Enzymes that oxidize beta-hydroxybutyrate (BHB), test strips, and sensors that use them.

Abstract

This disclosure relates to enzymes capable of oxidizing beta-hydroxybutyrate (BHB). In embodiments, the enzymes are modified to optimize BHB activity. Modified enzymes, including amino acid substitutions, shortenings, and sequence deletions, are further provided. This disclosure further provides the use of the enzymes described herein in, for example, test strips, BHB sensors, devices, and systems for detecting and measuring BHB concentration.

Description

Technical Field

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 580,620, filed on September 5, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy was created on August 26, 2024, is named 081906-1457969-253310PC_SL.xml, and has a size of 61,977 bytes.

[0003] The present disclosure relates to enzymes capable of oxidizing beta-hydroxybutyrate (BHB) and methods thereof. In some aspects, the present disclosure relates to use of the enzymes described herein in various applications, for example test strips and additional detection platforms, BHB sensors, and systems for detecting and measuring BHB concentration. Background Art

[0004] Ketogenic diets are an effective way to promote weight loss and wellness. Ketogenic diets minimize carbohydrate intake, while often concurrently increasing the consumption of fat and / or protein. This type of diet causes the body to burn fat and results in the production of ketone bodies (ketones) as the body breaks down fat. For optimized dietary management, it is important that subjects undergoing a ketogenic diet can monitor their ketone levels. The present disclosure provides a solution to this industrial and medical need.

[0005] Beta-hydroxybutyrate (BHB) is the conjugate base of beta-hydroxybutyrate. BHB is synthesized through fatty acid metabolism, and high levels of BHB indicate that the body is using fat as its primary fuel source. Therefore, it is necessary to accurately and reliably measure BHB levels in a subject. This can be achieved by invasive, minimally invasive, or non-invasive mechanisms. This disclosure addresses this need by describing novel enzymes for assisting in the measurement of BHB. Useful systems, devices, and related methods are further provided herein. [Overview of the project]

[0006] This disclosure relates to enzymes having oxidase activity. In one embodiment, the enzymes described herein are engineered and / or modified from natural enzymes. In the embodiments described herein, the enzymes can oxidize beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate. In some embodiments, beta-hydroxybutyrate (BHB) is (R)-beta-hydroxybutyrate. In some embodiments, beta-hydroxybutyrate (BHB) is (S)-beta-hydroxybutyrate. In some embodiments, beta-hydroxybutyrate (BHB) is a mixture of (R)-beta-hydroxybutyrate and (S)-beta-hydroxybutyrate.

[0007] In some embodiments, the enzyme, for example, the modified enzyme, contains (e.g., contains, essentially consists of, or comprises) an amino acid sequence derived from an oxidase of the EC 1.1.3.6 family. In some embodiments, the oxidase from the EC 1.1.3.6 family is a cholesterol oxidase.

[0008] In some embodiments, the enzyme is non-natural and / or modified and includes one or more amino acid substitutions, deletions, or shortenings compared to the natural enzyme (e.g., includes, essentially consists of, or comprises).

[0009] In one embodiment, the enzyme described herein comprises modified oxidase 8 derived from the genus Scytonema sp. In another embodiment, the enzyme is a modified version of SEQ ID NO: 8, comprising one or more amino acid substitutions, deletions, or shortenings. In one embodiment, SEQ ID NO: 8 is optimized to yield improved BHB detection activity.

[0010] In some embodiments, the enzyme comprises an amino acid sequence containing one or more mutations corresponding to N137G, Y235Q and / or A455Y of SEQ ID NO: 8 (e.g., includes, essentially comprises, or consists of). In other embodiments, the enzyme for use herein comprises SEQ ID NOs: 1-43. In some aspects, the enzyme for use herein comprises either SEQ ID NO: 24 or SEQ ID NOs: 40-43, a fragment thereof, or a modified protein therein (e.g., includes, essentially comprises, or consists of).

[0011] In some embodiments, the enzyme contains an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to any of SEQ ID NOs: 1-43 (e.g., contains, essentially consists of, or comprises). In some embodiments, the enzyme contains an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to any of SEQ ID NOs: 24 or any of SEQ ID NOs: 40-43, a fragment thereof, or a modified protein thereof (e.g., contains, essentially consists of, or comprises).

[0012] In further embodiments, the enzyme comprises any of the amino acid sequences of SEQ ID NOs: 1-43 (e.g., comprises, essentially comprises, or consists of), and the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, ten, or fifteen or more amino acid substitutions. In some embodiments, the enzyme comprises any of the amino acid sequences of SEQ ID NOs: 24 or 40-43, and the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, ten, or fifteen or more amino acid substitutions (e.g., comprises, essentially comprises, or consists of). In embodiments, the amino acid substitutions are conservative substitutions. In embodiments, substitutions are made only to portions of the protein involved in binding and / or propagation activity.

[0013] In one embodiment, the Disclosure provides a protein containing (e.g., containing, essentially consisting of, or comprising) an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to any of SEQ ID NOs: 1 to 43. In some embodiments, the Disclosure provides an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to any of SEQ ID NOs: 24 or any of SEQ ID NOs: 40 to 43, a fragment or modified protein therein.

[0014] In further embodiments, the disclosure provides a protein comprising (e.g., comprising, essentially comprising, or consisting of) any amino acid sequence of SEQ ID NOs: 1-43, wherein the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, ten, or fifteen or more amino acid substitutions (e.g., comprising, essentially comprising, or consisting of). In some embodiments, the disclosure provides a protein having the amino acid sequence of SEQ ID NOs: 24 or any of SEQ ID NOs: 40-43, wherein the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, ten, or fifteen or more amino acid substitutions. In embodiments, the amino acid substitutions are conservative substitutions.

[0015] In further embodiments, the modified or altered enzymes described herein exhibit increased BHB activity compared to the corresponding wild-type enzyme. As used herein, wild-type, wildtype, wild type, and WT are all synonymous and refer to naturally occurring enzymes (i.e., in their wild or natural state). As used herein, mutant, mut, and MUT are all synonymous and refer to modified and / or mutant enzymes, e.g., modified and / or mutant enzymes produced by enzyme design and / or enzyme modification. Directed evolution, rational design, and semi-rational design are major in silico-assisted approaches widely used in enzyme modification. In one embodiment, BHB activity is measured by peroxide strips, acetoacetate strips, and / or liquid chromatography-mass spectrometry (LCMS). In one embodiment, the peroxide strip is a Bartovation strip, and the acetoacetate strip is a Bayer Ketostix® strip. In some embodiments, the BHB activity of the modified or altered enzyme is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10,000, 100,000, 100,000, or more greater than that of the corresponding unmodified wild type, as measured by LC-MS.

[0016] In another embodiment, SEQ ID NOs: 24 or 40-43 and their derivatives exhibit increased BHB activity compared to SEQ ID NO: 8. In one embodiment, BHB activity is measured by peroxide strip, acetoacetic acid strip, or liquid chromatography-mass spectrometry (LCMS). In one embodiment, the peroxide strip is a Bartovation strip, and the acetoacetic acid strip is a Bayer Ketostix® strip. In some embodiments, the BHB activity of sequence numbers 24 or 40-43 and their derivatives, as measured by LC-MS, is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10,000, 100,000, 100,000 or more greater than that of sequence number 8.

