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WO2025054130A1 - Enzymes pour l'oxydation de bêta-hydroxybutyrate (bhb), bandelettes de test et détecteurs les utilisant - Google Patents

Enzymes pour l'oxydation de bêta-hydroxybutyrate (bhb), bandelettes de test et détecteurs les utilisant Download PDF

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Publication number
WO2025054130A1
WO2025054130A1 PCT/US2024/045047 US2024045047W WO2025054130A1 WO 2025054130 A1 WO2025054130 A1 WO 2025054130A1 US 2024045047 W US2024045047 W US 2024045047W WO 2025054130 A1 WO2025054130 A1 WO 2025054130A1
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bhb
sensor
enzyme
wearable
amino acid
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PCT/US2024/045047
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English (en)
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Justin B. Siegel
Augustine ARREDONDO
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The Regents Of The University Of California
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Publication of WO2025054130A1 publication Critical patent/WO2025054130A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03006—Cholesterol oxidase (1.1.3.6)
    • 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
    • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones

Definitions

  • the present disclosure relates to enzymes capable of oxidizing beta- hydroxybutyrate (BHB) and methods thereof. In an aspect, the present disclosure relates to using enzymes described herein for various applications, such as use in test strips and additional detection platforms, BHB sensors, and systems for detecting and measuring BHB concentration.
  • BHB beta- hydroxybutyrate
  • BACKGROUND [0004]
  • the ketogenic diet is an effective way to propagate weight loss and wellness. The ketogenic diet encompasses minimizing the intake of carbohydrates while oftentimes increasing the consumption of fat and/or protein in tandem.
  • Beta-hydroxybutyrate is the conjugate base of beta-hydroxybutyric acid. BHB is synthesized via the metabolism of fatty acids, and a high level of BHB indicates that the body is using fats as its main fuel source. Therefore, there is a TOWNSEND 787450941 need to accurately and reliably measure BHB levels in a subject. This can be through invasive, minimally invasive, or non-invasive mechanisms.
  • the present disclosure solves this need by describing novel enzymes for aiding in the measurement of BHB.
  • Useful systems, devices, and associated methods are further provided herein.
  • BRIEF SUMMARY OF THE DISCLOSURE [0006]
  • the instant disclosure relates to enzymes with oxidase activity.
  • enzymes described herein are engineered and/or modified relative to native enzymes.
  • enzymes are capable of oxidizing beta- hydroxybutyrate (BHB) to produce 3-oxobutanoate.
  • beta- hydroxybutyrate (BHB) is (R)-beta-hydroxybutyrate.
  • beta- hydroxybutyrate (BHB) is (S)-beta-hydroxybutyrate.
  • the enzyme is non-naturally occurring and/or engineered and includes (e.g., comprises, consists essentially of or consists of) one or more amino acid substitutions, deletions, or truncations relative to a native enzyme.
  • the enzyme described herein covers modified Oxidase 8, derived from Scytonema sp.
  • the enzyme is a modified version of SEQ ID NO: 8 which includes one or more amino acid substitutions, deletions, or truncations.
  • SEQ ID NO: 8 is optimized to yield improved BHB detection activity.
  • the disclosure further provides for sensors capable of detecting and/or measuring BHB concentration, including utilizing an enzyme described herein, for continuous, continual, or on-demand detection or measurement.
  • Continuous is meant to be uninterrupted; unbroken; not intermittent or occasional; so persistently repeated at short intervals as to constitute virtually an unbroken series.
  • Continual means repeated regularly and frequently in a steady suggestion.
  • On demand means the detection or measurement can readily be obtained when the sensor or display of sensor data is actuated by the user.
  • the BHB sensor capable of sensing, detecting, and/or measuring BHB concentration includes: a sensor, comprising a sensing electrode; a sensing reagent, wherein the sensing reagent composition includes an enzyme described herein; and a reference electrode.
  • Body fluid refers to all body fluid including but not limited to whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, and/or ascites.
  • CSF cerebrospinal fluid
  • ISF interstitial fluid
  • the body fluids can either be from direct contact within body tissue internal to the body or from body fluids that are sampled either non-invasively or invasively from the body and sensed “on-body”.
  • the disclosure further relates to a system for detecting and measuring BHB concentration by utilizing an enzyme described herein.
  • the system for detecting and measuring BHB concentration includes: a BHB sensor, wherein the BHB sensor is configured to continuously, continually, or on-demand measure a BHB concentration of a subject and output a data stream; a sensor can use any method of BHB-measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetric, iontophoretic, radiometric, immunochemical, and the like; and a device connected to the BHB sensor, wherein the device comprises: a processor configured to process the data stream from the BHB sensor; and an interface configured to display and/or communicate measured BHB concentration values.
  • the disclosure further relates to a method of detecting and measuring BHB concentration by utilizing an enzyme described herein.
  • the method of detecting and measuring BHB concentration includes: obtaining a body fluid from a subject; subjecting the body fluid to a BHB sensor; determining a single point BHB concentration in the body fluid; and displaying and/or communicating the single point BHB concentration on an interface.
  • the enzymes, devices, or systems described herein are used in methods of improving health and wellness in a subject in need thereof.
  • the enzymes, devices, or systems described herein are used in methods of weight loss.
  • enzymes, devices, or systems described herein are used for improving mental and/or metabolic health.
  • enzymes, devices, or systems described herein are used in methods of controlling intake or monitoring of carbohydrates.
