ii. an enzyme having non- lactate hydroxy acid oxidizing activity is used. Thereby, the enzyme having non-lactate hydroxy acid oxidizing activity is herein also referred to as enzyme B.
According to a specific embodiment, enzyme B has non-lactate hydroxy acid oxidizing activity but may also be able to oxidize lactate. Lactate oxidizing activity of enzyme B does not disturb the determination of the non-lactate hydroxy acid as lactate is removed in step i.
According to a specific embodiment, enzyme B is selected from the group consisting of non-lactate hydroxy acid dehydrogenases, non-lactate hydroxy acid oxidases, and non-lactate hydroxy acid monooxygenases. Thereby, enzymes such as LDH, FCb2, LOx, or hydroxy acid oxidase may be used as enzyme B.
In general, the use of e.g., LDH, FCb2, and LOx as enzyme A or as enzyme B depends on the specific substrate specificity of the specific enzyme and the specific non- lactate hydroxy acid to be determined in the sample. According to one embodiment of the invention, enzyme A is an enzyme capable of modifying lactate to such an extent that the enzyme B cannot catalyze a reaction with the modified lactate.
According to a specific embodiment of the invention, enzyme A is an enzyme capable of selectively oxidizing lactate but does not substantially oxidize the non-lactate hydroxy acid.
According to one embodiment of the invention, enzyme A is an enzyme having lactate oxidizing activity.
In general, the term “oxidizing” in the context of an oxidizing agent such as an enzyme having oxidizing activity, refers to an agent that oxidizes a substance and gains or “accepts” an electron from said substance. Thereby, the enzyme has “substance oxidizing activity”. Such a substance may also be referred to as substrate. Therefore, an enzyme having lactate oxidizing activity catalyzes the oxidation of lactate. An enzyme having non-lactate hydroxy acid oxidizing activity catalyzes the oxidation of non-lactate hydroxy acid. Herein, the term “capable of oxidizing” may be alternatively used for the term “oxidizing” in the context of an enzyme having oxidizing activity.
In general, an enzyme having substance oxidizing activity gains or accepts one or more electrons from the substance. Thereby, the enzyme itself or a cofactor of the enzyme, gets reduced. In the reduced state, an enzyme cannot catalyze another oxidation reaction of a substance. Therefore, the enzyme or the cofactor of the enzyme needs to be re-oxidized by transferring the gained electrons to an electron acceptor before another oxidation reaction of a substance can be catalyzed.
According to one embodiment, the enzyme or the cofactor may be re-oxidized by transferring the gained electrons to oxygen, to a molecule with electrochemical activity such as a redox mediator, or to an electrode.
In general, the preference or degree of electron transfer of an enzyme to an electron acceptor differs depending on the specific enzyme and on the specific electron acceptor used.
In general, an enzyme having substance oxidizing activity is known as “oxidase” if the enzyme uses dioxygen as preferred electron acceptor for the reoxidation of the enzyme. If an enzyme having substance oxidizing activity uses an electron acceptor different than dioxygen as preferred electron acceptor for the reoxidation of the enzyme, then such an enzyme is generally known as a “dehydrogenase”. According to a specific embodiment of the invention, enzyme A may be an enzyme having lactate oxidase activity or alternatively an enzyme having lactate dehydrogenase activity.
The term “lactate oxidase activity” refers to the activity of an enzyme catalyzing the oxidation of lactate with dioxygen as electron acceptor forming pyruvate and hydrogen peroxide as products. Thereby, two electrons are transferred from lactate to the cofactor of the enzyme e.g., to FAD, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide.
According to a specific embodiment of the invention, if an enzyme having lactate oxidase activity is used as enzyme A, dioxygen may be used as electron acceptor for the re-oxidation of the enzyme.
According to one embodiment, enzyme A is a lactate oxidase (LOx). Lactate oxidases belong to the enzyme family of E.C 1.1.3.2. Specifically, various lactate oxidases may be used in the method described herein e.g., lactate oxidase from Aerococcus viridans, Nostoc sp. (PCC7120), Lactobacillus jensenii, Lysinibacillus sphaericus, Chlamydomonas reinhardtii, Alicycliphilus denitrificans, Lacticaseibacillus rhamnosus, Lentilactobacillus hilgardii, Roseobacter sp. (strain GAI101), Streptococcus iniae, and Pediococcus acidilactici. Specifically, the lactate oxidase may be a functional variant of any one of the foregoing lactate oxidases having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the respective amino acid sequence of the foregoing lactate oxidases.
According to another embodiment, enzyme A may be a lactate monooxygenase. Therefore, various lactate monooxygenases may be used in the method described herein e.g., lactate 2-monooxygenase from Mycolicibacterium smegmatis.
According to a specific embodiment, the lactate oxidase is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L-lactate.
According to a specific embodiment of the invention, enzyme A may be an engineered variant of an enzyme. For example, an engineered variant may be an oxidase engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.
According to an alternative embodiment of the invention, enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate. For example, an oxidase naturally oxidizing a different molecule than lactate may have been engineered towards using lactate as substrate. In a specific embodiment, the LOx described herein is a functionally active variant of a LOx peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LOx sequence, compared to the respective 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, specifically resulting in one or more amino acid substitutions, additions, or deletions, or the like. Specifically, the functional variant of the LOx peptide sequence is a full-length LOx peptide sequence comprising point mutations, or it is a fragment of the full-length LOx peptide sequence with retained enzymatic activity. Specifically, depending on the use of the LOx as described elsewhere herein as enzyme A or as enzyme B, a variant of a LOx sequence is functionally active if it is capable of converting L-lactate to pyruvate or capable of converting a non-lactate hydroxy acid to the respective oxidized non-lactate hydroxy acid. Specifically, a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence with lactate or non-lactate hydroxy acid. Specifically, a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence, wherein said enzymatic activity is determined with the following assays.
The lactate oxidase activity of an enzyme can be determined for example by the Amplex Red assay. Thereby, oxidase activity is measured using a peroxidase-coupled reaction containing 7.1 U/mL horseradish peroxidase (181 U/mg; Sigma) and 0.05 mM AmplexRed (resorufin: £560 nm = 54.0 mM-1 cm-1). Oxygen is present at ambient concentrations of ~250 pM (Kadowaki, M. A. S. et al. (2020)). The oxidase activity of an enzyme with non-lactate hydroxy acid as substrate can be determined using the method for determining lactate oxidase activity and using the non-lactate hydroxy acid instead of lactate.
The term “lactate dehydrogenase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of lactate with an electron acceptor different from dioxygen forming an oxidized lactate molecule e.g., pyruvate, and the respective reduced electron acceptor as products. Such a reaction is e.g., performed by a lactate dehydrogenase (LDH) or by a flavocytochrome b2 (FCb2).
According to a specific embodiment of the invention, if an enzyme having lactate dehydrogenase activity is used as enzyme A, a molecule different from dioxygen is used as electron acceptor for the re-oxidation of the enzyme. In even more specific embodiments, a system for regeneration of the used electron acceptor may be implemented in the method described herein.
According to a specific embodiment, a system for regeneration of an electron acceptor may comprise an enzymatic, chemical, electrochemical, homogeneous catalytic, photocatalytic, or heterogeneous catalytic regeneration system.
The term “lactate dehydrogenase” is abbreviated herein as LDH. In general, a LDH is an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN or FAD, and the so gained electrons are subsequently transported towards a suitable electron acceptor like DCIP. Thereby, the LDH is commonly irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
According to one embodiment, a LDH is used in the method described herein as enzyme A. Specifically, various LDHs may be used in the method described herein such as but not limited to NAD+ dependent LDH e.g., from Sus scrofa, Homo sapiens, Mus musculus, Rattus norvegicus, Lactobacillus easel, Geobacillus stearothermophilus, Lactiplantibacillus pentosus, Deinococcus radiodurans, Thermus caldophilus, Thermotoga maritima, Bacillus subtilis, and Thermus thermophilus; or FAD- or FMN- dependent LDH e.g., from Pediococcus acidilactici. Specifically, the LDH may be a functional variant of any one of the foregoing LDHs having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the respective amino acid sequence of the foregoing LDHs.
According to a specific embodiment of the invention, enzyme A may be an engineered variant of an enzyme. For example, an engineered variant of a dehydrogenase e.g., a lactate dehydrogenase, engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.
According to an alternative embodiment of the invention, enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate. For example, a dehydrogenase naturally oxidizing a different molecule than lactate may be engineered towards using lactate as substrate and thus, an engineered variant may also be an enzyme having lactate dehydrogenase activity.
According to a specific embodiment, the LDH used as enzyme A is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L- lactate. In a specific embodiment, the LDH described herein is a functionally active variant of a LDH peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LDH sequence, compared to the respective 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 resulting in one or more amino acid substitutions, additions or deletions, or the like. Specifically, the functional variant of the LDH peptide sequence is a full-length LDH peptide sequence comprising point mutations, or it is a fragment of the full-length LDH peptide sequence with retained enzymatic activity. Specifically, depending on the use of the LDH as described elsewhere herein as enzyme A or as enzyme B, a variant of a LDH sequence is functionally active, if it is capable of converting L-lactate to pyruvate or capable of converting a non-lactate hydroxy acid to the respective oxidized non-lactate hydroxy acid. Specifically, a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence with lactate or non-lactate hydroxy acid. Specifically, a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence, wherein said enzymatic activity is determined with the following assays.