[0017] In another embodiment, SEQ ID NO: 24 shows increased BHB activity compared to SEQ ID NO: 8. In one embodiment, BHB activity is measured by a peroxide strip, an acetoacetic acid strip, or liquid chromatography-mass spectrometry (LCMS). In one embodiment, the peroxide strip is a Bartovation strip, and the acetoacetic acid strip is a Bayer Ketostix® strip. In some embodiments, the BHB activity of SEQ ID NO: 24, when measured by LC-MS, is approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 90x, 1000x, 10,000x, 100,000x, or more than 100,000x.

[0018] In embodiments, the enzymes described herein are non-natural, modified, isolated, and / or purified.

[0019] This disclosure further provides a sensor for non-wearable sensing systems that can detect and / or measure BHB concentration and includes utilizing the enzyme described herein. An exemplary format of the non-wearable sensing system is a test strip for detecting and / or measuring BHB concentration. The test strip may be for single use or multiple use.

[0020] In some embodiments, the test strip comprises a substrate layer and one or more sensing reagents applied on at least a portion of the substrate layer, wherein at least one of the sensing reagents comprises an enzyme.

[0021] In some embodiments, at least one of the sensing reagents further comprises a cofactor, a mediator, and / or another adjuvant or excipient. In some embodiments, the cofactor comprises flavin adenine dinucleotide (FAD). In some embodiments, the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complex, and / or organometallic ruthenium complex.

[0022] In some embodiments, the material of the substrate layer includes one or more composite materials, fibrous materials, woven fabrics, nonwoven fabrics, polymers, adhesives, films, gels, PTFE, and / or silicones.

[0023] A sensor, for example a non-wearable sensor, can be used to detect or measure BHB in any body fluid. The fluid can be obtained by non-invasive, minimally invasive, or invasive means, for example, finger puncture, blood collection, or spinal puncture as examples of invasive means, or collection of sweat or saliva as examples of non-invasive or minimally invasive means. Body fluid refers to all body fluids including but not limited to whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cyst contents and / or ascites.

[0024] The present disclosure further provides a sensor capable of detecting and / or measuring BHB concentration for continuous, continual, or on-demand detection or measurement, the sensor comprising utilizing the enzyme described herein. Continuous means uninterrupted, unbroken, not intermittent or sporadic, and repeated persistently at short intervals so as to form a substantially unbroken sequence. Continual means repeated regularly and frequently in a steady progression. On-demand means that detection or measurement can be readily obtained when the sensor or a display of sensor data is activated by a user.

[0025] In some embodiments, a BHB sensor capable of sensing, detecting, and / or measuring BHB concentration comprises a sensor including a sensing electrode, a sensing reagent, wherein the sensing reagent composition comprises the enzyme described herein, and a reference electrode. In some embodiments, the sensing reagent is on or adjacent to the sensing electrode. In some embodiments, the sensing reagent is provided to be in contact with the sensing electrode during use.

[0026] In some embodiments, the sensing reagent composition further comprises a cofactor and / or a mediator.

[0027] In some embodiments, the BHB sensor is configured as a wearable sensor that measures a subject's BHB concentration and outputs a data stream in a continuous, sustained, or on-demand manner. In some embodiments, part or all of the sensor is placed in body tissue to measure bodily fluid within the tissue (an in vivo sensor). The body tissue may include any tissue including, but not limited to, epidermis, dermis, subcutaneous tissue, muscle, and the intraperitoneal space. Exemplary embodiments of in vivo sensors include, but are not limited to, continuous BHB sensors in needle-type sensors, microneedle-based sensors, or implantable sensors. In other embodiments, the BHB sensor is configured to measure BHB in a bodily fluid sampled from the body. The sample may be obtained by non-invasive, minimally invasive, or invasive means. Bodily fluid includes, but is not limited to, whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, chyle, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cyst contents, and / or ascites. Bodily fluid may be either from direct contact within body tissue inside the body, or from bodily fluid collected non-invasively or invasively from the body and detected "on the body".

[0028] The present disclosure further relates to systems for detecting and measuring BHB concentration by utilizing the enzymes described herein.

[0029] In some embodiments, a system for detecting and measuring BHB concentration includes a BHB sensor and a device connected to the BHB sensor, wherein the BHB sensor is configured to measure the BHB concentration of interest continuously, on a continuous or on-demand basis and output a data stream, and the sensor can use any method of BHB measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarization measurement, calorimetry, iontophoresis, radiometric, immunochemistry, etc., wherein the device includes a processor configured to process the data stream from the BHB sensor and an interface configured to display and / or communicate the measured BHB concentration value.

[0030] This disclosure further relates to a method for detecting and measuring BHB concentration using the enzyme described herein.

[0031] In some embodiments, a method for detecting and measuring BHB concentration includes obtaining body fluid from a subject, providing the body fluid to a BHB sensor, determining a single-time point in time BHB concentration in the body fluid, and displaying and / or communicating the single-time point in time BHB concentration on an interface.

[0032] In other embodiments, the enzymes, devices, or systems described herein are used in methods to improve health and wellness in subjects requiring improvement. In other embodiments, the enzymes, devices, or systems described herein are used in methods for weight loss. In other embodiments, the enzymes, devices, or systems described herein are used to improve mental and / or metabolic health. In yet another embodiment, the enzymes, devices, or systems described herein are used in methods for controlling or monitoring carbohydrate intake. In other embodiments, the enzymes, devices, or systems described herein are used in methods for monitoring ketone body levels to improve cognitive function; for treating various neurological disorders, such as epilepsy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), traumatic brain injury, Alzheimer's disease, and dementia; for treating mental health, mental illness, psychiatric problems, and mental disorders, such as depression, bipolar disorder, and schizophrenia; for improving cardiac metabolism; for improving the response to immunotherapy, chemotherapy, and radiotherapy to cancer treatment; for regulating inflammatory pathways and immune function; for treating obesity and diabetes; for quantifying and monitoring cholesterol; and for monitoring and detecting alcoholic or diabetic ketoacidosis.

[0033] In other embodiments, the enzymes, devices, or systems described herein are used in combination with other weight loss or wellness methods or therapies. In other embodiments, the enzymes, devices, or systems described herein are used in combination with foods or supplements containing low amounts of carbohydrates or sugar alcohols in the formulation.

[0034] In some embodiments, the food or supplement comprises a ketone supplement or additive. In other embodiments, the food or supplement comprises an exogenous ketone and / or ketone-producing supplement. In some embodiments, the exogenous ketone and / or ketone-producing supplement comprises ketone bodies and / or ketone body precursors. In some embodiments, the ketone bodies and / or ketone body precursors comprise one or more of acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium-chain triglycerides (MCTs) containing fatty acids with hydrocarbon side chains of 6 to 12 carbon atoms. In some embodiments, the ketone bodies and / or ketone body precursors are in the form of salts and / or esters. In some embodiments, the MCTs comprise one or more of caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12).

[0035] In some embodiments, the devices, systems, or methods described herein are prescribed by a medical professional to subjects requiring prescription. In other embodiments, the patients requiring prescription are obese or have an underlying medical condition, such as heart disease. [Brief explanation of the drawing]

[0036] [Figure 1] This shows an initial screening of a sequence similarity network (SSN) composed of 4000 bacterial oxidases.

[0037] [Figure 2] This paper compares oxidase 8 (SEQ ID NO: 8) with modified cholesterol oxidase (ShCOb) derived from Streptomyces hygrospinosus, using an Alphafold model.

[0038] [Figure 3]This shows a visual representation of the design of the modified enzyme (8_MUT) predicted by Alphafold based on wild-type oxidase 8 (8_WT).