  • enzymes, devices, or systems described herein are used in methods of monitoring ketone body levels for improvement of cognitive function, to treat various neurological disorders, such as epilepsy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), traumatic brain injury, Alzheimer’s disease, and dementias, to treat mental health, mental illness, psychiatric problems, and psychiatric disorders such as depression, bi-polar disorders, schizophrenia, to improve cardiac metabolism, improve immunotherapeutic, chemotherapeutic and radiotherapeutic response to cancer treatments, modulate inflammatory pathways and immune function, to treat obesity and diabetes, for cholesterol quantification and monitoring, and to monitor and detect alcoholic or diabetic ketoacidosis.
  • neurological disorders such as epilepsy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), traumatic brain injury, Alzheimer’s disease, and dementias
  • ALS amyotrophic lateral sclerosis
  • MS multiple
  • enzymes, devices, or systems described herein are used in conjunction with other weight loss or wellness methods or therapies. In other embodiments, enzymes, devices, or systems described herein are used in conjunction with food or supplements which contain a low carbohydrate amount or sugar alcohols in the formulation.
  • the food item or supplement comprises a ketone supplement or additive. In another embodiment, the food item or supplement comprises an exogenous ketone and/or ketogenic supplement. In some embodiments, the exogenous ketone and/or ketogenic supplement comprises ketone bodies and/or precursors of ketone bodies.
  • the ketone bodies and/or precursors of ketone bodies comprise one or more of acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium chain triglycerides (MCT) containing fatty acids with hydrocarbon side chains in the length of 6-12 carbons.
  • BHB beta-hydroxybutyrate
  • MCT medium chain triglycerides
  • the ketone bodies and/or precursors of ketone bodies are in the form of salts and/or esters.
  • the MCT comprises one or more of the following: caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12).
  • devices, systems, or methods described herein are prescribed to a subject in need thereof by a medical professional.
  • the patient the patient in need thereof is obese or afflicted with an underlying medical condition such as heart disease.
  • Figure 1 Initial screening of sequence similarity networks (SSNs) composed of 4000 bacterial oxidases.
  • Figure 2 Comparison between Oxidase 8 (SEQ ID NO 8) and an engineered cholesterol oxidase from Streptomyces hygrospinosus (ShCOb) using the Alphafold model.
  • Figure 3 Visual display of the design of the engineered enzyme (8_MUT) predicted by Alphafold based on wild type Oxidase 8 (8_WT).
  • Figure 4 Detection results of hydrogen peroxide (H2O2; Peroxide) produced by the engineered enzyme containing amino acid sequence SEQ ID NO: 24 (Oxidase8_mut) and the wild type Oxidase 8 (Oxidase8_WT, SEQ ID NO: 8).
  • H2O2 hydrogen peroxide
  • Figure 5 Detection results of acetoacetate produced by the engineered enzyme containing amino acid sequence SEQ ID NO: 24 (Oxidase8_mut) and the wild type Oxidase 8 (Oxidase8_WT, SEQ ID NO: 8).
  • Bayer Ketostix® urine reagent test strips were used.
  • Figure 6 The HPLC-MS profile of the reaction mixture of the engineered enzyme containing amino acid sequence SEQ ID NO: 24 (Oxidase8_mut).
  • Figure 7 Comparing the HPLC-MS profile of the Oxidase8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS profile of the commercially available synthetic acetoacetate (Acetoacetate).
  • Figure 8 Comparing the HPLC-MS profile of the Oxidase8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS profile of the wild type Oxidase 8 (Oxidase8_WT, SEQ ID NO: 8) (8_WT) reaction mixture.
  • FIG. 9 Comparing the HPLC-MS profile of the Oxidase8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS profile of the Substrate Control. DETAILED DESCRIPTION OF THE DISCLOSURE [0045]
  • the disclosure provides for novel proteins and enzymes capable of exhibiting BHB activity.
  • the novel proteins or enzymes are capable of being used with methods, systems, devices, and kits described herein.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • R groups e.g., norleucine
  • modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer,
  • sequence that is “at least 80% identical to a reference sequence” is a sequence having, over its entire length, 80%, or more, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.5%, 99.6%, 99.7%, 99.8% sequence identity with the entire length of a reference sequence.
  • Proteins consisting of an amino acid sequence “at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical” to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence.
  • the protein consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.
  • the “percentage of identity” can be calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art.
  • the “needle” program which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol.48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used.
  • the needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp.276—277).
  • the percentage of identity between two polypeptides is calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.
  • Other algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol.
  • At least one herein refers to one or more of the specified objects such as 1, 2, 3, 4, 5 or 6 or more of the specified objects.
  • at least one amino acid substitution herein refers to 1, 2, 3, 4, 5 or 6 or more amino acid substitutions.
  • amino acid substitutions may be conservative or non-conservative. In an aspect, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties.
  • conservative substitution leads to the same or similar functional properties.
  • conservative substitutions may include those, which are described by Dayhoff in “The Atlas of Protein Sequence and Structure. Vol.5”, Natl. Biomedical Research, the contents of which are incorporated by reference in their entirety.
  • amino acids which belong to one of the following groups, can be exchanged for one another, thus, constituting a conservative exchange: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine (C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E).
  • a conservative amino acid substitution may be selected from the following of T ⁇ A, G ⁇ A, A ⁇ I, T ⁇ V, A ⁇ M, T ⁇ I, A ⁇ V, T ⁇ G, and/or T ⁇ S.