The lactate dehydrogenase activity of an enzyme or the enzymatic activity of a LDH or a variant thereof can be determined by a DCIP assay assessing the enzymatic activity from the colorimetric reduction of 2,6-dichlorophenol-indophenol sodium salt hydrate (DCIP) at 30 °C and 520 nm or alternatively at 600 nm (molar extinction coefficient £520nm= 6.8 mM-1 cm-1 ; molar extinction coefficient £6oonm= 8.98 mM-1 cm-1), e.g. as previously described (W.J. Bao, S.N. et al. (1993), Krondorfer, L, et al. (2014), Harreither, W. et al. (2011)). The assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCI, 3 mM KCI and contains 10 mM lactate and 120 pM DCIP, which acts as an electron acceptor. One unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of lactate per min under the assay conditions. The reaction stoichiometry of lactate: DCIP is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single DCIP molecule. For the detection of activity with other substrates, lactate can be exchanged for other compounds. For the detection of the non-lactate hydroxy acid dehydrogenase activity, the lactate is exchanged for this specific non-lactate hydroxy acid. For example, if the 2-HBA dehydrogenase activity of an enzyme is measured, the lactate is exchanged for 2-HBA. The enzymatic activity of LDH variants can also be determined by assessing the colorimetric reduction of 500 pM 1 ,4-benzoquinone (1-4-BQ) (molar extinction coefficient £290 nm = 2.24 mM"1 cm-1) or 160 pM ferrocenium hexafluorophosphate (FcPFe) (molar extinction coefficient £300 nm = 4.3 mM-1 cm-1). The assay mixture is formulated as is described for the DCIP assay but contains 500 pM 1 ,4-benzoquinone or 160 pM ferrocenium hexafluorophosphate instead of DCIP. The reaction stoichiometry of lactate: 1 ,4-benzoquinone is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single molecule of 1 ,4-benzoquinone. The reaction stoichiometry of lactate : ferrocenium hexafluorophosphate is 1 : 2, since two electrons are gained per lactate molecule and transferred individually to two molecules ferrocenium hexafluorophosphate. For the detection of activity with other substrates, lactate can be exchanged for other compounds (Brugger, D, et al. (2014), Sygmund, C. et al. (2011)).
The determination of the enzymatic activity of a LDH or a variant thereof can be also determined for oxygen as electron acceptor. Thereby, specifically the oxidase activity of a LDH is measured. The oxidase activity might not be detectable if the capability of the LDH to transfer electrons to oxygen is very low. As an example of a suitable method, the Amplex Red assay can be used. Thereby, oxidase activity is measured using a peroxidase-coupled reaction containing 7.1 U/mL horseradish peroxidase (181 U/mg; Sigma) and 0.05 mM AmplexRed (resorufin: £560 nm = 54.0 mM-1 cm-1). Oxygen is present at ambient concentrations of ~250 pM (Kadowaki, M. A. S. et al. (2020)).
According to a specific embodiment, the enzyme having lactate oxidizing activity such as e.g., lactate oxidase activity or lactate dehydrogenase activity, used as enzyme A in the method described herein does not substantially alter the concentration or amount of the non-lactate hydroxy acid. For example, the enzyme having lactate oxidase or dehydrogenase activity used as enzyme A does not have or does substantially not have 2-HBA oxidase or 2-HBA dehydrogenase activity.
According to a specific embodiment, enzyme A has a specific activity with the non-lactate hydroxy acid of less than 10, 5, 4, 3, 2, 1 % relative to the specific activity of said enzyme with lactate. Specifically, enzyme A has a specific activity with the non- lactate hydroxy acid of 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 % relative to the specific activity of said enzyme with lactate. According to a specific embodiment, the sample is treated 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 even longer with the enzyme A.
According to a specific embodiment, the sample is treated at room temperature with the enzyme A.
According to a specific embodiment, the sample is treated at 10, 15, 20, 25, 30, 35, or 40 °C with the enzyme A.
According to a specific embodiment, the enzyme A is added to a final concentration in the sample of at least 0.01 mg/mL. Specifically, the enzyme A is added to a final concentration in the sample of 0.01 to 0.1 mg/mL or even higher.
According to a specific embodiment, the duration of selectively removing lactate (pretreatment step, step i.) depends on the specific activity of the enzyme with lactate and on the final concentration of the enzyme in the sample. Thus, the duration of the pretreatment step may be adapted depending on the specific enzyme and on the specific concentration of enzyme in the sample.
According to a specific embodiment, the enzyme A and optionally the catalase may be inactivated after selectively removing lactate and prior to incubation with the enzyme B. For example, the sample may be heated up to 100 °C for 5, 10, 15, or 20 minutes for inactivating the enzyme(s).
According to a specific embodiment, the determination of the non-lactate hydroxy acid as described herein may be corrected by a pre-determined factor. Such a predetermined factor may be derived from the side-reactivity of enzyme A with the specific non-lactate hydroxy acid.
According to a specific embodiment, enzyme A does not substantially change the concentration of non-lactate hydroxy acid in the sample. Thereby, the degree of this substantially change of the concentration of non-lactate hydroxy acid may be predetermined and a correction factor for the determination of non-lactate hydroxy acid may be determined.
According to one embodiment of the invention, hydrogen peroxide may be produced as a by-product in the step of selectively removing lactate from the sample. Therefore, an agent capable of removing hydrogen peroxide may be added in the method described herein. Such an agent may be added in step i. of the method described herein i.e., in the step of selectively removing lactate from the sample. Alternatively, such an agent may be added after step i. but before step ii., or simultaneously to step ii.
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 catalyzing the reaction of decomposing hydrogen peroxide to water and molecular oxygen. Non-limiting examples of such enzymes are catalases, e.g., catalase from Aspergillus niger, bovine liver, human erythrocytes, and any other known catalase.
The catalase activity of an enzyme or the enzymatic activity of a catalase or a variant thereof can be determined spectrophotometrically by continuous spectrophotometric rate reduction determination at 240 nm. Thereby, the rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm. One unit of catalase will decompose 1 .0 pmole of H2O2 per minute at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase is described by Beers, R. F. Jr, & Sizer, I. W. (1952).
According to a specific embodiment, catalase is added to the sample in step i.. Thereby, catalase decomposes the hydrogen peroxide produced by the lactate oxidase to water and molecular oxygen. The produced molecular oxygen may again serve as electron acceptor for the lactate oxidase.
According to one embodiment, catalase is used to boost treatment and/or detection reactions with lactate oxidase.
According to one embodiment of the invention, enzyme B is an enzyme having non-lactate hydroxy acid oxidizing activity. Enzyme B may be an enzyme having nonlactate hydroxy acid dehydrogenase activity or an enzyme having non-lactate hydroxy acid oxidase activity.
The term “non-lactate hydroxy acid oxidizing activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid. Alternatively, the term “hydroxy acid oxidizing activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
The term “non-lactate hydroxy acid dehydrogenase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid with an electron acceptor different from dioxygen and thereby forming an oxidized hydroxy acid and the respective reduced electron acceptor as products. Such enzymes having dehydrogenase activity are commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor. Alternatively, the term “hydroxy acid dehydrogenase activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
In general, the enzyme having hydroxy acid dehydrogenase activity or hydroxy acid oxidase activity may also have the respective lactate dehydrogenase or lactate oxidase activity. Since the lactate present in the sample is selectively removed from the sample in step i., there is no lactate present anymore in step ii. and thus, the enzymatic reaction of step ii. is not disturbed by lactate irrespective if the used enzyme has the theoretical capability of oxidizing lactate.
According to one embodiment of the invention, the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-HBA dehydrogenase activity. For example, flavocytochrome b2 (FCb2) and lactate dehydrogenase (LDH) are enzyme having 2-HBA dehydrogenase activity.
According to one embodiment, the enzyme having non-lactate hydroxy acid dehydrogenase activity is flavocytochrome b2 (FCb2) or lactate dehydrogenase (LDH).
According to another embodiment of the invention, such an enzyme having hydroxy acid dehydrogenase activity may also be a functionally active variant of e.g., FCb2 or LDH.
The term “lactate dehydrogenase” (LDH) is described elsewhere herein as an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor. However, in the herein described invention, if the term “LDH” or “lactate dehydrogenase” is used in the context of enzyme B, it is referred to an enzyme catalyzing the oxidation of the non-lactate hydroxy acid e.g., 2-HBA, to its corresponding oxidized form whereby two electrons are transferred from the non-lactate hydroxy acid to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.
According to a specific embodiment, several different lactate dehydrogenases are known and described elsewhere herein. Thereby, the lactate dehydrogenase may 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 lactate oxidase (see Ashok, Y., et al., 2020).
According to one embodiment of the invention, a LDH is used in the method described herein as enzyme B.
According to a specific embodiment, a LDH is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.
According to an alternative embodiment of the invention, a FCb2 is used in the method described herein as enzyme B.
According to a specific embodiment, a FCb2 is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.
The term “FCb2” refers to a L-lactate-cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b2, FCb2, L-lactate cytochrome c oxidoreductase). In general, FCb2 catalyzes the 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 herein referred to as “Flavocytochrome b2” or "FCb2". Native yeast flavocytochrome b2 (FCb2) has two functional domains that are connected via a “hinge” linker (57 kDa monomer). The FCb2 from S. cerevisiae is the best studied representative and has been crystallized (PDB 1 FCB). However, in the herein described invention, if the term “FCb2” or a synonym thereof is used in the context of an enzyme having the capability of oxidizing the non-lactate hydroxy acid to be determined with the method described herein, these terms refer to an enzyme catalyzing the oxidation of the non- lactate hydroxy acid e.g., 2-HBA, to its corresponding oxidized form whereby the FCb2 catalyzes the electron transfer from non-lactate hydroxy acid to cytochrome c.
According to a specific embodiment, the FCb2 described herein may comprise a sequence based on the mature form of FCb2 naturally found in the yeast mitochondrial intermembrane space, which comprises a cytochrome b2 domain, a flavin domain, a hinge region connecting the cytochrome b2 domain and the flavin domain and a tail region at its C-terminus. A mature FCb2 peptide sequence is the sequence of an FCb2 peptide as it is naturally found in the yeast mitochondrion, specifically in the mitochondrial intermembrane space.