[0039] [Figure 4] This report shows the detection results for hydrogen peroxide (H2O2; peroxide) produced by a modified enzyme containing the amino acid sequence of SEQ ID NO: 24 (oxidase 8_mut) and wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8). A commercially available Bartovation hydrogen peroxide strip was used.

[0040] [Figure 5] The results of detecting acetacetate produced by a modified enzyme containing the amino acid sequence of SEQ ID NO: 24 (oxidase 8_mut) and wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) are shown. Commercially available Bayer Ketostix® urine test strips were used.

[0041] [Figure 6] The HPLC-MS profile of the reaction mixture of modified enzymes containing the amino acid sequence of SEQ ID NO: 24 (oxidase 8_mut) is shown.

[0042] [Figure 7] This shows a comparison of the HPLC-MS profile of the oxidase 8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS profile of commercially available synthetic acetacetate (acetacetate).

[0043] [Figure 8] This shows a comparison of the HPLC-MS profiles of the oxidase-8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture and the wild-type oxidase-8 (oxidase-8_WT, SEQ ID NO: 8) (8_WT) reaction mixture.

[0044] [Figure 9]This shows a comparison of the HPLC-MS profile of the oxidase 8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS profile of the substrate control. [Modes for carrying out the invention]

[0045] This disclosure provides novel proteins and enzymes capable of exhibiting BHB activity. In some embodiments, the novel proteins or enzymes can be used in conjunction with the methods, systems, devices, and kits described herein.

[0046] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, namely hydrogen, a carboxyl group, an amino group, and a carbon atom bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as natural amino acids.

[0047] Various methods exist in the literature that enable the site-specific incorporation of non-natural amino acid derivatives or analogs into polypeptide chains; see, for example, International Publication No. 02 / 086075, whose entire content is incorporated by reference.

[0048] Amino acids may be referred herein by either a commonly known three-letter code or a single-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred by a commonly accepted single-letter code.

[0049] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural and non-natural amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, where the amino acid residues are linked by covalent peptide bonds.

[0050] In the context of two or more nucleic acid or polypeptide sequences (e.g., two proteases and the encoding polynucleotide of this disclosure), the terms “identical” or “identity” percentage mean two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, which is measured using one of the following sequence comparison algorithms or by visual inspection.

[0051] For sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0052] In the context of this application, a sequence that is "80% or more identical to the reference sequence" is a sequence that, over its entire length, has sequence identity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more with respect to the full-length reference sequence. A protein consisting of an amino acid sequence that is "80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more" with respect to the reference sequence may include mutations, such as deletions, insertions, and / or substitutions. In the case of substitution, a protein consisting of an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more identical to the reference sequence may correspond to a homologous sequence originating from a different species than the reference sequence.

[0053] In the context of this application, the “percentage of identity” can be calculated using global pairwise alignment (i.e., two sequences are compared over their entire lengths). Methods for comparing the identity of two or more sequences are well known in the art. For example, the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J.Mol.Biol.48:443-453) can be used to find the optimal alignment (including gaps) of two sequences considering their entire lengths using the “Needle” program. The Needle program is available, for example, on the ebi.ac.uk World Wide Web site and is further described in publications (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). According to the present invention, 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 suitable for determining sequence identity percentage and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0054] In this specification, “at least one” means one or more of the specified subjects, for example, one, two, three, four, five, or six or more of the specified subjects. For example, “at least one amino acid substitution” in this specification means one, two, three, four, five, or six or more amino acid substitutions.

[0055] An "amino acid substitution" may be conservative or non-conservative. In some embodiments, the substitution is a conservative substitution, in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties. In some embodiments, a conservative substitution results in the same or similar functional properties.

[0056] In one embodiment, the conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure, Vol. 5," Natl. Biomedical Research, whose entire contents are incorporated by reference. For example, in a certain embodiment, amino acids belonging to one of the following groups can be exchanged with each other, 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). In some embodiments, the conservative amino acid substitution may be selected from T->A, G->A, A->I, T->V, A->M, T->I, A->V, T->G, and / or T->S.

[0057] In further embodiments, conservative amino acid substitutions may include substitutions with other amino acids of the same class, such as substitutions with (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. Other conservative amino acid substitutions can also be made such as (1) aromatic: Phe, Tyr, His; (2) proton donors: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptors: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, whose entire contents are incorporated by reference).

[0058] In another embodiment, conservative substitutions may be performed according to Table 1. Methods for predicting resistance to protein modification can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the entire contents of which are incorporated by reference. [Table 1]

[0059] In another embodiment, conservative substitutions may be those shown under the heading “Conservative Substitutions” in Table 2. If such substitutions result in changes in biological activity, more substantial changes, referred to as “Exemplary Substitutions” in Table 2, may be introduced, and the products may be screened as necessary. [Table 2]

[0060] enzyme The enzymes or proteins described herein may be modified, isolated, or purified. In some embodiments, the enzymes or proteins described herein may be non-natural. In other embodiments, the enzymes or proteins described herein may be modified by amino acid substitution, deletion, and / or shortening. In other embodiments, the enzymes described herein are optimized for the detection or analysis of BHB. In other embodiments, the enzymes described herein are designed for use with the systems, devices, kits, and methods described herein.

[0061] In some embodiments, the disclosure relates to an enzyme having oxidase activity capable of oxidizing beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate. In other embodiments, the enzyme comprises, consists of, or essentially comprises an amino acid sequence comprising one or more mutations corresponding to N137G, Y235Q and / or A455Y of SEQ ID NO: 8. In some embodiments, the enzyme described herein comprises only the combination of N137G, Y235Q and / or A455Y mutations of SEQ ID NO: 8. In other embodiments, the enzyme described herein comprises, consists of, or essentially comprises an N137G, Y235Q and / or A455Y mutation combination of SEQ ID NO: 8 linked to an N-terminal shortening at amino acid positions 2-32 of SEQ ID NO: 8. In other embodiments, the enzymes described herein include, essentially consist of, or consist of, a combination of the N137G, Y235Q, and / or A455Y mutations of SEQ ID NO: 8, linked to an N-terminal shortening of an amino acid position selected from the group consisting of amino acid positions 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. In other embodiments, the enzymes described herein include, essentially consist of, or consist of, a combination of the N137G, Y235Q, and / or A455Y mutations of SEQ ID NO: 8, linked to an N-terminal shortening 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.

[0062] In some embodiments, SEQ ID NO: 24 shows increased BHB activity compared to SEQ ID NO: 8 under similar test conditions. In other embodiments, SEQ ID NO: 24 or SEQ ID NOs: 40-43 show increased BHB activity compared to SEQ ID NO: 8 under the same test conditions or under the same test conditions. In other embodiments, SEQ ID NO: 24 or SEQ ID NOs: 40-43 show increased BHB activity compared to SEQ ID NO: 8 under similar test conditions or under similar test conditions.

[0063] In some embodiments, the amino acid sequence is derived from an oxidase of the EC 1.1.3.6 family. In some embodiments, the oxidase of the EC 1.1.3.6 family is a cholesterol oxidase. In some embodiments, the amino acid sequence is derived from a cholesterol oxidase originating from the genus Scytonema, Rhodococcus erythropolis (ReCO), Brevibacterium sterolicum (BsCO), or Streptomyces hygrospinosus (ShCO).