  • a conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His.
  • conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, the contents of which are incorporated by reference in their entirety). [0058] In another embodiment, conservative substitutions may be made in accordance with Table 1. Methods for predicting tolerance to protein modification
  • TOWNSEND 787450941 may be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205- 9210 (2004), the contents of which are incorporated by reference in their entirety.
  • Table 1 Representative Conservative Amino Acid substitutions [0059] In another embodiment, conservative substitutions may be those shown in Table 2 under the heading of “conservative substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 2, may be introduced and the products screened if needed.
  • Enzymes or proteins described herein may be engineered, isolated, or purified. In an aspect, enzymes or proteins described herein may be non-naturally occurring. In other aspects, enzymes or proteins described herein may be modified via amino acid substitution, deletion, and/or truncation. In other aspects, enzymes described herein are optimized to detect or analyze BHB. In other aspects, Enzymes described herein are designed for use with the systems, devices, kits, and methods described herein.
  • the disclosure relates to enzymes with oxidase activity, wherein the enzyme is capable of oxidizing beta-hydroxybutyrate (BHB) to produce 3- oxobutanoate.
  • the enzyme comprises, consists of, or consists essentially of an amino acid sequence comprising one or more mutations
  • an enzyme described herein only comprises a combination of N137G, Y235Q, and/or A455Y mutations of SEQ ID NO: 8.
  • an enzyme described herein comprises, consists essentially of, or consists of a combination of N137G, Y235Q, and/or A455Y mutations of SEQ ID NO: 8 coupled with a N-terminal truncation at amino acid positions 2-32 of SEQ ID NO: 8.
  • an enzyme described herein comprises, consists essentially of, or consists of a combination of N137G, Y235Q, and/or A455Y mutations of SEQ ID NO: 8 coupled with a N-terminal truncation of the amino acid positions selected from the group consisting of amino acid position numbers 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, or 2-32 of SEQ ID NO: 8.
  • an enzyme described herein comprises, consists essentially of, or consists of a combination of N137G, Y235Q, and/or A455Y mutations of SEQ ID NO: 8 coupled with a N-terminal truncation of 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, or 1 amino acid of SEQ ID NO: 8.
  • SEQ ID NO: 24 exhibits increased BHB activity over SEQ ID NO: 8 in similar testing conditions.
  • SEQ ID NO: 24 or SEQ ID NO: 40 – 43 exhibits increased BHB activity over SEQ ID NO: 8 in or under the same testing conditions.
  • amino acid sequences described herein comprise a truncation at the N-terminal and/or at the C-terminal.
  • the truncation has a length of at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, or at least 31 amino acids.
  • the truncation has a length of at most 3 amino acids, at most 5 amino acids, at most 10 amino acids, at most 15 amino acids, at most 20 amino acids, at most 25 amino acids, at most 26 amino acids, at most 27 amino acids, at most 28
  • the truncation has a length of from 5 to 50 amino acids, from 10 to 50 amino acids, from 15 to 50 amino acids, from 20 to at least 50 amino acids, from 10 to 40 amino acids, from 10 to 35 amino acids, from 15 to 35 amino acids, from 20 to 35 amino acids, from 25 to 32 amino acids, from 28 – 32 amino acids, from 2 to 10 amino acids, or from 2 to 5 amino acids from N-terminal and/or at the C-terminal of an enzyme or protein described herein.
  • the truncations described herein are in one or more of SEQ ID NO: 1 – 43.
  • the truncations described herein are relative to SEQ ID NO: 8, SEQ ID NO: 24, or SEQ ID NO: 40 – 43.
  • the enzyme comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to any of SEQ ID NO: 1 – 43.
  • the enzyme comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to any of SEQ ID NO: 24 or SEQ ID NO: 40 – 43.
  • enzymes described herein comprise N137G, Y235Q, and/or A455Y mutations relative to SEQ ID NO: 8, wherein the enzyme further comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% to SEQ ID NOs: 8, 24, or 40 – 43.
  • the enzyme comprises an amino acid sequence of any of SEQ ID NO: 1 – 43, wherein the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, or ten or more amino acid substitutions.
  • the enzyme comprises an amino acid sequence of any of SEQ ID NO: 1 – 43, wherein the amino acid sequence comprises at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten or more amino acid substitutions.
  • the enzyme comprises an amino acid sequence of any of SEQ ID NO: 24 or 40 – 43, wherein the amino acid sequence includes one, two, three, four, five, six, seven, eight, nine, or ten or more amino acid substitutions.
  • the amino acid substitution(s) are conservative substitutions.
  • the enzyme comprises an amino acid sequence of any of any of
  • a biosensor is a device that measures biological or chemical reactions by generating signals proportional to the concentration of an analyte in the reaction.
  • a biosensor is a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounts.
  • the disclosure provides for a sensor, such as a nonwearable sensor, capable of detecting and/or measuring BHB concentration including utilizing enzymes described herein.
  • a sensor such as a nonwearable sensor
  • Such sensors may be in the format of a test strip or a one-touch fingertip sweat sensor.
  • the enzyme comprises one or more of SEQ ID NO: 1 – 43, fragments, or modified enzymes of SEQ ID NO: 1 – 43 as described herein.
  • the nonwearable sensor can be used to detect or measure BHB in body fluid, in which the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, and/or ascites.