According to a specific embodiment, the FCb2 described herein comprises a FCb2 peptide sequence comprising at least a yeast heme domain and a yeast flavin domain. In a specific embodiment, the FCb2 described herein is a functionally active variant of a FCb2 peptide sequence found in the yeast mitochondrial intermembrane space and comprises one or more point mutations in the nucleotide sequence encoding the FCb2 sequence, compared to the respective 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, specifically resulting in one or more amino acid substitutions, additions or deletions, or the like. Specifically, the functional variant of the FCb2 peptide sequence is a full-length mature FCb2 peptide sequence comprising point mutations, or it is a fragment of the full-length mature FCb2 peptide sequence with retained enzymatic activity. Specifically, a variant of a FCb2 sequence is functionally active if it is capable of converting the non-lactate hydroxy acid to be determined with the method to the corresponding oxidized form. Specifically, a functionally active variant of a FCb2 sequence has at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence with the non-lactate hydroxy acid as substrate. Specifically, a functionally active variant of a FCb2 sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence, wherein said enzymatic activity is determined with the CytC assay and the respective non-lactate hydroxy acid, e.g., 2-HBA as substrate.
The enzymatic activity of a flavocytochrome b2 variant can be readily determined by assays known in the art, such as assays determining the colorimetric reduction of cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP). Specifically, the FCb2 described herein is determined by the CytC assay described by Diep Le et al. (2009) using the respective non-lactate hydroxy acid or alternatively lactate as substrate.
According to a specific embodiment, the FCb2 may comprise the amino acid sequence of a FCb2 from S. cerevisiae, W. anomalus, K. marxianus, O. parapolymorpha, Candida glabrata, Kluyveromyces lactis, Lachancea thermotolerans, Saccharomycodes ludwigii, Naumovozyma castelli, Zygosaccharomyces bailii, Zygosaccharomyces parabalii, Lachancea mirantina, Tetrapisispora phaffii, 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, Cyberlindnera fabianii, Cyberlindnera jadinii, Kazachstania africana, Lachancea quebecensis, Kuraishia capsulata, Torulaspora delbrueckii, Komogatella pastoris, Komagatella phaffii, Lachancea nothofagi, or Naumovomyces dairenensis. Specifically, the FCb2 may be a functional variant of any one of the foregoing and comprise an amino acid sequence having 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% with the amino acid sequence of an FCb2 of any one of the foregoing.
Specifically, the recombinant FCb2 described herein comprises a peptide sequence derived from the FCb2 of Saccharomyces cerevisiae, Kluyveromyces marxianus, Wickerhamomyces anomalus, Naumovozyma castelli or Cyberlindera fabianii.
Amino acid sequences of polypeptides derived from organisms may be readily derived from publicly available databases such as e.g., from databases provided by the National Center for Biotechnology Information (NCBI).
According to another alternative embodiment, enzyme B may also be an enzyme having non-lactate hydroxy acid oxidase activity.
The term “non-lactate hydroxy acid oxidase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a non-lactate hydroxy acid with dioxygen as electron acceptor forming the respective oxidized non-lactate hydroxy acid and hydrogen peroxide as products. Thereby, two electrons are transferred from the non-lactate hydroxy acid to the cofactor of the enzyme e.g., to a FAD cofactor, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide. Alternatively, the term “hydroxy acid oxidase activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.
According to a specific embodiment of the invention, an enzyme having non- lactate hydroxy acid oxidase activity may be an enzyme selected from the enzyme class EC: 1.1.3.15.
According to a specific embodiment, an enzyme having non-lactate hydroxy acid oxidase activity may be hydroxy acid oxidase from Mus musculus, Rattus norvegicus, Homo sapiens, Arabidopsis thaliana, or any other known enzyme having hydroxy acid oxidase activity.
According to a specific embodiment, the enzymes used in the method described herein may be active at acidic, neutral, or alkaline pH ranges. Specifically, the enzymes of the invention may 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 at any pH of a body fluid e.g., of sweat or blood. Usually, blood has a pH between 7.35 and 7.45. According to a specific embodiment, in the method described herein a lactate dehydrogenase-based detection system (flavocytochrome b2, Fcb2) is applied in combination with a lactate oxidase (LOx) dependent lactate removal. Specifically, the enzymes are characterized either by high turnover rates with the analyte of interest (2- HBA) or high specificity to the otherwise interfering substances such as lactate.
According to one embodiment, the combination of an oxygen-dependent enzyme and an oxygen-independent enzyme for unaffected detection or unaffected colorimetric reaction is described herein. For example, the colorimetric reaction with FCb2 is unaffected by lactate removal with LOx.
According to another embodiment, the method described herein may comprise the combination of an engineered lactate oxidase variant with specificity for L-lactate and an engineered flavocytochrome b2 variant with improved reactivity towards 2-HBA. Thereby, the lactate oxidase variant is used in a pre-treatment step to specifically oxidize L-lactate. The improved FCb2 variant is used to oxidize 2-HBA and colorize a reagent concomitantly to translate 2-HBA concentration into a quantifiable signal (e.g., absorbance, fluorescence).
The term “incubating” as used herein refers to contacting the sample with an enzyme to allow the enzyme to react with the non-lactate hydroxy acid. Thereby, incubating may be performed directly in the sample e.g., after adding the enzyme. Alternatively, an aliquot of the sample may be taken from the sample and mixed with a suitable liquid e.g., a buffer, prior to adding the enzyme. Depending on the specific determination method, such a suitable liquid may also comprise other compounds such as e.g., a compound necessary for the colorimetric detection or an electron acceptor.
According to one embodiment of the invention, the step of incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity may be sequentially or simultaneously performed with the step of determining the non-lactate hydroxy acid in the sample.
According to one embodiment of the invention, the enzyme having non-lactate hydroxy acid oxidizing activity may be any enzyme capable of oxidizing the non-lactate hydroxy acid and transferring the so gained electrons to a suitable electron acceptor. Such an electron acceptor may be any suitable molecule which can be used for the colorimetric, photometric, fluorimetric, or electrochemical detection of the electron transfer reaction. Thereby, the signal obtained from detecting the electron transfer reaction is used for the determination of the non-lactate hydroxy acid in the sample. According to an alternative embodiment, if an enzyme having hydroxy acid oxidase activity is used, such an electron acceptor may also be dioxygen. In this case, hydrogen peroxide is produced through the electron transfer from non-lactate hydroxy acid to dioxygen. Hydrogen peroxide can also be detected by suitable methods such as e.g., by colorimetric, photometric, fluorimetric, bioelectrochemical, electrochemical methods, possibly using enzyme based assays. Thereby, the presence or amount of non-lactate hydroxy acid can be calculated from the detection of hydrogen peroxide.
According to yet another alternative embodiment of the invention, the electron acceptor used in the oxidation reaction of the non-lactate hydroxy acid may also be a polypeptide. Such a polypeptide may be naturally attached to the enzyme capable of oxidizing the non-lactate hydroxy acid or synthetically attached to said enzyme. For example, the enzyme FCb2 comprises a heme domain which accepts electrons from the cofactor of the catalytically active domain of the FCb2 i.e. , flavin domain, capable of oxidizing the non-lactate hydroxy acid. Furthermore, a polypeptide used as electron acceptor may transfer the so gained electrons to a terminal electron acceptor. Examples of such a terminal electron acceptor are molecules such as cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP) which can be used in colorimetric and photometric detection methods. Another example of such a terminal electron acceptor is an electrode surface. The presence or amount of non-lactate hydroxy acid in the sample can be derived from a signal obtained by the specific detection method.
According to one embodiment of the invention, determining the non-lactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
The term “colorimetrically” as used herein refers to the application of a colorimetric analysis for the determination of the presence or the amount of non-lactate hydroxy acid in the sample. In a colorimetric analysis the concentration of a chemical element or chemical compound in a solution is determined with the aid of a color reagent. The color reagent may be visually detected or detected with suitable equipment e.g., a colorimeter. In enzymatic analysis as described herein, the color reaction is preceded by a reaction catalyzed by an enzyme such as the enzyme capable of oxidizing a non- lactate hydroxy acid. A general example of such a colorimetric determination is the detection of a colored complex formed by a peroxidase from hydrogen peroxide and ABTS. The term “photometrically” as used herein refers to the usage of a photometer or a spectrophotometer for enzymatically determining a substance in a sample, or alternatively also for determining the enzyme activity, by following the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution. A colorimetric detection may be coupled or performed by a photometer.
The term “fluorimetrically” as used herein refers to the detection of fluorescence by determining the difference in the fluorescence spectra of substrate from product to measure the enzyme reaction or determine the substrate or product concentration. Thereby, the substrate and product may be different from the direct substrates and products of enzymes described herein e.g., different from non-lactate hydroxy acid and oxidized non-lactate hydroxy acid. The Amplex red assay which is elsewhere described herein is an example of an assay based on a fluorimetric detection method.
The term “electrochemically” as used herein refers to the usage of an electrochemical biosensor based on the measurement of biological binding eventdependent changes in conductance, resistance, or capacitance of the biosensor surface. In such an electrochemical biosensor, one of the electrodes is immobilized with a biological recognition molecule. The binding of the analyte to the biological recognition molecule triggers a change in the electrical properties due to oxidation and reduction reactions taking place as a result of biological interaction activity, thus providing the sensor signal. Electrochemical biosensors rely mostly on enzyme-catalyzed reactions to produce current/potential difference which is then detected.
According to one embodiment of the invention, the electron acceptor used in the oxidation reaction of the non-lactate hydroxy acid for the determination of the amount of non-lactate hydroxy acid in the sample may also be an electrode equipped with an enzyme capable of oxidizing the non-lactate hydroxy acid. Such an electrode may be part of a biosensor.