[0064] In some embodiments, the amino acid sequences described herein include shortenings at the N-terminus and / or C-terminus. In some embodiments, the shortenings have lengths of 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 26 or more amino acids, 27 or more amino acids, 28 or more amino acids, 29 or more amino acids, 30 or more amino acids, or 31 or more amino acids. In other embodiments, the shortenings have lengths of 3 or fewer amino acids, 5 or fewer amino acids, 10 or fewer amino acids, 15 or fewer amino acids, 20 or fewer amino acids, 25 or fewer amino acids, 26 or fewer amino acids, 27 or fewer amino acids, 28 or fewer amino acids, 29 or fewer amino acids, 30 or fewer amino acids, or 31 or fewer amino acids. In other embodiments, the abbreviations have lengths of 5–50 amino acids, 10–50 amino acids, 15–50 amino acids, 20–at least 50 amino acids, 10–40 amino acids, 10–35 amino acids, 15–35 amino acids, 20–35 amino acids, 25–32 amino acids, 28–32 amino acids, 2–10 amino acids, or 2–5 amino acids from the N-terminus and / or C-terminus of the enzyme or protein described herein. In another embodiment, the abbreviations described herein are present in one or more of SEQ ID NOs: 1–43. In a certain embodiment, the abbreviations described herein are for SEQ ID NOs: 8, SEQ ID NOs: 24, or SEQ ID NOs: 40–43.

[0065] In some embodiments, the enzyme contains an amino acid sequence that is 80% or more identical to any of SEQ ID NOs: 1-43, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more. In some embodiments, the enzyme contains an amino acid sequence that is 80% or more identical to SEQ ID NOs: 24 or any of SEQ ID NOs: 40-43, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more. In embodiments, the enzymes described herein include N137G, Y235Q, and / or A455Y mutations relative to SEQ ID NO: 8, and further include amino acid sequences that are identical by 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more to any of SEQ ID NOs: 8, 24, or 40-43.

[0066] In a further embodiment, the enzyme comprises any amino acid sequence of SEQ ID NOs: 1 to 43, and the amino acid sequence comprises one, two, three, four, five, six, seven, eight, nine, or ten or more amino acid substitutions. In another embodiment, the enzyme comprises any amino acid sequence of SEQ ID NOs: 1 to 43, and the amino acid sequence comprises one or fewer, two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, or ten or fewer amino acid substitutions.

[0067] In further embodiments, the enzyme comprises the amino acid sequence of either SEQ ID NO: 24 or SEQ ID NOs: 40-43, wherein the amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions. In embodiments, the amino acid substitutions are conservative substitutions. In further embodiments, the enzyme comprises the amino acid sequence of either SEQ ID NO: 24 or SEQ ID NOs: 40-43, wherein the amino acid sequence comprises 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, or 10 or fewer amino acid substitutions.

[0068] BHB sensor and method for detecting and measuring BHB concentration Generally, a biosensor is a device that measures a biological or chemical reaction by generating a signal proportional to the concentration of an analyte during the reaction. According to the IUPAC definition, a biosensor is a device that uses a specific biochemical reaction mediated by an isolated enzyme, immune system, tissue, organelle, or whole cell to detect a chemical compound. For an overview of biosensors, see, for example, Bhalla et al., Introduction to biosensors, June 30, 2016, Essays Biochem. 60(1):1-8, which is incorporated herein in its entirety by reference.

[0069] In other embodiments, the Disclosure provides a sensor, e.g., a non-wearable sensor, that can detect and / or measure BHB concentration and utilize the enzymes described herein. Such a sensor, e.g., a non-wearable sensor, may be in the form of a test strip or a one-touch fingertip sweat sensor. In other embodiments, the enzymes include one or more of the SEQ ID NOs: 1-43, fragments thereof, or modified enzymes of SEQ ID NOs: 1-43 described herein. A detailed description of suitable applications of the enzymes in various non-wearable sensors can be found in Yin et al. (International Publication No. 2022 / 170361), the entirety of which is incorporated herein by reference.

[0070] Non-wearable sensors can be used to detect or measure BHB in body fluids, which include one or more of the following: whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites.

[0071] In some embodiments, the test strip comprises a substrate layer and one or more sensing reagents applied on at least a portion of the substrate layer, wherein at least one of the sensing reagents comprises an enzyme.

[0072] In some embodiments, at least one of the sensing reagents further comprises a cofactor, a mediator, and / or another adjuvant or excipient. In some embodiments, the cofactor comprises flavin adenine dinucleotide (FAD). In some embodiments, the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complex, and / or organometallic ruthenium complex.

[0073] In other embodiments, the test strip further comprises one or more tetrazolium salts. In some embodiments, one or more of 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).

[0074] The material of the substrate layer may include one or more of composite materials, fibrous materials, woven fabrics, nonwoven fabrics, polymers, adhesives, films, gels, PTFE, and / or silicones. The Disclosure further provides a BHB sensor capable of sensing, detecting, and / or measuring BHB concentration by utilizing the enzymes described herein in a continuous, ongoing, or on-demand manner. In other embodiments, the enzymes include one or more of SEQ ID NOs: 1-43, fragments thereof, or modified enzymes of SEQ ID NOs: 1-43 as described herein.

[0075] In some embodiments, a BHB sensor capable of sensing, detecting, and / or measuring BHB concentration comprises a sensor containing a sensing reagent (wherein the sensing reagent composition includes a modified enzyme) and a reference electrode.

[0076] In some embodiments, the sensing reagent composition further comprises one or more cofactors, mediators, adjuvants, or excipients.

[0077] In some embodiments, the wearable BHB sensor is configured to measure the concentration of BHB in question continuously, continuously, or on demand and output a data stream. In some embodiments, the continuous BHB sensor is an implantable or non-implantable device. In some embodiments, the wearable BHB sensor is a needle-type sensor or a microneedle-based sensor. In some embodiments, the sensor can use any method of BHB measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetry, iontophoresis, radiometric, and immunochemical methods. In some embodiments, the wearable BHB sensor is configured to measure the concentration of BHB in body fluids. In other embodiments, the body fluids include one or more of whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites.

[0078] This disclosure further provides a system for detecting and measuring BHB concentration by utilizing the enzymes described herein. In other embodiments, the enzymes include one or more of the SEQ ID NOs: 1-43, fragments thereof, or modified enzymes of SEQ ID NOs: 1-43 described herein.

[0079] In some embodiments, a system for detecting and measuring BHB concentration includes a BHB sensor and a device connected to the BHB sensor, wherein the BHB sensor is configured to measure the BHB concentration of interest and output a data stream, and the device includes a processor configured to process the data stream from the BHB sensor and an interface configured to display and / or communicate the measured BHB concentration value.

[0080] In some embodiments, BHB sensors or BHB sensing systems are fabricated by integrating modified enzymes into various sensing platforms and / or devices. Platforms and / or devices that may employ modified enzymes include, but are not limited to, pH change sensors, e.g., those described in Chodavarapu et al., U.S. Patent No. 7,794,584; electrochemical sensors, e.g., those described in Simpson et al., U.S. Patent No. 7,081,195, Lebel et al., U.S. Patent No. 6,915,147, and Jina et al., U.S. Patent No. 2010 / 0049021; optical sensors, e.g., those described in Petrich et al., U.S. Patent No. 10,724,943; implantable sensor platforms, e.g., those described in Jain, European Patent No. 2079358; chip-shaped blood analyzer devices, e.g., those described in Ogawa et al., U.S. Patent No. 7,582,259; and monitoring systems with remotely connected control terminals, e.g., those described in Karan et al., U.S. Patent No. 2012 / 0245447. The contents of each of these patents and applications are incorporated herein by reference in their entirety. In some embodiments of this disclosure, the BHB sensors, BHB sensing systems, BHB devices, and applicable technologies are non-invasive or minimally invasive, wearable biosensing and / or chemical monitoring sensors, systems, devices, and technologies.