  • body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritone
  • the test strip includes: a substrate layer; and one or more sensing reagents dispensed upon at least a portion of the substrate layer, wherein at least one of the sensing reagents includes the enzyme.
  • the at least one of the sensing reagents further includes a co-factor, a mediator, and/or another adjuvant or excipient.
  • the co-factor includes flavin adenine dinucleotide (FAD).
  • the mediator includes one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green/blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and/or organometallic ruthenium complexes.
  • the test strip further comprises one or more tetrazolium salts.
  • the tetrazolium salts are selected from the group consisting of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5- phenyltetrazolium chloride (INT) and 3-(4,5-dimethylthiazolyl-1-2)-2,5- diphenyltetrazolium bromide (MTT).
  • the material of the substrate layer may comprise one or more of a composite material, a fibrous material, a woven textile, a non-woven textile, a polymer, an adhesive, a film, a gel, PTFE, and /or silicone.
  • the disclosure further provides for a BHB sensor capable of sensing, detecting, and/or measuring BHB concentration by utilizing enzyme described herein in a continuous, continual, or on- demand manner.
  • the enzyme comprises one or more of SEQ ID NO: 1 – 43, fragments, or modified enzymes of SEQ ID NO: 1 – 43 as described herein.
  • the BHB sensor capable of sensing, detecting, and/or measuring BHB concentration comprises: a sensor comprising a sensing reagent, wherein the sensing reagent composition comprises said engineered enzyme; and a reference electrode.
  • the sensing reagent composition further comprises one or more of a co-factor, mediator, adjuvant, or excipient.
  • the wearable BHB sensor is configured to continuously, continually, or on-demand measure a BHB concentration of a subject and output a data stream.
  • the continuous BHB sensor is an implantable or non-implantable device.
  • the wearable BHB sensor is a needle-based sensor or a micro-needle-based sensor.
  • a sensor can use any method of BHB-measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetric, iontophoretic, radiometric, immunochemical, and the like.
  • the wearable BHB sensor is configured to measure a BHB concentration in a body fluid.
  • the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma,
  • the disclosure further provides for a system for detecting and measuring BHB concentration by utilizing enzymes described herein.
  • the enzyme comprises one or more of SEQ ID NO: 1 – 43, fragments, or modified enzymes of SEQ ID NO: 1 – 43 as described herein.
  • the system for detecting and measuring BHB concentration comprises: a BHB sensor, wherein the BHB sensor is configured to measure a BHB concentration of a subject and output a data stream; and a device connected to the BHB sensor, wherein the device comprises: a processor configured to process the data stream from the BHB sensor; and an interface configured to display and/or communicate measured BHB concentration values.
  • a BHB sensor or BHB sensing system is created by integrating said engineered enzyme into a variety of sensing platforms and/or devices.
  • Platforms and/or devices that can adopt said engineered enzyme include, but are not limited to, pH-change sensors such as the ones described in Chodavarapu et al., US 7,794,584, electrochemical sensors such as the ones described in Simpson et al., US 7,081,195, Lebel et al., US 6,915,147, and Jina et al., US 2010/0049021, optical sensors such as the ones described in Petrich et al., US 10,724,943, implantable sensor platforms such as the ones described in Jain, EP 2079358, chip-shaped blood analysis devices such as the ones described in Ogawa et al., US 7,582,259, and monitoring systems where the control terminal is remotely coupled such as the ones described in Karan et al., US 2012/0245447.
  • pH-change sensors such as the ones described in Chodavarapu et al., US 7,794,584
  • electrochemical sensors such as the ones described in Simpson et al., US 7,081,195, Le
  • the BHB sensors, BHB sensing systems, BHB devices, and applicable techniques are non- invasive or minimally invasive, wearable biosensing and/or chemical monitoring sensors, systems, devices and techniques.
  • the electrochemical biosensors or chemical sensors used to measure BHB qualitativley and/or quantatively are fingertip sensors.
  • the BHB sensor or BHB sensing system is a one- touch fingertip sweat sensor and personalized data processing method, system, or device such as those described in WIPO International Patent Application No. PCT/2022/070554, filed on February 07, 2022, and published as Publication No. WO/2022/170361 on August 11, 2022; or in U.S. Patent Application No.18/264,755, filed on August 08, 2023, and published as Publication No. US 2024/0049994 A1 on February 15, 2024.
  • the content of each of these applications is hereby incorporated by reference in their entireties.
  • the BHB sensor or BHB sensing system utilizes reverse iontophoresis, which is a non-invasive or minimally invasive process of extraction of biomarkers.
  • Reverse iontophoresis is a technique by which a small current flow is applied to the skin with the effect of extracting polar and non-polar molecules to the anode or cathode, where they can be sensed by the electrochemistry.
  • Reverse iontophoresis is a technique by which a small current flow is applied to the skin with the effect of extracting polar and non-polar molecules to the anode or cathode, where they can be sensed by the electrochemistry.
  • the electrochemical biosensors or chemical sensors are wearable, epidermal electrochemical sensor devices for detecting BHB in sweat.
  • epidermal electrochemical sensors devices see, e.g., U.S. Patent No.9,820,692 and U.S. Patent No.11,185,286.
  • the content of each of these patents is hereby incorporated by reference in their entireties.
  • the disclosure further provides for methods of detecting and measuring BHB concentration by utilizing an enzyme described herein.