According to one embodiment of the invention, detecting the oxidation of non- lactate hydroxy acid by the enzyme capable of oxidizing the non-lactate hydroxy acid may be carried out by a sensor, specifically a bio-electrochemical sensor, configured to detect and/or quantify non-lactate hydroxy acid in a sample via (bio)electrochemical redox reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of the analyte non-lactate hydroxy acid. Electrochemical biosensors can be impedimetric, potentiometric, or amperometric. In an amperometric biosensor, a biochemical signal is transduced into a quantifiable amperometric signal.
As described in Rocchitta G. et al. (2016) amperometric biosensors are commonly divided into three main generations depending on the electron transfer method used for the measurement of the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediators, and cofactors). First-generation biosensors measure the concentration of analytes and/or products of enzymatic reactions that diffuse to the transducer surface and generate an electrical response. They are also called mediatorless amperometric biosensors. Commonly, oxidases are used in first-generation biosensors. Oxidases need molecular oxygen as a second substrate so the oxidase-based biosensors are oxygen dependent. Second-generation biosensors require an electron mediator for the transfer of electrons obtained from enzymatic reactions to the transducer surface and thereby generate an electrical response. In third-generation biosensors, direct electron transfer is enabled between the redox-active biomolecule i.e., the enzyme, and the electrode surface.
According to one embodiment of the invention, for the determination of nonlactate hydroxy acid by electrochemical means, an electrode comprising an enzyme capable of oxidizing the non-lactate hydroxy acid is contacted with the sample. This contact between electrode and sample can be performed by any approach which brings the electrode and the sample in contact in order that the enzyme is allowed to react with the non-lactate hydroxy acid or with the sample suspected to contain non-lactate hydroxy acid.
The term “electrode” refers to any suitable surface for accepting electrons from the enzyme via mediatorless, mediated, or direct electron transfer. Thereby the electrode is of a material capable of accepting electrons. Furthermore, the electrode may be of any material suitable or modified with any material to adsorb or immobilize the enzyme capable of oxidizing the non-lactate hydroxy acid. Non-limiting examples of such a material are platinum, gold, boron doped diamond and carbons such as graphite, pyrolytic graphite and glassy carbon where all of them can additionally be modified with carbon nanotubes (single or multi-walled), carbon fibers, nanoparticles e.g. gold nanoparticles or promoters as e.g., thiols. The electrode may be also of any material to increase the specific surface are of the electrode. According to one embodiment of the invention, the electrode may be used as single electrode or as a stack of electrodes of e.g., 2, 3, 4, 5, or more electrodes.
According to one embodiment of the invention, the electrode provided herein is a working electrode.
According to one embodiment, the electrode comprising the enzyme capable of oxidizing the non-lactate hydroxy acid of the invention enables the detection and/or quantification of non-lactate hydroxy acid based on mediatorless, mediated, or direct electron transfer.
Mediatorless electron transfer typically employs the transfer of electrons from hydrogen peroxide, produced through the enzymatic reaction, to the electrode.
Mediated electron transfer in biosensors typically employs a two-step procedure in which the enzyme takes part in a first redox reaction with the substrate and is in turn re-oxidized by a redox mediator. Finally, the redox mediator is oxidized by the electrode.
Redox mediators are artificial electron transferring agents that can readily participate in the redox reaction with the biological component and thus help in rapid electron transfer to the electrode. A "redox mediator" is an electron-transfer agent for carrying electrons between an analyte, an analyte-reduced or analyte-oxidized enzyme, and an electrode, either directly, or via one or more additional electron-transfer agents. A redox mediator that includes a polymeric backbone may also be referred to as a redox polymer.
Specifically, said redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.
According to one embodiment of the invention, the oxidation of non-lactate hydroxy acid is performed in the presence of a redox mediator. The method of the invention can also be performed in the presence of more than one redox mediator e.g., in the presence of two or more different redox mediators. Thereby, the redox mediator may be present on the electrode, in an enzyme composition comprising the enzyme and the redox mediator, or may be present in the sample.
According to one embodiment, the redox mediator may be any molecule or material able to carry electrons between the enzyme capable of oxidizing the non-lactate hydroxy acid and electrode. Specifically said redox mediator is selected from the group consisting of any one of organic redox mediators, soluble redox mediators, insoluble redox mediators, redox polymers, transition metal complexes, polymeric transition metal complexes, wired redox mediators, sandwich compounds, and derivatives of these redox mediators.
Polymeric transition metal complexes comprise a polymeric backbone, spacers, and transition metal complexes.
Specifically, redox polymers are polymers comprising redox species. Non-limiting examples of such redox species used in redox polymers are osmium (Os), ruthenium (Ru), iron (Fe), cobalt (Co), or any transition metal. Non-limiting examples of polymers used for redox polymers are poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). An example of a redox polymer is Os-containing poly(vinylpyridine).
According to a specific embodiment, in the case the redox mediator comprises osmium the redox mediator may be an osmium transition metal complex with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2'- bipyridine, 1 ,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole, or derivatives thereof. The redox mediator may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. One example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2' -bipyridine, 1 ,10- phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. Some derivatives of 2,2' -bipyridine for complexation with the osmium cation include but are not limited to 4,4'-dimethyl-2,2'- bipyridine and mono-, di-, and polyalkoxy-2, 2'-bipyri dines, including 4,4'-dimethoxy-2,2'- bipyridine. Derivatives of 1 , 10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl- 1 , 10- phenanthroline and mono, di-, and polyalkoxy-1 , 10- phenanthrolines, such as 4,7- dimethoxy- 1 ,10-phenanthroline. Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1 -vinyl imidazole) and poly(4-vinyl pyridine). Suitable copolymer substituents of poly(1 -vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1 -vinyl imidazole). An example of a redox polymer is also derived from poly(l-vinylimidazole) or a copolymer of (1 -vinyl imidazole) bound to a metal ion selected from the group consisting of Os.sup.3+/2+, Ru.sup.3+/2+, and Fe.sup.3+/2+. The term “transition metal” refers to an element whose atom has a partially filled d sub-shell, or which can give rise to cations with an incomplete d sub-shell. Thereby, transition metals are elements in the d-block of the periodic table and also lanthanides and actinides.
Non-limiting examples of transition metal complexes include complexes comprising 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 ferricyanide, ruthenium hexamine, metalloporphyrins such as heme b or heme c. In these complexes, the transition metal is coordinatively bound to one or more ligands, which are typically mono-, di-, tri-, or tetradentate.
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.
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.
A sandwich compound is a chemical compound featuring a metal bound by haptic covalent bonds to two arene ligands. The arenes have the formula CnHn, substituted derivatives (for example Cn(CH3)n) and heterocyclic derivatives (for example BCnHn+i). A special class of sandwich complexes are metallocenes. A metallocene contains a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure, i.e. , the two cyclopentadienyl anions are on parallel planes with equal bond lengths and strengths. Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1 ,1'-dimethyl ferrocene [DMF], and ferrocene monocarboxylic acid.
Organic redox compounds are organic molecules capable to act as a redox mediator. Non-limiting examples of organic redox mediators are organic molecules such as quinones, compounds having a quinoid structure such as benzoquinones or phenanthroline quinones, phenazine such as 1 -methoxyphenazine methosulfate, tetracyanoquinodimethane (TCNQ), N,N,N', N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivative of these molecules. According to one embodiment, a direct electron transfer biosensor may be used for the determination of non-lactate hydroxy acids, wherein the biosensor comprises an electrode comprising an enzyme capable of oxidizing the non-lactate hydroxy acid and said enzyme is able to transfer the electrons gained from the oxidation of the non-lactate hydroxy acid directly to the electrode surface. An example of such an enzyme capable of oxidizing the non-lactate hydroxy acid enabling direct electron transfer is FCb2.
According to one embodiment of the invention, the enzyme capable of oxidizing the non-lactate hydroxy acid is immobilized on the electrode by adsorption, physical entrapment in a polymer, complex formation, preferably via an additional complexing linker, covalent binding, in particular cross-linking, or ionic binding and/or the immobilized enzyme can be cross-linked, in particular by bifunctional agents, to increase stability or activity. Cross-linking agents are e.g., dialdehydes such as glutaraldehyde.
According to one embodiment of the invention, the electrode of the invention is part of a biosensor. Thereby, a specific use of the electrodes of the invention is in the provision of a biosensor, more specifically a first, second, or third-generation non-lactate hydroxy acid biosensor using mediatorless, mediated, or direct electron transfer properties to detect non-lactate hydroxy acid and/or to measure the non-lactate hydroxy acid concentration. The biosensor may be suitable for use at acidic, neutral, or alkaline pH. The biosensor may be suitable for use at room temperature or at body temperature. Specifically, the biosensor may be suitable for the detection and/or quantification at 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.
According to another embodiment, the biosensor may have one or more electrodes comprising the enzyme capable of oxidizing the non-lactate hydroxy acid. In further embodiments, the non-lactate hydroxy acid biosensor includes: a working electrode comprising a conductive material, wherein the enzyme capable of oxidizing the non-lactate hydroxy acid is in proximity to the conductive material. One or more other electrodes may be included such as one or more counter electrodes, one or more reference electrodes and/or one or more counter/reference electrodes.
The particular configuration of the biosensor may depend on the use for which the biosensor is intended and the conditions under which it will operate. In a specific embodiment of the present invention, the biosensor may be a single use biosensor for the detection of non-lactate hydroxy acid. Thereby, the biosensor may be a biosensor strip.
According to another embodiment of the invention, a kit for determining, e.g., detecting and/or quantifying, non-lactate hydroxy acid in a sample is provided comprising an enzyme capable of selectively removing lactate from the sample and an enzyme capable of oxidizing the non-lactate hydroxy acid. These enzymes are described elsewhere herein.