[0081] In some embodiments of this disclosure, the electrochemical biosensor or chemical sensor used to qualitatively and / or quantitatively measure BHB is a fingertip sensor.

[0082] In some embodiments, the BHB sensor or BHB sensing system is a one-touch fingertip sweat sensor and a personalized data processing method, system, or device, for example, described in International Publication 2022 / 070554, filed on 7 February 2022 and published on 11 August 2022 as International Publication 2022 / 170361, or described in U.S. Patent No. 18 / 264,755, filed on 8 August 2023 and published on 15 February 2024 as U.S. Patent 2024 / 0049994(A1). The contents of each of these applications are incorporated herein by reference in their entirety.

[0083] In some embodiments, BHB sensors or BHB sensing systems utilize reverse iontophoresis, a non-invasive or minimally invasive process for the extraction of biomarkers. Reverse iontophoresis is a technique in which a small electric current is applied to the skin, with the effect of extracting polar and nonpolar molecules to the anode or cathode, where they can be sensed electrochemically. For a detailed description of applicable iontophoresis techniques, systems, and devices, see, for example, U.S. Patent No. 10,722,160 issued on 28 July 2020, U.S. Patent Application Publication No. 2021 / 0076988 published on 18 March 2021, and U.S. Patent Application Publication No. 2015 / 063836, filed on 31 May 2017 and published as International Publication No. 2016 / 090189 on 9 June 2016. The contents of each of these patents and applications are incorporated herein by reference in their entirety.

[0084] In some embodiments, the electrochemical biosensor or chemical sensor is a wearable skin electrochemical sensor device for detecting BHB in sweat. For a detailed description of applicable skin electrochemical sensor devices, see, for example, U.S. Patent No. 9,820,692 and U.S. Patent No. 11,185,286. The contents of each of these patents are incorporated herein by reference in their entirety.

[0085] This disclosure further provides a method for detecting and measuring BHB concentration by utilizing the enzymes described herein. In other embodiments, the enzymes include one or more of the SEQ ID NOs: 1-43, fragments thereof, or modified enzymes of SEQ ID NOs: 1-43 described herein.

[0086] In some embodiments, a method for detecting and measuring BHB concentration includes obtaining body fluid from a subject, providing the body fluid to a BHB sensor, determining a single-time point in time BHB concentration in the body fluid, and displaying and / or communicating the single-time point in time BHB concentration on an interface.

[0087] Test kit This disclosure further relates to test kits suitable for use in, for example, automated analyzers, systems, and devices.

[0088] In some embodiments, the test kit described herein comprises reagent A and reagent B. In some embodiments, the test kit is activated by mixing reagent A with reagent B.

[0089] In some embodiments, reagent A comprises the enzyme described herein. In some embodiments, the enzyme described herein is dissolved in a buffer. In some embodiments, reagent A comprises NaCl and / or KCl. In some embodiments, the concentration of NaCl and / or KCl is 0.1 to 5 M. In some embodiments, reagent A comprises citric acid, acetic acid, KH2PO4, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphoric acid, and / or tris(hydroxymethyl)aminomethane. In some embodiments, reagent A has a pH of 3 to 11, optionally 4 to 10, optionally 5 to 9, and optionally 6 to 8.

[0090] In some embodiments, reagent B comprises a cofactor, mediator, excipient, adjuvant, or carrier. In one embodiment, the enzyme-containing composition is soluble in water. In some embodiments, the cofactor comprises one or more of flavin adenine dinucleotide (FAD), semiquinone-type flavin adenine dinucleotide (FADH), and / or quinone-type flavin adenine dinucleotide (FADH2). In some embodiments, the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complex, and / or organometallic ruthenium complex. In some embodiments, reagent B comprises NaCl and / or KCl. In some embodiments, the concentration of NaCl and / or KCl is 0.1 to 5 M. In some embodiments, reagent B comprises citric acid, acetic acid, KH2PO4, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphoric acid, and / or tris(hydroxymethyl)aminomethane. In other embodiments, reagent B has a pH of 3 to 11, optionally 4 to 10, optionally 5 to 9, and optionally 6 to 8. In some embodiments, reagent B comprises one or more tetrazolium salts. In some embodiments, one or more of 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).

[0091] Pharmaceutical composition In another embodiment, the present invention relates to the use of enzymes as components of a composition.

[0092] In some embodiments, the composition comprises one or more active ingredients. In some embodiments, the active ingredients further comprise one or more other enzymes, such as glucose oxidase, glucosyltransferase, fructosyltransferase, catalase, amylase, lactase, lipase, and / or protease.

[0093] In some embodiments, 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 art can be found, for example, in Margolin et al. (U.S. Patent No. 7,718,169), the contents of which are incorporated herein by reference.

[0094] In some embodiments, one or more excipients include microcrystalline cellulose, maltrin, crospovidone, colloidal silicon dioxide, magnesium stearate, talc, sucrose, trehalose, lactose, sorbitol, lactitol, mannitol, inositol, sodium and potassium salts (e.g., acetate, phosphate, citrate, and borate), glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, and methoxypolyethylene glycol. Gelatin, hydroxypropyl-β-cyclodextrin, polylysine, polyarginine, amino acids (e.g., glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline), carbohydrates (e.g., glucose, fructose, galactose, mannose, arabinose, xylose, ribose, lactose, trehalose, maltose, sucrose, maltodextrin, dextran, starch, glycogen), argitol (e.g., mannitol, xylitol, lactitol, sorbitol) Cyclodextrins (e.g., methylcyclodextrin, hydroxypropyl-β-cyclodextrin, etc.), inorganic molecules (e.g., sodium chloride, potassium chloride, magnesium chloride, sodium and potassium phosphates, boric acid, ammonium carbonate, and ammonium phosphate), organic molecules (e.g., acetates, citrates, ascorbic acid, lactates, glucuronic acid, galacturonic acid), emulsifiers or solubilizers / stabilizers (e.g., acacia, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, The material comprises one or more of the following: oleyl alcohol, poloxamer, polysorbate, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, other sorbitan derivatives, polyoxyl derivatives, waxes, polyoxyethylene derivatives), thickening agents (e.g., agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives), propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol, or salts of these compounds.Further examples of excipients are listed in the Handbook of Pharmaceutical Excipients, jointly published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain.

[0095] In some embodiments, one or more adjuvants include one or more of water-in-oil and oil-in-water emulsions, aluminum salt adjuvants, liposomes, and / or CpG oligodeoxynucleotides adsorbed to aluminum salts.

[0096] In some embodiments, one or more carriers comprise one or more polymers used for encapsulating protein crystals for protein delivery, including controlled-release biological delivery. In some embodiments, the polymers comprise biocompatible and biodegradable polymers, or mixtures thereof. In some embodiments, the enzyme dissolution rate, and therefore the delivery rate, is determined by the specific encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer stability, the geometric shape of the enzyme crystals, and the degree of enzyme crosslinking, if present.

[0097] In some embodiments, one or more active ingredients may be present in the composition in association with a polymer carrier.

[0098] Useful polymer carriers include, for example, polymers used for encapsulating protein crystals for protein delivery, including controlled-release biological delivery. Such polymers include biocompatible and biodegradable polymers, or mixtures thereof. Preferably, the polymer carrier is a biodegradable polymer. The rate of enzyme dissolution, and therefore delivery, is determined by the specific encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer stability, the geometric shape of the enzyme crystal, and the degree of enzyme crosslinking (if present). Another useful carrier is water.