  • the enzyme comprises one or more of SEQ ID NO: 1 – 43, fragments, or modified enzymes of SEQ ID NO: 1 – 43 as described herein.
  • the method of detecting and measuring BHB concentration comprises: obtaining a body fluid from a subject; subjecting the body fluid to a BHB sensor; determining a single point BHB concentration in the body fluid; and displaying and/or communicating the single point BHB concentration on an interface.
  • the reagent A comprises citric acid, acetic acid, KH 2 PO 4 , N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N- morpholino)ethanesulfonic acid (MES), phosphate, and/or tris(hydroxymethyl) ainomethane.
  • the reagent A has a pH of 3-11, optionally 4-10, optionally 5-9, optionally 6-8.
  • the reagent B comprises a co-factor, a mediator, an excipient, an adjuvant, or a carrier.
  • the composition containing the enzyme is dissolved in water.
  • the co-factor comprises one or more of flavin adenine dinucleotide (FAD), semiquinone form flavin adenine dinucleotide (FADH), and/or quinone form flavin adenine dinucleotide (FADH 2 ).
  • the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green/blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and/or organometallic ruthenium complexes.
  • the reagent B comprises NaCl and/or KCl. In some embodiments, the concentration of NaCl and/or KCl is 0.1-5 M. In some embodiments, the reagent B comprises citric acid, acetic acid, KH2PO4, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N- morpholino)ethanesulfonic acid (MES), phosphate, and/or tris(hydroxymethyl) ainomethane. In other embodiments, the reagent B has a pH of 3-11, optionally 4-10, optionally 5-9, optionally 6-8. In some embodiments, the reagent B comprises one or more tetrazolium salts. In some embodiments, wherein one or more of the tetrazolium salts are selected from the group consisting of 2-(p-iodophenyl)-3-(p-
  • compositions [0091] In another embodiment, the invention relates to applications of the enzyme as an ingredient of a composition.
  • the composition comprises one or more of active ingredients.
  • the active ingredients further comprise one or more of other enzymes such as glucose oxidase, glucosyl transferase, fructosyl transferase, catalase, amylase, lactase, lipase, and/or protease.
  • the composition may further comprise one or more excipients, one or more adjuvants, and/or one or more carriers.
  • Excipients, adjuvants, and/or carriers commonly known in the field may be found from, for example, Margolin et al. (US 7,718,169), the content of which is incorporated herein by reference.
  • the one or more excipients comprises one or more of microcrystalline cellulose, Maltrin, Crospovidone, colloidal silcon dioxide, magnesium stearate, talc, sucrose, trehalose, lactose, sorbitol, lactitol, mannitol, inositol, salts of sodium and potassium, such as acetate, phosphates, citrates and borate, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxy polyethylene glycol, gelatin, hydroxypropyl- ⁇ -cyclodextrin, polylysine, polyarginine, amino acids such as glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline, carbohydrates such as glucose, fructose, galactose, mannose, arabinose, xylose, rib
  • the one or more adjuvants comprises one or more of water-in-oil and oil-in-water emulsions, aluminum salt adjuvants, liposomes, and/or CpG oligodeoxynucleotide adsorbed to aluminum salt.
  • the one or more carriers comprises one or more of polymers used for encapsulation of protein crystals for delivery of proteins, including controlled release biological delivery.
  • the polymers include biocompatible and biodegradable polymers, or mixtures thereof.
  • polymer composition wherein a rate of dissolution and, therefore, delivery of enzymes will be determined by the particular encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer stability, enzyme crystal geometry and degree, if any, of enzyme crosslinking.
  • the one or more active ingredient(s) may be present in the composition in association with a polymeric carrier.
  • Useful polymeric carriers include, for example, polymers used for encapsulation of protein crystals for delivery of proteins, including controlled release biological delivery. Such polymers include biocompatible and biodegradable polymers, or mixtures thereof.
  • the polymeric carrier is a biodegradable polymer.
  • ketogenic diets and methods of treating diseases [0099] The disclosure further provides for ketogenic diets and methods of treating diseases via the use of devices, systems, methods, or kits integrated with enzymes described herein. In an embodiment, ketogenic diets are used in tandem with the devices and systems described herein. A representative description of nutritional ketosis and diseases treatable with the same may be found in, for example, at
  • Ketosis [0100]
  • ketone and/or “ketone body” include the following non-exhaustive compounds: acetone (2-propanone, dimethyl ketone, or beta- ketopropane), acetoacetic acid (3-oxobutanoic acid, acetonecarboxylic acid, or diacetic acid), acetoacetate, beta-hydroxybutyric acid (3-hydroxybutyric acid), beta- hydroxybutyrate (BHB, 3HB, or 3-hydroxybutyrate), beta-ketopentanoate (3- oxopentanoate, 3-oxovaleric acid, or 3-ketovaleric acid), and beta- hydroxypentanoate (3-hydroxyvalerate, 3-hydroxy valeric acid, or beta-hydroxyvaleric acid).
  • BHB is an anionic small molecule acid metabolite with a hydroxyl group and is the major ketone body that is distributed in the human brain and its primary energy source when glucose is absent.
  • BHB see, e.g., J.C. Newman and E. Verdin, August 21, 2017, ⁇ -Hydroxybutyrate, Ann Rev Nutr.37:51-76.
  • ketosis refers to the conjugate base of acetoacetic acid and is used interchangeably with the term “3-oxobutanoate.”