According to one embodiment, a kit is provided for determining 2-HBA in a sample.
According to one embodiment, the kit may further comprise an agent for selectively removing hydrogen peroxide, specifically an enzyme, specifically a catalase.
According to one embodiment, in the kit described herein, the enzyme capable of oxidizing the non-lactate hydroxy acid may be part of an electrode. Specifically, the electrode may be part of a biosensor.
According to one embodiment, the kit described herein further comprises an instruction manual.
According to one embodiment, the kit may also comprise auxiliary substances, like buffers, molecules necessary for detection e.g., electron acceptors, and containers such as a sample holding means, and/or non-lactate hydroxy acid standards. Non- lactate hydroxy acid standards may be used to calibrate the assay. The kit may also comprise a reader for a signal, especially an electrochemical signal such as a potentiostat, a computer readable memory device with software for calibration and/or measurement calculations.
According to one embodiment of the present invention, the enzymes described herein may be recombinantly expressed by methods commonly known in the art. For example, the enzymes described herein may be expressed using standard methods for cloning, transformation, and recombinant production in suitable host organisms e.g., in Escherichia coli or in Pichia pastoris.
The terms "increase in activity", "increased activity", or the like used herein may refer to a detectable increase in activity of an enzyme. The terms "increase in activity", or "increased activity" used herein may mean that a modified enzyme (variant) shows higher activity than a comparable enzyme of the same type, like an enzyme that does not have the particular modification. As another example, the modified enzyme may comprise sequence alterations in the polypeptide or the nucleotide sequence encoding the enzyme. For example, activity of a modified or engineered enzyme may be higher than activity of a non-engineered enzyme of the same type, for example, a wild-type enzyme 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 a protein or enzyme of the same type in a parent cell, for example, a non-engineered cell 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. Increased activity of an enzyme or protein in a cell may be verified by any methods known in the art. Similarly, the term “decrease in activity”, “decreased activity”, or the like used herein may refer to a detectable decrease in activity of an enzyme.
An increase or decrease in activity may also target only a specific substrate of interest. For example, an enzyme used herein may have a decreased activity with nonlactate hydroxy acid as a substrate but a substantially non-altered or even increased activity with lactate.
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 is known in the art, an allelic variant, or also referred to as homologue, is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may comprise a substitution, deletion and/or addition 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 thereof. Specifically, substitutions, deletions and/or additions may be conservative modifications. Specifically, substitutions, deletions and/or additions do not decrease the enzyme’s specific activity. Specifically, a functionally active variant of the enzyme capable of oxidizing non-lactate hydroxy acid as described herein comprises specific enzymatic activity towards a non-lactate hydroxy acid of at least 1 ll/rng, as determined by the respective assay as described herein.
Specifically, a functional variant as described herein comprises no more than or up to 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 substitutions, deletions and/or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity. Specifically, a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5, amino acid substitutions, deletions and/or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity.
Specifically, a functionally active variant described herein comprises at least 40, 50, 60, 70, 80 or 90% or even more of the enzymatic activity of the respective wild type enzyme.
Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence e.g., by one or more point mutations, wherein the sequence alterations retain or improve a feature of the enzyme, such as its stability or activity for example. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.
A point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the nonengineered amino acid sequence in the substitution, or exchange, deletion, or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.
According to a specific embodiment, the enzymes described herein may comprise one or more tag sequences, specifically N-terminal tag sequences. Specifically, such tag sequence is C-terminal of the N-terminal methionine of the enzymes described herein. Such tag sequence may comprise any number of amino acids of more than 2, 4, 5, 6 or 10 amino acids and up to 20 or 50 or more amino acids. Specifically, tag sequences used herein may be any tag sequence known to the person skilled in the art. Specifically, tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.
Affinity tags are amino acid sequences that can be used for example for the purification of proteins where they are attached to. These affinity tags have high affinity to appropriate ligands of a solid support, like chromatography resins or directly to the resins. By selectively binding of the protein having the affinity tag to the particular resin the protein can be purified highly effective by only one chromatography step. According to a specific embodiment, affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, poly-arginine tag, FLAG tag, Strep tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP) tag, S-tag, HA tag, c-Myc tag, and SUMO tag, or any other tag known to be useful for the efficient purification of a protein it is fused to. Preferably, the tag is a His tag comprising one or more H, specifically a hexahistidine tag. Specifically, proteins comprising a poly-, or hexa-histidine tag (His-tag) can be captured and purified using chromatography, e.g. by Immobilized Metal Affinity Chromatography (IMAC).
Solubility enhancement tags can be fused N-terminal to the enzymes described herein. Solubility enhancement tags can increase the titer of the soluble protein when expressed in a host cell, e.g. in the cytosol of P. pastoris, compared to expression of the proteins without the tag. According to a further specific embodiment, solubility enhancement tag sequences used herein are selected from calmodulin-binding peptide (CBP), poly Arg, poly Lys, protein D tag (dTAG), Z domain of Staphylococcal protein A, and thioredoxin or any other tag known to improve the solubility of the protein it is fused to e.g. during expression in a host cell. Specifically, the solubility enhancement tag is a T7 tag, preferably selected from the group consisting of T7A, T7A1 , T7A2, T7A3, T7A4, T7A5, T7B, T7B1 , T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11 , T7B12, T7B13, and T7C.
According to a further specific embodiment, the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin or any other tag useful for detection or quantification of the recombinant enzyme during production steps including fermentation, isolation and purification by simple in-situ, inline, online or atline detectors, like UV, IR, Raman, fluorescence and the like.
The term “sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.
Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.
Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry. Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.
To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.
“Percent (%) identity” with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared. In case of percentages determined for sequence identities, it is possible that arithmetical decimal places may result which are not possible with regard to full nucleotides or amino acids. In this case, the percentages shall be rounded up to whole nucleotides or amino acids.
For purposes described herein, the sequence identity between two amino acid sequences is determined using standard methods, e.g. using the NCBI BLAST program version 2.2.29 (Jan-06-2014) or online using the multiple sequence alignment tool EMBL-EBI Clustal Omega (Sievers, F. et al. (2011)).
"Percent (%) identity" with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared.
Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, MAFFT based algorithms: multiple alignment using fast fourier transform, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).
In a structure alignment the maximal set of corresponding pairs of amino acid residues that gives a good structural match when the structures are overlaid, i.e., superposed, is identified. Thereby, the positions of the protein’s backbone C-alpha atoms and/or location of secondary structural elements are considered in this alignment. Tools for performing a structure alignment are available, e.g., the protein data bank provides a tool for pairwise structure alignment. Specifically, structure superposition is also a tool for determining corresponding amino acid positions in different enzymes. Structure superposition can be performed using the Molecular Graphics System PyMOL, (Schrodinger) using the command “align”.
The invention further provides the following ITEMS:
1. A method for determining a non-lactate 2-hydroxy acid in a sample comprising the non-lactate 2-hydroxy acid and optionally lactate, said method comprising the steps of: i. optionally selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate 2-hydroxy acid oxidizing activity; and iii. determining the non-lactate 2-hydroxy acid in the sample.
2. The method of item 1 , wherein the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C1-6 alkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
3. The method of item 2, wherein R1 denotes -CH3.
4. The method of item 2, wherein R2 denotes phenyl.
5. The method of any one of items 1 to 4, wherein the non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
6. The method of any one of items 1 to 5, wherein the enzyme in ii. is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity.
7. The method of item 6, wherein the enzyme having non-lactate 2-hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
8. The method of any one of items 1 to 7, wherein the enzyme in ii. is FCb2 or LDH.
9. The method of any one of items 1 to 8, wherein in i. an enzyme is used.
10. The method of any one of items 1 to 9, wherein in i. an enzyme having lactate oxidizing activity is used.
11 . The method of item 10, wherein an enzyme having lactate oxidase activity is used.
12. The method of any one of items 1 to 11 , further adding an agent removing hydrogen peroxide in i..
13. The method of item 12, wherein an enzyme having catalase activity is added. 14. The method of any one of items 1 to 13, wherein determining the nonlactate 2-hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
15. The method of any one of items 1 to 14, wherein 2-hydroxybutyric acid is determined in a sample comprising 2-hydroxybutyric acid and optionally lactate, said method comprising the steps of: a. optionally selectively removing lactate from the sample by an enzyme having lactate oxidase activity; b. incubating the sample with an enzyme having 2-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
16. The method of any one of items 1 to 15, wherein the sample is a human sample.
17. The method of any one of items 1 to 16, wherein an electrode comprising the enzyme having non-lactate 2-hydroxy acid oxidizing activity is used in ii..
18. The method of item 17, wherein the electrode is part of a biosensor.
19. Use of an enzyme having non-lactate 2-hydroxy acid oxidizing activity for determining a non-lactate 2-hydroxy acid in a sample in a method of any one of items 1 to 18.
20. The use of item 19, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity.
21. Use of an electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity for determining a non-lactate 2-hydroxy acid in a sample.
22. The use of item 21 , in a method of any one of items 1 to 18.
23. The use of item 21 or 22, wherein the enzyme having non-lactate 2- hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity.
24. A kit for determining a non-lactate 2-hydroxy acid in a sample comprising non-lactate 2-hydroxy acid and lactate, said kit comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
25. The kit of item 24, wherein the non-lactate 2-hydroxy acid is 2- hydroxybutyric acid. 26. The kit of item 24 or 25, wherein the enzyme having lactate oxidizing activity is lactate oxidase.