[0099] Ketogenic diet and methods for treating diseases This disclosure further provides a ketogenic diet and a method for treating diseases through the use of a device, system, method, or kit integrated with the enzymes described herein. In one embodiment, the ketogenic diet is used in conjunction with the devices and systems described herein. A representative description of nutritional ketosis and the diseases treatable thereby can be found, for example, in D'Agostino et al. (U.S. Patent Application Publication No. 2020 / 0268701), the entire content of which is incorporated by reference.

[0100] Ketosis The terms “ketone” and / or “ketone body” refer to water-soluble molecules or compounds containing a ketone group derived from fatty acids. As is commonly known in the art, a ketone group contains one or more carbonyl groups -C(=O)-. In the context of this application, “ketone” and / or “ketone body” include, but are not exhaustive, compounds of acetone (2-propanone, dimethyl ketone, or beta-ketopropane), acetoacetic acid (3-oxobutanoic acid, acetonecarboxylic acid, or diacetic acid), acetacetate, beta-hydroxybutyrate (3-hydroxybutyrate), beta-hydroxybutyrate (BHB, 3HB, or 3-hydroxybutyrate), beta-ketopentanoate (3-oxopentanoate, 3-oxovaleric acid, or 3-ketovaleric acid), and beta-hydroxypentanoate (3-hydroxyvalerate, 3-hydroxyvaleric acid, or beta-hydroxyvaleric acid). For a review of ketones, see, for example, L. Laffel, Nov.-Dec. 1999, Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes, Diabetes Metab Rev. 15(6):412-426, which is incorporated in its entirety herein.

[0101] BHB is an anionic small acid metabolite containing a hydroxyl group, the major ketone body distributed in the human brain, and its primary energy source in the absence of glucose. For a review of BHB, see, for example, JC Newman and E. Verdin, August 21, 2017, β-Hydroxybutyrate, Ann Rev Nutr. 37:51-76.

[0102] The term "acetoacetate" refers to the conjugate base of acetoacetic acid and is used interchangeably with the term "3-oxobutanoate."

[0103] In one embodiment, the enzymes, methods, devices, and systems described herein may be used in relation to ketosis, for example, in nutritional or therapeutic ketosis. Nutritional or therapeutic ketosis is a physiological state of elevated blood ketone body levels (typically greater than 0.5 mmol / L) resulting from a ketogenic diet, calorie restriction, therapeutic fasting, and / or supplementation with ketone precursors. Ketone bodies represent alternative energy substrates for both peripheral tissues and the central nervous system. The two most abundant and physiologically important ketone bodies are acetacetate and beta-hydroxybutyrate (BHB), while a third ketone body, acetone, is produced as a byproduct of lung exhalation. The body produces ketone bodies in the range of about 0.3–16 mmol / L during nutritional or therapeutic ketosis. Ketone body metabolism is associated with anticonvulsant effects, enhanced cerebral metabolism, neuroprotection, muscle-sparing properties, and improvements in cognitive and physical abilities. The science of improving the efficiency of cellular metabolism managed through ketone supplementation may have beneficial effects on physical, cognitive, and psychological health, combatant resilience, and long-term health effects on common preventable diseases such as obesity, neurodegenerative diseases, autoimmune diseases, diabetes, and cancer.

[0104] Under normal conditions of a standard diet, the brain relies solely on glucose metabolism to supply its metabolic energy. Although the brain accounts for only 2% of body weight, it accounts for 25% of total glucose consumption. Ketones can replace glucose to supply a large portion (>50%) of the brain's metabolic energy needs during periods of limited glucose availability resulting from starvation / fasting, calorie restriction, or carbohydrate restriction, as in the case of a ketogenic diet. During carbohydrate deficiency, glucose availability decreases, leading to a metabolic shift to fatty acid beta-oxidation and the production of ketone bodies for energy homeostasis.

[0105] Dietary carbohydrates (carbs) include simple sugars, such as table sugar (sucrose), and complex carbohydrates (starch) found in foods like potatoes and pasta. Carbohydrate and sugar consumption has increased dramatically in Western societies over the past two centuries. When sugars and carbohydrates are consumed by humans, the pancreas secretes insulin, a hormone used to convert sugars and carbohydrates into glucose. Glucose is then used by the body as fuel. In most Western diets, glucose is the body's primary fuel source.

[0106] During periods of fasting, extreme exercise, and / or low carbohydrate intake, glucose stores in the body can be rapidly used and rapidly depleted. As glucose stores are depleted and cannot be replenished, the body shifts to an alternative method of generating energy by producing ketones. Ketones can be used by any cell in the body as an alternative fuel to meet the body's energy needs, including those of the brain. For example, during prolonged fasting, blood ketone levels rise to 2 mmol / L, 3 mmol / L, or higher. When blood ketones rise above 0.5 mmol / L, it is conventionally understood and agreed that the heart, brain, and peripheral tissues are using ketones (beta-hydroxybutyrate and acetacetate) as a primary fuel source. This condition is called ketosis or "nutritional ketosis." This is distinguished from diabetic or alcoholic ketoacidosis, which is a runaway accumulation of ketones and associated blood pH drops. Diabetic ketoacidosis is associated with insulin deficiency, as it occurs in people with type 1 diabetes. Ketoacidosis typically results in blood ketone levels above 15 mmol / L, accompanied by metabolic disorders and electrolyte imbalances.

[0107] In ketosis, the body essentially burns fat for fuel. This is achieved because fat stores in the body are utilized to produce water-soluble ketone bodies beta-hydroxybutyrate (BHB) and acetacetate (also known as acetylacetonate). These ketone bodies are then used by the body as its primary energy source.

[0108] The body enters a state of ketosis when it lacks a food source of glucose or sugar and glycogen stores are depleted. This typically occurs during fasting, exercise, and / or the practice of a carbohydrate-restricted ketogenic diet. Once in ketosis, the body begins to break down fat into fatty acids and glycerol, converting fatty acids into acetyl-CoA molecules, which are then ultimately converted into ketone bodies in the liver. In other words, during ketone production metabolism in the liver, the body uses dietary fat and body fat as its primary energy source. As a result, once in ketosis, it is easy to induce a reduction in body fat by reducing dietary fat intake and adjusting carbohydrate intake to be low enough to maintain ketosis.

[0109] In one embodiment, the enzymes, methods, devices, and systems described herein relate to and may assist in methods of weight loss, regulation and / or monitoring of weight loss, and regulation and / or monitoring of carbohydrate intake. In other embodiments, the enzymes, methods, devices, and systems described herein relate to the regulation and / or monitoring of ketosis over periods of hours, days, weeks, months, or years. In some embodiments, ketone concentrations are monitored for the purpose of detecting and preventing ketoacidosis.

[0110] Ketogenic diet and weight loss This disclosure provides a method for treating weight loss in subjects in need by utilizing the enzymes, methods, devices, or systems described herein.

[0111] A ketogenic diet is high in dietary fat, low in carbohydrates, and moderate in protein (approximately 1-2 g / kg). A classic ketogenic diet consists of a strict regimen of 4 parts fat to 1 part protein, containing less than 25-50 grams of carbohydrates per day. The ideal macronutrient ratio for maintaining a ketogenic diet has been suggested to be 65-85% of calories from fat, 10-20% from protein, and 5% from carbohydrates. In embodiments, this disclosure provides a method for assisting a subject in monitoring and / or calibrating carbohydrate intake of approximately 10-30 grams, 15-40 grams, 20-50 grams, 30-60 grams, or 40-80 grams per day by utilizing the enzymes, systems, and devices described herein. This dietary protocol can be used in conjunction with the devices and systems described herein.