  • enzymes, methods, devices, and systems described herein are related to ketosis and may be used in, for example, nutritional or therapeutic ketosis.
  • Nutritional, or therapeutic, ketosis is the physiological state of elevated blood ketone body levels (typically above 0.5 mmol/L) resulting from ketogenic diets, calorie restriction, therapeutic fasting and/or supplementation with ketogenic precursors.
  • Ketone bodies represent alternative energy substrates for both peripheral tissues and the central nervous system.
  • ketone bodies are acetoacetate and beta-hydroxybutyrate (BHB), while the third ketone body, acetone, is produced as a byproduct that the lungs breathe off.
  • BHB beta-hydroxybutyrate
  • acetone is produced as a byproduct that the lungs breathe off.
  • the body produces ketone bodies during nutritional or therapeutic
  • Ketones can replace glucose to supply most of the brain's metabolic energy needs (>50%) during periods of limited glucose availability resulting from starvation/fasting, caloric restriction or carbohydrate restriction as in ketogenic diets. During carbohydrate deprivation, glucose availability decreases causing a metabolic shift towards fatty acid beta-oxidation and the production of ketone bodies for energy homeostasis.
  • Dietary carbohydrates include simple sugars, such as table sugar (sucrose) and complex carbohydrates (starch) found in foods like potatoes and pasta. Carbohydrate and sugar consumption have dramatically increased in the last two centuries in Western societies.
  • ketone levels will increase to as high as 2 or 3 mmol/L, or even higher. It is conventionally understood and agreed that when blood ketones rise above 0.5 mmol/L, the heart, brain and peripheral tissues are using ketone bodies (beta hydroxybutyrate and acetoacetate) as the primary fuel source. This condition is
  • ketosis 25 TOWNSEND 787450941 referred to as ketosis, or “nutritional ketosis.” This is distinguished from diabetic or alcoholic ketoacidosis, which is the runaway accumulation of ketone bodies and associated drop in blood pH. Diabetic ketoacidosis is associated with the absence of insulin as occurs in those suffering from type 1 diabetes. Ketoacidosis typically results in blood ketone levels more than 15 mmol/L in combination with metabolic derangement and electrolyte imbalance. [0107] When in ketosis, the body essentially burns fat for fuel. This is accomplished because fat stores in the body are utilized to create the water-soluble ketone bodies beta-hydroxybutyrate (BHB) and acetoacetate (also known as acetylacetonate).
  • BHB beta-hydroxybutyrate
  • acetoacetate also known as acetylacetonate
  • ketone bodies are then used by the body as its primary energy source.
  • the body enters a state of ketosis when it has no dietary source of glucose or sugar and its stores of glycogen have been depleted. This typically occurs during fasting, exercise, and/or pursuing a carbohydrate restricted ketogenic diet.
  • ketosis the body begins cleaving fats into fatty acids and glycerol and transforms the fatty acids into acetyl CoA molecules which are then eventually transformed into ketone bodies in the liver.
  • the body uses dietary and bodily fats as its primary energy source.
  • enzymes, methods, devices, and systems described herein are related to and may aid in methods of weight loss, regulation and/or monitoring of weight loss and regulation and/or monitoring of carbohydrate intake. In other embodiments, enzymes, methods, devices, and systems described herein are related to regulation and/or monitoring of ketosis over hours, days, weeks, months, or years. In some embodiments, the ketone concentration is monitored for the purpose of detecting and preventing ketoacidosis.
  • Ketogenic diets and weight loss The disclosure provides for methods of treating weight loss in subjects in need thereof by utilizing the enzymes, methods, devices, or systems described herein.
  • a ketogenic diet is one that is high in dietary fat and low in carbohydrates with moderate levels of protein (approximately 1-2 g/kg). The classical ketogenic diet
  • ketogenic diet may result in loss of fat stores while maintaining and protecting muscle mass.
  • Some studies have suggested that the muscle sparing properties of a ketogenic diet result in improvement in physical performance. Athletes who maintain nutritional ketosis maintain lower insulin levels and can better utilize fatty acids and ketones for fuel, effectively sparing blood glucose, which optimizes and prolongs physical and mental performance. This state is referred to as being “keto adapted.” Keto adaptation occurs when the body adjusts to ketosis by building up the necessary fat-burning enzymes, hormone levels are changed to accommodate ketosis, glycogen stored in muscles and liver is reduced, and the body is carrying less water.
  • a ketogenic diet comprises about 5 – 10% carbohydrates; about 55 – 85% fats; and about 10 – 40% proteins.
  • the ketogenic diet comprises: 15-50 g carbohydrates; 60-400 g fats; and 40-100 g proteins.
  • the ketogenic diet provides 1500 – 3000 kcal per day.
  • the ketogenic diet includes intermittent or prolonged fasting. In aspects, these parameters may be modulated and/or monitored via methods, systems, and devices described herein.
  • the ketogenic diet is consumed each day for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.
  • ketosis monitoring is performed after 1 day, after 2 days, after 3 days, after 4 days, after 5 days, after 6 days, after 7 days, after 8 days,
  • ketosis monitoring is performed by using a beta- hydroxybutyrate (BHB) sensor to detect a BHB concentration.
  • BHB concentration is a blood BHB concentration or an interstitial fluid (ISF) BHB concentration.