27. The kit of any one of items 24 to 26, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity.
28. The kit of item 27, wherein the enzyme having non-lactate 2-hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
29. The kit of any one of items 24 to 28, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is FCb2 or LDH.
30. The kit of any one of items 24 to 29, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is part of an electrode.
31. An electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
32. The electrode of item 31 , wherein the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C i-ealkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
33. The electrode of item 32, wherein R1 denotes -CH3.
34. The electrode of item 32, wherein R2 denotes phenyl.
35. The electrode of any one of items 31 to 34, wherein the non-lactate 2- hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2- hydroxy valeric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, and 2-hydroxy-isobutyric acid.
36. The electrode of any one of items 31 to 35, wherein enzyme having non- lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity.
37. The electrode of item 36, wherein the enzyme having non-lactate 2- hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
38. The electrode of any one of items 31 to 37, wherein the enzyme is FCb2 or LDH.
39. The electrode of any one of items 31 to 38, wherein the electrode is part of a biosensor.
40. A method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
41. The method of item 40, wherein the non-lactate hydroxy acid is 2- hydroxybutyric acid, 3-hydroxybutyric acid, 2-hydroxyvaleric acid, mandelic acid, 2- hydroxyoctanoic acid, 2-hydroxypalmitic acid, or glycolic acid.
42. The method of item 40 or 41 , wherein the enzyme in ii. is an enzyme having non-lactate hydroxy acid dehydrogenase activity, or an enzyme having non-lactate hydroxy acid oxidase activity.
43. The method of item 42, wherein the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity, preferably FCb2 or LDH.
44. The method of any one of items 40 to 43, wherein in i. an enzyme having lactate oxidizing activity is used, specifically an enzyme having lactate oxidase activity.
45. The method of any one of items 40 to 44, further adding an agent removing hydrogen peroxide in i., preferably adding an enzyme having catalase activity.
46. The method of any one of items 40 to 45, wherein determining the non- lactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically. 47. The method of any one of items 40 to 46, wherein 2-hydroxybutyric acid is determined in a sample comprising 2-hydroxybutyric acid and lactate, said 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-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
48. Use of an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non-lactate hydroxy acid in a sample in a method of any one of items 40 to 47.
49. Use of an electrode comprising an enzyme having non-lactate hydroxy acid oxidizing activity, specifically an enzyme having non-lactate hydroxy acid dehydrogenase activity, for determining a non-lactate hydroxy acid in a sample in a method of any one of items 40 to 47.
50. A kit for determining a non-lactate hydroxy acid, specifically 2- hydroxybutyric acid, in a sample comprising non-lactate hydroxy acid and lactate, said kit comprising an enzyme having lactate oxidizing activity and an enzyme having non- lactate hydroxy acid oxidizing activity.
51. The kit of item 50, wherein the enzyme having lactate oxidizing activity is lactate oxidase.
52. The kit of item 50 or 51 , wherein the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity, preferably having 2-hydroxybutyric acid dehydrogenase activity.
53. The kit of any one of items 50 to 52, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is FCb2 or LDH.
54. The kit of any one of items 50 to 53, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is part of an electrode.
The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention. EXAMPLES
Example 1 : Enzymatic assay principle for the determination of 2-HBA in two steps
In this example, the enzyme lactate oxidase (LOx) is used as an example of an enzyme for the removal of L-lactate. Flavocytochrome b2 is used as an example of an enzyme for the detection of the non-lactate hydroxy acid. 2-HBA is used as an example for the non-lactate hydroxy acid. A general scheme of the reaction is shown in Fig. 1.
According to the example, using the assay a specific detection of 2-HBA is performed in samples, where interfering L-lactate can be removed using a dedicated enzymatic pre-treatment step with lactate oxidase (LOx). Thereby, as in this example an enzyme is used for removal which uses oxygen as electron acceptor, H2O2 accumulates from O2 reduction simultaneous to the oxidation of L-lactate, and can be partly recovered (half-stoichiometrically) employing an enzymatic conversion with catalase: 2 H2O2 2 H2O + O2. This regeneration using catalase may aid in maintaining a steady oxygen concentration for the LOx reaction. Subsequently to the removal step, the 2-HBA analyte can be detected without interference using an enzyme like FCb2.
In general, in physiological relevant samples L-lactate is present at >10-fold higher concentrations (1-2 mM) than the 2-HBA analyte (0.05-0.10 mM).
This example provides the general proof of principle that the method described herein can be used for the determination of a non-lactate hydroxy acid in samples comprising L-lactate. Furthermore, results of spiking the non-lactate hydroxy acid in physiological samples is shown.
Materials
Enzymes:
Amino acid sequences of enzymes used for removal or detection in the examples provided herein:
AvLQx - amino acid sequence (SEQ ID NO: 1):
MGSSHHHHHHNNNDIEYNAPSEIKYIDWNTYDLEEEASKWPHGGFNYIAGA SGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHT TKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDE AKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGAS KQKISPRDIEEIAGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGS FDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADWALGRPVLFGLALGGWQG AYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY CanqFCb2 - amino acid sequence (SEQ ID NO: 2):
MHHHHHHHHDAKFDSSKPKISPSEVIKHNTPEDCWWIDGYVYDLTNFIALHP
GGPDIIKTNAGKDVTAIFDPIHPPDAIEKYIKPEQHVGPLDGKLDAEYICPPYAPGETPD
DIARKAALRARLPPLSSIMNLYDFEYLASQILSKQAWAYYSSASDDEVSYRENHNAYH
RIFFNPKVLVDVSKVDTSTEMLGHKVDVPFYVTATALCKLGNPKEGEKDIARGCGQG
PNKTPQMISTLASCSVDEIVNAAPSKDQVIWYQLYVNSDRKITENLIKHVEDLGVKAIF
VTVDAPSLGSREKDKKVKFNNTMSGPKSMKKSDVGESEGAAQTLSKFIDPSLSWQDI
KILRKKTKLPIVIKGVQRVQDWKAAEIGCNGWLSNHGGRQLDFARAPIEVLAETMPV
LKEKKLDKNFEVFVDGGVRRGTDVIKALCLGASGVGLGRPFLYANSCYGKDGVQKAI
DLLKTEIEMNMRLLGVTSIKDMNPELLDLSSLHGRTVNVPKDSLYVNVYNKPELAEFL DDASD
Catalase from Corynebacterium glutamicum (CgCat), Sigma #02071 Chemicals:
• Equine Cytochrome C, Sigma #C2506
• Sodium L-Lactate, > 99.0 % (NT), Sigma #71718
• Na 2-Hydroxybutyrate > 97 %, racemic Sigma #220116 (CAS 5094-24-6, 126.09 g/mol)
• Phosphate buffer saline (PBS), pH 7.4, Sigma #P3813
• Human plasma (SIGMA #P9523): lyophilized, 4 % Citrate
• Human Serum H3667-20 mL (Sigma, Source SLCL8404), heat inactivated: frozen
• Multititer plate 96-well (Greiner, Polystyrole, Merck # M2936)
Instrumentation:
Photometer (TECAN Infinite M Nano Plate reader); for measurement at 550 nm
Proof of principle
Methods:
Reagent 1 is used for the pretreatment step. Reagent 1 : Aerococcus viridans
Lactate Oxidase at 0.1 mg/mL and Corynebacterium glutamicum Catalase solution 1 pL/mL in 11 mM PBS, pH 7.4 Reagent 2 is used for the measurement step. Reagent 2: Candida glabrata FCb2 at 50 pg/mL and 80 pM equine Cytochrome C (Sigma-Aldrich, #C2506) in 11 mM PBS, pH 7.4
The determination of 2-HBA concentrations is performed at room temperature (22 °C), including the 5 min pre-treatment reaction and the 3 min measurement using a photometric plate reader.
At time zero of the reaction, 10 pL of the sample (serum, plasma, synthetic controls) are transferred into the well of a 96-well plate. The pre-treatment reaction is started by adding 90 pL of reagent 1 . After 5 minutes, 100 pL of the reagent 2 is added and the photometric measurement is started immediately. The reaction (absorbance change) is followed at 550 nm for 3 min in the photometer. The increase in optical absorbance at 550 nm per minute (AAbs550 min-1) for this measurement setup can be calculated into an enzymatic activity, ll/mL or ll/rng, or an apparent 2-HBA concentration according to the following equations (which only apply for the mentioned technical setup):
Equation 1 - calculation of volumetric activities (Ll/mL) from changes in optical absorbance at 550 nm: volumetric activity [U mL 1] = Abs min 1 * 0.98 * df
Equation 2 - calculation of 2-HBA concentration from changes in optical absorbance at 550 nm (this calculation only applies for the given technical setup):
&Abs min 1 — 0.00101
2 - HBA [jiM] =
0.000121
The following applies for the equations shown:
0.98: enzyme factor uniting dilution, extinction coefficient and pathlength df: sample dilution factor (10pL in 200pL, equals 0.05)
Samples contained different combinations of 5 mM L-lactate, 0.1 mM 2-HBA, 0.1 mg/mL LOx but contained catalase solution at 0.1 % (v/v) in all assays. Pre-treatment reactions were carried out for 5 minutes at 22 °C prior to sample measurement of 2-HBA concentrations, relying on a 3-minute Cytochrome C (CytC) assay with FCb2. The increase in absorbance at 550 nm originating from CytC reduction translated to a substrate concentration dependent enzyme activity (Ll/mL). Results:
The results of the proof of principle are shown in Fig. 2.
(1) The sample containing 2-HBA (analyte) and L-lactate (interferent) without addition of LOx yields high activities (CytC) in the subsequent measurement with CangFCb2 due to the presence of high concentrations of L-lactate.
(2) When LOx is present, the sample containing the 2-HBA analyte and L- lactate interferent yields low activities since L-lactate is oxidized by LOx and hence cannot be accepted by the FCb2 during the colorimetric measurement. The residual activity originates solely from 2-HBA.
(3) In contrast, no detectible signals were yielded from the FCb2 reaction when L-lactate, but no 2-HBA was present.