[0112] The advantage of pursuing weight loss through a ketogenic diet is that it can lead to a reduction in fat storage while maintaining and protecting muscle mass. Several studies suggest that the muscle-conserving properties of the ketogenic diet lead to improved physical performance. Athletes who maintain nutritional ketosis can maintain lower insulin levels, better utilize fatty acids and ketones as fuel, effectively conserve blood glucose, and optimize and prolong physical and mental performance. This state is called "keto adaptation." Ketogenic adaptation occurs when the body adapts to ketosis by accumulating the fat-burning enzymes it needs, hormone levels change to adapt to ketosis, glycogen stored in muscles and the liver decreases, and the body retains less water.

[0113] Individuals on a standard US diet can expect to reach peak ketone formation while exercising at 60-65% of their maximum oxygen consumption (VO2max), with subsequent depletion of glycogen stores due to the high level of exercise. Keto-adapted individuals can draw proportionally more substrates from fat and ketones (to conserve glycogen), shifting the peak to much higher VO2 levels and thus allowing them to sustain effort for longer periods. Transitioning to a keto-adapted state (blood ketones >0.5 mmol / L) typically requires 1-2 weeks of severe carbohydrate restriction (<25g / day) and moderate protein restriction (1g / kg / day) with the remaining macronutrients derived from fat. Sustained physiological reductions in glucose and insulin are necessary for sustained hepatic ketone formation, which is extremely difficult for most people.

[0114] Vlahakos (U.S. Patent No. 6,613,356), whose entire content is incorporated by reference, provides a weight-loss composition using n-butyrate ions from potassium butyrate or related compounds. Butyrate stimulates receptors in the stomach that indicate the stomach is full and food is lingering in the stomach. Therefore, consuming a butyrate precursor before eating reduces food consumption. Studies have shown that the composition improves patients' ability to withstand strenuous exercise, improves hypercholesterolemia and hypertriglyceridemia, and reduces fatigue.

[0115] Another advantage of pursuing weight loss through a ketogenic diet is that being in a state of ketosis reduces hunger. In fact, hunger is often cited as a major obstacle to being unable to stick to a traditional calorie-restricted diet.

[0116] The presence of blood ketones can be easily measured using one of the many ketone test strips or devices available on the commercial market, so those who wish to pursue a state of ketosis can easily measure their progress. Just as those on a conventional diet can get positive feedback by measuring their weight and measuring weight loss, those pursuing a state of ketosis can also be encouraged by measuring their blood ketone levels. However, when transitioning to ketosis, it may take several days to more than two weeks for a measurable increase in blood ketone levels to appear through a urine test.

[0117] A method for implementing a ketogenic diet and monitoring ketosis using a BHB sensor. In some embodiments, the ketogenic diet contains about 5–10% carbohydrates, about 55–85% fat, and about 10–40% protein. In some embodiments, the ketogenic diet contains 15–50 g of carbohydrates, 60–400 g of fat, and 40–100 g of protein. In some embodiments, the ketogenic diet provides 1500–3000 kcal per day. In some embodiments, the ketogenic diet includes intermittent or prolonged fasting. In some embodiments, these parameters can be adjusted and / or monitored via the methods, systems, and devices described herein.

[0118] In some embodiments, the ketogenic diet is consumed daily for a period of 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, or 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks or more, 11 weeks or more, or 12 weeks or more.

[0119] In some embodiments, ketosis monitoring is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after starting the ketogenic diet.

[0120] In some embodiments, ketosis is monitored by detecting BHB concentration using a beta-hydroxybutyrate (BHB) sensor. In some embodiments, the BHB concentration is either blood BHB concentration or interstitial fluid (ISF) BHB concentration. In some embodiments, ketosis is monitored at least once daily, at least twice daily, at least three times per day, at least four times per day, at least five times per day, at least once every two days, at least once every three days, at least once every four days, at least once every five days, at least once every six days, or at least once every seven days, or at least once every two weeks, at least once every three weeks, or at least once every four weeks. In some embodiments, ketosis monitoring is performed at 7:00 a.m., 8:00 a.m., 9:00 a.m., 10:00 a.m., 11:00 a.m., 12:00 p.m., 1:00 p.m., 2:00 p.m., 3:00 p.m., 4:00 p.m., 5:00 p.m., 6:00 p.m., 7:00 p.m., 8:00 p.m., 9:00 p.m., and / or 10:00 p.m. In some embodiments, ketosis monitoring is performed 3 hours before breakfast, 2 hours before breakfast, 1 hour before breakfast, immediately before breakfast, immediately 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, immediately before lunch, immediately 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, immediately before dinner, immediately after dinner, 1 hour after dinner, 2 hours after dinner, or 3 hours after dinner.

[0121] The effects of ketosis on cognitive and physical abilities Performance studies have shown improved motor function, endurance, and cognitive function with ketone supplementation. Rats given ketone supplementation have achieved greater cardiopulmonary and neurological resilience under extreme oxidative stress (hyperoxygen) conditions. Many people on a ketogenic diet report greater mental clarity, improved multitasking ability, and a more favorable, balanced mood.

[0122] Other benefits of the ketogenic diet include anti-aging and mood-stabilizing effects. Other studies have demonstrated superior performance in terms of endurance, oxygen consumption, heart rate, blood lactate levels, and power output when blood ketone levels are elevated.

[0123] For this purpose, and in one embodiment, the enzymes, methods, devices, and systems described herein may relate to and assist in methods for increasing endurance, improving cognitive function, and improving mood.

[0124] Examples The following embodiments illustrate specific aspects of the Disclosure and are not intended to limit the Disclosure in any way.

[0125] Example 1: Enzyme Design (R)-Beta-hydroxybutyrate (R-BHB) can be converted to 3-oxobutanoate via oxidation of a secondary alcohol, as shown below. [ka]

[0126] There are naturally occurring alcohol oxidases that can promote the oxidation of secondary alcohols to ketones. For example, cholesterol oxidase, belonging to the oxidoreductase family EC 1.1.3.6, has the ability to convert cholesterol to cholesta-4-en-3-one. The reaction to convert cholesterol to cholesta-4-en-3-one is shown below. This ability makes cholesterol oxidase a potential candidate for developing optimized modified enzymes capable of converting (R)-beta-hydroxybutyrate to 3-oxobutanoate. [ka]

[0127] Any suitable assay for determining the presence and / or amount of hydrogen peroxide (H2O2) produced by the enzymatic reaction can be used in accordance with this disclosure. For example, the resulting hydrogen peroxide can be detected using a highly sensitive and stable fluorescent probe. See, for example, Allain et al., Clin Chem 1974, 20:470-475; Amundson et al., J Biochem Biophys Meth 1999, 38:43-52; and dos Santos Ferreira et al., Clin Chim Acta 2015, 446:263-266. The contents of each of these publications are incorporated herein by reference in their entirety.

[0128] To identify suitable candidate oxidases for further modification, a sequence similarity network (SSN) consisting of 4000 bacterial oxidases was screened. The SSN method is described in Atkinson et al., PLoS ONE 2009 Feb., 4(2), e4345, and is incorporated herein by reference. The initial screening results are shown in Figure 1. Thirty-nine amino acid sequences were identified as potential candidates. These amino acid sequences are shown in Table 3.