  • the ketosis monitoring is performed at least once every day, at least twice every day, at least 3 times a day, at least 4 times a day, at least 5 times a day, at least once every 2 days, at least once every 3 days, at least once every 4 days, at least once every 5 days, at least once every 6 days, or at least once every 7 days, or at least once every 2 weeks, at least once every 3 weeks, or at least once every 4 weeks.
  • the ketosis monitoring is performed at 7:00 am, at 8:00 am, at 9:00 am, at 10:00 am, at 11:00 am, at 12:00 pm, at 1:00 pm, at 2:00 pm, at 3:00 pm, at 4:00 pm, at 5:00 pm, at 6:00 pm, at 7:00 pm, at 8:00 pm, at 9:00 pm, and/or at 10:00 pm.
  • the ketosis monitoring is performed 3 hours before breakfast, 2 hours before breakfast, 1 hour before breakfast, right before breakfast, right after breakfast, 1 hour after breakfast, 2 hours after breakfast, 3 hours after breakfast, 3 hours before lunch, 2 hours before lunch, 1 hour before lunch, right before lunch, right after lunch, 1 hour after lunch, 2 hours after lunch, 3 hours after lunch, 3 hours before dinner, 2 hours before dinner, 1 hour before dinner, right before dinner, right after dinner, 1 hour after dinner, 2 hours after dinner, or 3 hours after dinner. Effects of ketosis on cognitive and physical performance [0121] Performance studies have shown improved motor function, endurance, and cognitive function with ketone supplementation.
  • cholesterol oxidase which belongs to the oxidoreductase family EC 1.1.3.6, has the ability to convert cholesterol into cholest-4-en-3-one.
  • the reaction of converting cholesterol into cholest-4-en-3-one is illustrated below.
  • This ability makes cholesterol oxidase a potential candidate for developing optimized engineered enzymes able to convert (R)-beta-hydroxybutyrate into 3-oxobutanoate.
  • hydrogen peroxide (H 2 O 2 ) produced by the enzymatic reactions can be utilized according to the present disclosure.
  • the resultant hydrogen peroxide can be detected using sensitive and stable fluorescent probes. See, e.g., Allain et al., Clin Chem
  • the initial screening result is shown in Fig.1.39 amino acid sequences were identified as potential candidates. These amino acid sequences are shown in Table 3.
  • a 31 amino acid N- terminus truncation (amino acid positions 2-32 of SEQ ID NO: 8 [oxidase 8 wt]) was introduced.
  • a visual display of the design of the engineered enzyme as predicted by Alphafold is shown in Fig.3.
  • the amino acid sequences of the engineered enzymes are also provided in Table 3.
  • SEQ ID NO: 24 (aka 8_MUT or Oxidase8_mut) comprises all three of these amino acid substitutions as well as the truncation of amino acid positions 2-32 when compared to SEQ ID NO: 8 (aka herein as 8_WT).
  • the three mutations correspond to N106G, Y204Q, and/or A424Y in the resultant SEQ ID 24.
  • the ‘M’ at amino acid position number 1 i.e., methionine or Met
  • Patent Publication No.2022/0348970 A1 the contents of each which are hereby incorporated herein by reference in their entireties.
  • Overnight cultures of BLR cells suspended in a 2 mL volume were transformed with a pet29b+ plasmid (encoding polypeptides of interest with a C- terminal His-tag) and grown in Terrific Broth with 50 ⁇ g/ml kanamycin. Cultures were diluted 1:1.000 in 500 ml of Terrific Broth with 1 mM MgSO4, 1% glucose and 50 ⁇ g/ml antibiotic and then grown at 37°C. for 24 hours.
  • Cultures were pelleted down at 5,000 G for 10 minutes and resuspended in auto-induction media (TB broth, 1 mM MgSO4, 1 ⁇ NPS and 1 ⁇ 5052) for induction at 18°C for 24 hours. At the end of induction, cells were centrifuged, the supernatant was removed and cells were resuspended in 40 mL lysis buffer (1x PBS, pH 7.5, 5 mM Imidazole) and 1 mM phenylmethylsulphonyl fluoride. The cell lysate suspension was sonicated for 2 min and followed by centrifugation at 5,000 G.
  • auto-induction media TB broth, 1 mM MgSO4, 1 ⁇ NPS and 1 ⁇ 5052
  • the engineered enzyme containing amino acid sequence SEQ ID NO: 24 (Oxidase8_mut) generated hydrogen peroxide with the presence of 2-pentanol, (R)-2-pentanol, BHB, or (R)-beta-hydroxybutyrate (R-BHB), as the test strip turned blue when contacting with the reaction mixture of these reactants.
  • the wild type Oxidase 8 (Oxidase8_WT, SEQ ID NO: 8) exhibited a lesser activity in oxidizing BHB, as the brighter blue color indicated; and did not oxidize R- BHB, as the test strip did not turn blue.
  • Fig.6 shows the HPLC-MS profile of the reaction mixture of the engineered enzyme containing amino acid sequence SEQ ID NO: 24 (Oxidase8_mut);
  • Fig.7 compares the HPLC-MS profile of the Oxidase8_mut reaction mixture with the HPLC-MS profile of the commercially
  • Table 4 Review of representative activities SEQ ID: Peroxide strip Acetoacetate HPLC-MS activity activity activity activity Example 4: Further Enzyme Design [0144] Additional enzymes suitable for use in the current invention and associated methods include those set forth in Tables 5 and 6.