(4) Similar levels of enzyme activity, respective to (2), were reached when no L-Lactate was present.
These results provide the proof of principle of the method described herein, especially since the following aspects are confirmed:
• Lactate removal: Whether L-lactate is present or not; (2) vs. (4): the residual signals are comparable, indicating that the 2-HBA fraction alone leads to the signals after treatment.
• This is attributed to LOx. The (1) assay where LOx is absent, shows substantially higher signals.
• When 2-HBA is absent in sample mixtures, no signals remain after LOx treatment pointing towards apparent complete removal of L-lactate.
Analytical slope of 2-HBA with the FCb2 assay:
Methods:
• 2-HBA was diluted in PBS to yield the following pM concentrations: 6.25; 12.5; 25; 50; 100; 200; 300; 400
• 10 pL of these samples were measured in technical replicates of 6 with the CytC/Fcb2 assay procedure using CangFCb2 as is described above. The procedure was adapted to using 90 pL of PBS instead of Reagent 1 , but using 100 pL Reagent 2 with CangFCb2
• From the calculated FCb2 activities, data points were used to calculate a linear regression, yielding slope, intercept and goodness of fit (R2) Results:
In trials with pure 2-HBA and the FCb2/CytC assay, an apparently linear analytical range was obtained between 6.25 and 400 pM sample concentration. In this measurement setup, no interferant was present and the LOx catalyzed L-lactate removal step was omitted.
The following Table 1 gives the numerical data of the analytical range measurements:
Table 1 : Numerical data of the analytical range measurements
These numerical data were plotted and the following linear calibration function was determined: y = 1 E-04X + 0.0029
R2 = 0.9994
These results underline a broad linear range and low standard errors for the 2- HBA measurement in the setup with CangFCb2.
2-HBA spiking in plasma
Methods:
• Spiked samples were prepared using the aforementioned human plasma and serum sample alongside synthetic standard solutions containing 5 mM L-lactate and 0.1 mM 2-HBA. • 10 pL of the spiked samples were measurement according to the proof of principle experiments in technical replicates of 6, adding the removal reaction with 90 pL Reagent 1 and subsequent 2-HBA measurement with CangFCb2 in 100 pL Reagent 2
• Utilizing the analytical slope parameters of 2-HBA determination from above, FCb2 activities were averaged and translated to apparent 2-HBA concentrations, which were the opposed to spiked 2-HBA concentrations in the sample
• From the calculated FCb2 activities, data points were used to calculate a linear regression, yielding slope, intercept and goodness of fit (R2) where the slope, opposing apparent and spiked 2-HBA concentrations reflected the “recovery”.
Results:
From a series of 2-HBA standards in buffer, with known 2-HBA concentrations, enzymatic activity measurements were performed and yielded kinetic slopes, i.e. at points of a certain 2-HBA concentration and kinetic slope scaled linearly with increasing concentration. These data points were used to calculate a linear regression, characterized by an intercept (d) and slope (x) value according to y = kx +d which was referred to as analytical calibration.
After spiking commercial human plasma and serum with known amounts of 2- HBA and unknown physiological L-lactate interferant concentrations, the calibration curve parameters (determined in buffer) were used to calculate apparent concentrations of the 2-HBA analyte in serum and plasma. The correlation of spiked and measured 2- HBA concentrations in this experiment are visualized here and represent in-assay concentrations. Sample concentrations were diluted 1 in 20.
The results are shown in the following Tables 2 and 3: Table 2: Numerical data of spiking experiments in plasma
Table 3: Numerical data of spiking experiments in serum • Calibration curves of 2-HBA were recorded in buffer for an extended range of analyte 0-40 pM in assay (1 :20; 0-800 pM in sample). The slope and intercept were used to calculate signals into concentrations, which were opposed to the spiked concentrations.
• Serum and plasma were spiked with 2-HBA concentrations of 0-10 pM (in assay)
• Recovery range for plasma and serum similar (~ 94 %)
• Linear (detectable) range: 0.625 - 10 pM 2-HBA in assay, translating to 12.5 - 200 pM samples Example 2: Electrochemical determination of non-lactate hydroxy acid
In this example, the electrochemical detection of 2-HBA is described. KmFCb2 is used as an exemplary enzyme for the electrochemical detection of 2-HBA.
Materials: Enzyme:
KmFCb2 - amino acid sequence (SEQ ID NO: 3):
MHHHHHHHHATKEELNKPKVSPLEVAKHSSPDDCWWIDGFVYNLTEFISAHP GGPAIIENNAGKDVTAIFGPIHAPDVIEKYIAPENRIGPLDGKMPDDLICAPLTPGETPE DVARKEELRQNMPDLDSLVNIYDFEFLASQILTKQAWSYYSSAADDEVTHRENHAAY HRIFFKPRILVNVKEVDTSTTMLGEKVGVPFYVSATALCKLGNPKEGEKDIARGCGES DVKPIQMISTLASCSLQEIVEAAPSKDQIQWFQLYVNSDRKITEELIKNVEKLGLKAIFV TVDAPSLGNREKDAKVKFTNKDSSAKAMEKSNVKESKGASRALSTFIDPALCWDDIV TLKSKTKLPIVIKGVQCVEDVLKAAEIGAAGWLSNHGGRQLDFSRAPIEVLAETMPILK EKKLDDKIEIFIDGGVRRGTDILKALCLGAKGVGLGRPFLYANSCYGKEGVKKAIELLK DELEMSMRLLGVTSIDQLSEKYLDLSTLHGRTVSVPRDNLYNGVYVPHEPTDFKEN
Chemicals:
• Na 2-Hydroxybutyrate > 97 %, racemic Sigma #220116 (CAS 5094-24-6, 126.09 g/mol)
• Phosphate buffer saline (PBS), pH 7.4, Sigma #P3813
• DropSens Carbon screen printed electrodes DRP-C110
Instrumentation:
• Potentiostat: Palmsens EMstat Blue
Method:
• 2-HBA was diluted in PBS to yield the following mM concentrations: 1.0, 5.0, 10
• Biosensor electrodes were prepared as is described in Geiss et al. (2021) with the adaptation of using 2 pL of 10 mg/mL engineered KmFCb2 in 100 mM Phosphate buffer. Curing was done at 22 °C for 2 h under dry ambience.
• The electrode measurement was carried out in triplicates utilizing a potentiostat in a chronoamperometric measuring mode at an applied potential of 0.2 V vs a pseudo-Ag/AgCI reference electrode at 22°C. Electrodes were mounted horizontally, and samples were added/removed step wise to the sensing area to yield increased substrate concentrations over time. The change of currents was measured over time. • Data evaluation: Baseline subtracted currents were evaluated 10 s after each sample addition and plotted versus the substrate concentration. Non-linear regression fitting to Michaelis-Menten equation were calculated.
Results:
The analyte can be detected using the FCb2 enzyme without the need of a soluble electron acceptor when the FCb2 enzyme is contacted on an electrode. Dependent on the substrate concentration in the assay, the enzyme delivers proportional catalytic currents that can be measured using an electronic device, such as a potentiostat.
The following Table 4 shows the results of the example.
Table 4: Catalytic currents of electrochemical KmFCb2 measurement of 2-HBA
In trials with electrochemically contacted KmFCb2, 2-HBA could be detected in a relevant range. In this measurement setup, no interferant was present and the LOx catalyzed L-lactate removal step was omitted as the general proof of L-lactate removal and its influence on the general 2-HBA determination was shown in example 1.
These results underline that electrochemical measurement presents a viable option for the detection of 2-HBA from samples using FCb2. This might be especially important for biosensor applications.
Example 3: Determination of different non-lactate hydroxy acids
This example shows the determination of different non-lactate hydroxy acids using three different FCb2 enzymes.
The following enzymes were used:
KmFCb2 and CanqFCb2 as described above. WaFCb2 - amino acid sequence (SEQ ID NO: 4):
MHHHHHHHHDVPHWKDIELTPEIVSQHNKKDDLWWLNGQVYDLTDFLPNHP GGQKIIIRYAGKDATKIFVPIHPPDTIEKFIPPEKHLGPLVGEFEQEEEELSDEEIDRLERI ERKPPLSQMINLHDFETIARQILPPPALAYYCSAADDEVTLRENHNAYHRIFFNPKILID VKDVDISTEFFGEKTSAPFYISATALAKLGHPEGEVAIAKGAGREDWQMISTLASCSF DEIADARIPGQQQWYQLYVNADRSITEKAVRHAEERGMKGLFITVDAPSLGRREKDM KMKFEADSDVQGDDEDIDRSQGASRALSSFIDPSLSWKDIAFIKSITKMPIVIKGVQRK EDVLLAAEHGLQGVVLSNHGGRQLDYTRAPVEVLAEVMPILKERGLDQKIDIFVDGGV RRGTDVLKALCLGAKGVGLGRPFLYAMSSYGDKGVTKAIQLLKDEIEMNMRLLGVNKI EELTPELLDTRSIHNRAVPVAKDYLYEQNYQRMSGAEFRPGIED
The FCb2 enzyme activities were determined photometrically following the general principles of the Cytochrome C assay as described herein e.g., in example 1. Therefore, 300 mM stock solutions were prepared in 50 mM PPB, pH 6.5 for each substrate.