  • Table 5 Amino acid sequences of representative enzymes according to the disclosure SEQ ID Amino Acid Sequence Representative Modifications l h Q TOWNSEND 787450941 LAQPLDFSATAHPLGGATIGQVCNTYGQVYGYR template NLFVVDGSFIPGSTACTNPSFTIAALAERSMERFL sequence NRSA l h Q l h Q
  • Table 6 Amino acid sequences of representative enzymes according to the disclosure SEQ ID Amino Acid Sequence Representative Modifications in e
  • X 1 may be N, G, P, A, or S
  • X 2 may be Y, Q, N, K, E, or D
  • X 3 may be A, Y, W, F, or H.
  • LCMS liquid chromatography–mass spectrometry
  • a non-wearable BHB sensor capable of detecting and/or measuring BHB concentration comprising utilizing the enzyme of any one of embodiments 1 – 19.
  • the test strip of embodiment 21 or 22, wherein the at least one sensing reagents comprising a co-factor, a mediator, an adjuvant, a carrier, and/or an excipient.
  • the co-factor comprises flavin adenine dinucleotide (FAD).
  • a material of the substrate layer comprises one or more of a composite material, a fibrous material, a woven textile, a non-woven textile, a polymer, an adhesive, a film, a gel, PTFE, and /or silicone.
  • the non-wearable BHB sensor of embodiment 20, wherein the non- wearable sensor can be used to detect or measure BHB in any body fluid.
  • the non-wearable BHB sensor of embodiment 28, wherein the body fluid can be obtained through non-invasive, minimally invasive, or invasive means, optionally via finger stick, blood draw, spinal tap, sweat and/or saliva collection.
  • CSF cerebrospinal fluid
  • ISF interstitial fluid
  • a wearable BHB sensor capable of detecting, measuring, and/or monitoring BHB concentration, comprising: a sensor, comprising a sensing electrode; a sensing reagent, wherein the sensing reagent composition comprising the enzyme of any one of embodiments 1 – 19; and a reference electrode.
  • a sensor comprising a sensing electrode; a sensing reagent, wherein the sensing reagent composition comprising the enzyme of any one of embodiments 1 – 19; and a reference electrode.
  • 32. The wearable BHB sensor of embodiment 31, wherein the sensing reagent is on or adjacent to the sensing electrode.
  • 33. The wearable BHB sensor of embodiment 31, wherein the sensing reagent is dispensed to be in contact with the sensing electrode at the time of use.
  • 34. The wearable BHB sensor of embodiment 31, wherein the wearable BHB sensor is a continuous sensor, a continual sensor, or an on-demand sensor.
  • the wearable BHB sensor is a needle-based sensor, a micro-needle-based sensor, a reverse iontophoretic sensor, a sweat-based sensor, or an implantable sensor. 38.
  • the wearable BHB sensor of embodiment 37 wherein the wearable BHB sensor is located in body tissues.
  • the wearable BHB sensor of embodiment 39 wherein the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, and/or ascites. 41.
  • CSF cerebrospinal fluid
  • ISF interstitial fluid
  • a system for detecting, measuring, and/or monitoring BHB concentration comprising: the wearable BHB sensor of anyone of embodiments 31
  • the wearable BHB sensor is configured to continuously, continually, or on-demand measure a BHB concentration of a subject and output a data stream; and a device connected to the wearable BHB sensor, wherein the device comprises: a processor configured to process the data stream from the wearable BHB sensor; and an interface configured to display and/or communicate measured BHB concentration values.
  • a method of detecting, measuring, and/or monitoring BHB concentration comprising: obtaining a body fluid from a subject; subjecting the body fluid to the wearable BHB sensor of any one of embodiments 31 – 41; determining a single point BHB concentration in the body fluid; and displaying and/or communicating the single point BHB concentration on an interface. 44.
  • a method of improving health and/or wellness in a subject in need thereof comprising using a non-wearable BHB sensor, device, or apparatus of any one of embodiments 20 – 30.
  • a method of improving health and/or wellness in a subject in need thereof comprising using a test kit, wherein the test kit utilizes the enzyme of any one of embodiments 1 – 19.
  • 46. A method aiding weight loss in a subject in need thereof comprising using a non-wearable BHB sensor, device, or apparatus of any one of embodiments 20 – 30.
  • the method of embodiment 49, wherein the exogenous ketone supplement comprises ketone bodies and/or precursors of ketone bodies. 51.
  • ketone bodies and/or precursors of ketone bodies comprise one or more of acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-
  • MCT medium chain triglycerides
  • the ketone bodies and/or precursors of ketone bodies are in the form of salts and/or esters.
  • the MCT comprises one or more of the following: caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12).

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Abstract

La présente invention concerne des enzymes capables d'oxyder le bêtahydroxybutyrate (BHB). Dans certains aspects, les enzymes sont modifiées pour optimiser l'activité de BHB. L'invention concerne en outre des enzymes modifiées comprenant des substitutions d'acides aminés, des troncatures et des délétions de séquence. L'invention concerne en outre l'utilisation d'enzymes décrites ici, par exemple, dans des bandelettes réactives, des détecteurs de BHB, des dispositifs et des systèmes pour détecter et mesurer la concentration en BHB.
PCT/US2024/045047 2023-09-05 2024-09-03 Enzymes pour l'oxydation de bêta-hydroxybutyrate (bhb), bandelettes de test et détecteurs les utilisant WO2025054130A1 (fr)

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