The following substrates were used:
• D-lactate (#CAS: 920-49-0)
• Sodium glycolate (#CAS: 2836-32-0)
• (S)-2-hydroxybutyric acid (a-HB, L(S)-enantiomer) (#CAS: 3347-90-8)
• 2-hydroxybutyrate (a-HB, Racemate) (#CAS: 5094-24-6)
• (S)-2-hydroxyvaleric acid (#CAS: 41014-93-1)
• (S)-(+)-Mandelic acid (#CAS: 17199-29-0)
• 2-hydroxy-n-octanoic acid* (#CAS: 617-73-2)
• (C+) Sodium L-lactate (#CAS:867-56-1)
* Sonicate and heat to 40 °C to solubilize
Table 5 gives the relative enzyme activity with respect to L-lactate for each substrate of CangFCb2, KmFCb2, and WaFCb2. Table 5: Overview over relative substrate-dependent activities of FCb2s
Example 4: Determination of different non-lactate 2-hydroxy acids
This example shows the determination of different non-lactate 2-hydroxy acids using the invention described herein. Example 4 was performed as described in Example 3. The following Table 6 shows the relative substrate-dependent activities.
Table 6 REFERENCES
Alesi, S., et al. (2021). Metabolomic biomarkers in gestational diabetes mellitus: A review of the evidence. In International Journal of Molecular Sciences (Vol. 22, Issue 11 , p. 5512). Multidisciplinary Digital Publishing Institute.
Ashok, Y., et al. (2020). FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici. PloS one, 15(2), e0223870. https://doi.org/10.1371/journal.pone.0223870
Bao W.J., et al (1993), Purification and Characterization of Cellobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-Rot Fungus Phanerochaete chrysosporium, Archives of Biochemistry and Biophysics, 300(2) 705- 713.
Beers, R. F., Jr, & Sizer, I. W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of biological chemistry, 195(1), 133-140.
Brugger D, et al. (2014) Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity. PLOS ONE 9(10): e109242.
Chafran, Liana S., et al. "Preparation of PLA blends by polycondensation of D, L- lactic acid using supported 12-tungstophosphoric acid as a heterogeneous catalyst." Heliyon 5.5 (2019)
Cobb, J., et al. (2016). a-Hydroxybutyric acid is a selective metabolite biomarker of impaired glucose tolerance. Diabetes Care, 39(6), 988-995.
Diep Le, K. H., et al. 2009. “Interdomain Contacts in Flavocytochrome B2, a Mutational Analysis.” Biochemistry 48 (45): 10803-9.
Gall, W. E., et al. (2010). a-Hydroxybutyrate Is an Early Biomarker of Insulin Resistance and Glucose Intolerance in a Nondiabetic Population. PLOS ONE, 5(5), e10883.
Geiss, A. F., et al., 2021. Engineering the turnover stability of cellobiose dehydrogenase toward long-term bioelectronic applications. ACS Sustainable Chemistry & Engineering, 9(20), 7086-7100.
Harreither, W. et al. (2011) Catalytic Properties and Classification of Cellobiose Dehydrogenases from Ascomycetes, Applied and Environmental Microbiology, 77(5), 1804-1815. Kadowaki, M. A. S. et al. (2020) 'Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77', Biochimica et Biophysica Acta, 1864(10), 0304-4165.
Krondorfer I,, et al. (2014) Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity. PLOS ONE 9(3): e91145.
Khunnonkwao, Panwana, et al. "Purification of l-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification." Process Biochemistry 47.12 (2012): 1948-1956.
Lu, W., et al. (2021). Discovery of metabolic biomarkers for gestational diabetes mellitus in a Chinese population. Nutrition and Metabolism, 18(1), 1-16.
Lunt, James. "Large-scale production, properties and commercial applications of polylactic acid polymers." Polymer degradation and stability 59.1 -3 (1998): 145-152.
Maughan et al., “A simple, rapid method for the determination of glucose, lactate, pyruvate, alanine, 3-hydroxybutyrate and acetoacetate on a single 20-pl blood sample”, Clinica Chimica Acta, vol. 122(2), 1982, pp. 231-240
Roque L., et al., “Stability and characterization studies of Span 80 niosomes modified with CTAB in the presence of NaCI”, Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 601 , 2020, 124999, ISSN 0927-7757, https://doi.Org/10.1016/j.colsurfa.2020.124999
Roberts et al. (2022) The serum glycolate concentration: its prognostic value and its correlation to surrogate markers in ethylene glycol exposures, Clinical Toxicology, 60:7, 798-807
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Sygmund, C. et al. (2011) 'Reduction of Quinones and Phenoxy Radicals by Extracellular Glucose Dehydrogenase from Glomerella Cingulata Suggests a Role in Plant Pathogenicity' Microbiology, 157(11):3203-12.
Wang, L., et al. (2021). Metabolite Triplet in Serum Improves the Diagnostic Accuracy of Prediabetes and Diabetes Screening. Journal of Proteome Research, 20(J\ ), 1005-1014.

Claims

1. A method for determining a non-lactate hydroxy acid in a sample comprising the non-lactate hydroxy acid and lactate, said method comprising the steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having non-lactate hydroxy acid oxidizing activity; and iii. determining the non-lactate hydroxy acid in the sample.
2. The method of claim 1 , wherein the non-lactate hydroxy acid is a non- lactate 2-hydroxy acid.
3. The method of claim 2, wherein the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C1-6 alkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
4. The method of claim 3, wherein R1 denotes -CH3.
5. The method of claim 3, wherein R2 denotes phenyl.
6. The method of any one of claims 1 to 5, wherein the non-lactate 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-hydroxy-isobutyric acid, D-lactic acid, and the respective salt of any one of the foregoing.
7. The method of any one of claims 1 to 6, wherein the enzyme in ii. is an enzyme having non-lactate hydroxy acid dehydrogenase activity, or an enzyme having non-lactate hydroxy acid oxidase activity.
8. The method of claim 7, wherein the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
9. The method of any one of claims 1 to 8, wherein the enzyme in ii. is FCb2 or LDH.
10. The method of any one of claims 1 to 9, wherein in i. an enzyme is used.
11. The method of any one of claims 1 to 10, wherein in i. an enzyme having lactate oxidizing activity is used, specifically an enzyme having lactate oxidase activity.
12. The method of any one of claims 1 to 11 , further adding an agent removing hydrogen peroxide in i.
13. The method of claim 12, wherein an enzyme having catalase activity is added.
14. The method of any one of claims 1 to 13, wherein determining the nonlactate hydroxy acid is performed colorimetrically, photometrically, fluorimetrically, or electrochemically.
15. The method of any one of claims 1 to 14, wherein 2-hydroxybutyric acid is determined in a sample comprising 2-hydroxybutyric acid and lactate, said 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-hydroxybutyric acid dehydrogenase activity; and c. determining 2-hydroxybutyric acid in the sample.
16. The method of any one of claims 1 to 15, wherein the sample is a human sample.
17. The method of any one of claims 1 to 16, wherein an electrode comprising the enzyme having non-lactate hydroxy acid oxidizing activity is used in ii..
18. Use of an enzyme having non-lactate hydroxy acid oxidizing activity for determining a non-lactate hydroxy acid in a sample in a method of any one of claims 1 to 17.
19. The use of claim 18, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity.
20. Use of an electrode comprising an enzyme having non-lactate hydroxy acid oxidizing activity for determining a non-lactate hydroxy acid in a sample in a method of any one of claims 1 to 17.
21. The use of claim 20, wherein the enzyme having non-lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity.
22. The use of any one of claims 18 to 21 , wherein the sample is a human sample.
23. A kit for determining a non-lactate hydroxy acid in a sample comprising non-lactate hydroxy acid and lactate, said kit comprising an enzyme having lactate oxidizing activity and an enzyme having non-lactate hydroxy acid oxidizing activity.
24. The kit of claim 23, wherein the non-lactate hydroxy acid is a 2-hydroxy acid.
25. The kit of claim 23 or 24, wherein the non-lactate hydroxy acid is 2- hydroxybutyric acid.
26. The kit of any one of claims 23 to 25, wherein the enzyme having lactate oxidizing activity is lactate oxidase.
27. The kit of any one of claims 23 to 26, wherein the enzyme having non- lactate hydroxy acid oxidizing activity is an enzyme having non-lactate hydroxy acid dehydrogenase activity.
28. The kit of claim 27, wherein the enzyme having non-lactate hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
29. The kit of any one of claims 23 to 28, wherein the enzyme having non- lactate hydroxy acid oxidizing activity is FCb2 or LDH.
30. The kit of any one of claims 23 to 29, wherein the enzyme having non- lactate hydroxy acid oxidizing activity is part of an electrode.
31. An electrode comprising an enzyme having non-lactate 2-hydroxy acid oxidizing activity.
32. The electrode of claim 31 , wherein the non-lactate 2-hydroxy acid is a compound having the general formula I wherein
Ri denotes H or C i-ealkyl, and
R2 denotes H, Ce-s aryl, or C1-20 alkyl optionally substituted by -C(O)OH.
33. The electrode of claim 32, wherein Ri denotes -CH3.
34. The electrode of claim 32, wherein R2 denotes phenyl.
35. The electrode of any one of claims 31 to 34, wherein the non-lactate 2- hydroxy acid is selected from the group consisting of 2-hydroxybutyric acid, 2- hydroxy valeric acid, mandelic acid, 2-hydroxyoctanoic acid, 2-hydroxypalmitic acid, glycolic acid, malic acid, and 2-hydroxy-isobutyric acid.
36. The electrode of any one of claims 31 to 35, wherein the enzyme having non-lactate 2-hydroxy acid oxidizing activity is an enzyme having non-lactate 2-hydroxy acid dehydrogenase activity, or an enzyme having non-lactate 2-hydroxy acid oxidase activity.
37. The electrode of claim 36, wherein the enzyme having non-lactate 2- hydroxy acid dehydrogenase activity is an enzyme having 2-hydroxybutyric acid dehydrogenase activity.
38. The electrode of any one of claims 31 to 37, wherein the enzyme is FCb2 or LDH.
EP23817412.2A 2022-12-05 2023-12-01 Enzymatic detection of non-lactate hydroxy acids Pending EP4630571A1 (en)

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