[0129] The cholesterol oxidase Uniprot ID: A0A1Z4IKQ1 (oxidase 8) from the genus Scytonema sp. was identified by using the Alphafold protein structure database for structural prediction. The Alphafold prediction method is described in detail in Varadi et al., Nucleic Acids Res., Vol. 50, Issue D1, 7 January 2022, pages D439-D444, and Jumper et al., Nature, Vol. 596, pages 583-589 (2021), each of which is incorporated herein by reference. The Alphafold model was then visualized using PyMol (http: / / citebay.com / how-to-cite / pymol / ). Figure 2 shows a comparison of oxidase 8 and modified cholesterol oxidase derived from Streptomyces hygrospinosus (ShCOb, SEQ ID NO: 21, see Heath et al., Chembiochem. 2022 Apr 5; 23(7):e202200075) using the Alphafold model.

[0130] Next, the in silico design of the modified enzyme was analyzed. Mutant N137G was introduced to create space in the enzyme's active site against BHB, and mutants Y235Q and A455Y were introduced to form hydrogen bonds with the carboxylic acid of wild-type BHB (reference sequence = SEQ ID NO: 8). 31 amino acid N-terminal shortenings (amino acid positions 2-32 of SEQ ID NO: 8 [oxidase 8 wt]) were introduced. A visual representation of the modified enzyme design predicted by Alphafold is shown in Figure 3. The amino acid sequences of the modified enzymes are also provided in Table 3. As provided in Table 3, SEQ ID NO: 24 (also known as 8_MUT or oxidase 8_mut) includes all three of these amino acid substitutions, as well as the shortening of amino acid positions 2-32, when compared to SEQ ID NO: 8 (also known as 8_WT herein). Considering the 31 shortened amino acids, the three mutations correspond to N106G, Y204Q, and / or A424Y of the resulting SEQ ID NO: 24. Alternatively, if the "M" at amino acid position 1 (i.e., methionine or Met) is ignored and 31 amino acids are considered, the three mutations correspond to N105G, Y203Q, and / or A423Y in the resulting SEQ ID NO: 24. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18

[0131] Example 2: Enzyme expression and purification Methods for expressing and purifying the modified enzyme are described in their entirety in Yoshikuni et al., U.S. Patent Application Publication No. 2022 / 0348970(A1), which is incorporated herein by reference.

[0132] Overnight cultures of BLR cells suspended in 2 mL volumes were transformed with the pet29b+ plasmid (encoding the target polypeptide with a C-terminal His tag) and grown in Terrific broth containing 50 μg / ml kanamycin. The cultures were diluted 1:1.000 in 500 mL of Terrific broth containing 1 mM MgSO4, 1% glucose, and 50 μg / ml antibiotic, and then grown at 37°C for 24 hours. The cultures were pelleted at 5,000 G for 10 minutes and resuspended in autoinduction medium (TB broth, 1 mM MgSO4, 1 × NPS, and 1 × 5052) for induction at 18°C ​​for 24 hours. At the end of induction, the cells were centrifuged, the supernatant was removed, and the cells were resuspended in 40 mL of lysis buffer (1 × PBS, pH 7.5, 5 mM imidazole) containing 1 mM phenylmethylsulfonyl fluoride. The cell lysate suspension was sonicated for 2 minutes, followed by centrifugation at 5,000 G. The supernatant was loaded onto a gravity flow column containing 500 μL of cobalt beads and washed five times with 15 mL of lysis buffer. The protein was eluted with 1 mL of elution buffer (1 × PBS, pH 7.5, 200 mM imidazole). Protein concentration was determined by measuring the absorbance at 280 nm using a Synergy H1 spectrophotometer (Biotek) with the calculated extinction coefficient. Enzymes with concentrations of 0.1 mg / mL or higher were then assayed.

[0133] Example 3: Enzyme activity Enzyme activity was evaluated by measuring the production of hydrogen peroxide and acetoacetic acid. Commercial test strips were used to detect the presence of hydrogen peroxide and acetoacetic acid.

[0134] To prepare the substrate, 100 mM BHB was dissolved in 400 mM sodium phosphate and 100 mM NaCl at pH 7. In a 96-well plate, 100 μL of 0.5 mg / mL enzyme was combined with 100 μL of the dissolved substrate. The reaction was allowed to proceed at 21°C for 24 hours. After the incubation period, 10 μL of each reactant was applied to individual test strips for colorimetric analysis using hydrogen peroxide and acetoacetic acid detection strips.

[0135] Detection of hydrogen peroxide: Bartovation hydrogen peroxide strips (https: / / bartovation.com) were used to detect the presence of hydrogen peroxide. These strips were calibrated at 0, 1, 3, 10, 50, and 100 ppm. In the presence of hydrogen peroxide, the test strips turned blue, with bluer colors indicating higher hydrogen peroxide concentrations.

[0136] As shown in Figure 4, the modified enzyme containing the amino acid sequence of SEQ ID NO: 24 (oxidase 8_mut) produced hydrogen peroxide in the presence of 2-pentanol, (R)-2-pentanol, BHB, or (R)-beta-hydroxybutyrate (R-BHB), and the test strip turned blue upon contact with a reaction mixture of these reactants. In contrast, wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) showed lower activity when oxidizing BHB, which was indicated by a brighter blue color, and did not oxidize R-BHB, as the test strip did not turn blue. The mutant enzyme of SEQ ID NO: 24 yielded a positive test result, but none of SEQ ID NOs. 1-23 and 25-39 yielded a positive result.

[0137] Detection of acetoacetate: Bayer Ketostix® urine test strips were used to detect the presence of acetoacetic acid in urine. These test strips are calibrated at 0, 5, 15, 40, 80, and 100 mg / dL. If acetoacetic acid is present, the test strip turns red.

[0138] As shown in Figure 5, the modified enzyme containing the amino acid sequence of SEQ ID NO: 24 (oxidase 8_mut) produced acetoacetic acid when R-BHB was used as a reactant, as the test strip turned red. In contrast, wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) did not produce acetoacetic acid when R-BHB was used as a reactant, as the test strip did not turn red.

[0139] HPLC-MS analysis of acetacetate: A method for detecting acetacetate using high-performance liquid chromatography-mass spectrometry (HPLC-MS) is described in detail in U et al., Nat. Prod. Chem. Res. 2019 7(2):364, which is incorporated herein by reference.

[0140] 2,4-Dinitrophenylhydrazine (2,4-DNP) readily reacts with C=O carbonyl groups to form hydrazone compounds and is therefore commonly used to detect ketones and aldehydes. The reaction between R-BHB and 2,4-DNP is shown below. [ka]

[0141] The above reaction was used for HPLC-MS analysis. A derivatization mixture (100 mM 2,4-DNP in methanol) was prepared. After incubation at 21°C for 24 hours, the sample was prepared for HPLC-MS analysis using 200 μL of the reaction mixture, 100 μL of 4 M acetic acid, and 700 μL of the derivation mixture. This was reacted at 60°C for 1 hour. The reaction product was then centrifuged at 5000 G for 10 minutes. The supernatant was collected and used for HPLC-MS analysis.

[0142] For separation, an Agilent Eclipse Plus C18 RRHD 1.8 μm column was used. Analysis was performed at a flow rate of 0.3 mL / min using mobile phase A (H2O, 0.1% formic acid) and mobile phase B (100% ACN, 0.1% formic acid). Using a gradient process, the analysis was started over 10 minutes at 80% MPA / 20% MPB to 0% MPA / 100% MPB. To detect acetoacetate, the derivatization product was searched for using MS in single-ion mode. Figure 6 shows the HPLC-MS profile of the reaction mixture of the modified enzyme (oxidase 8_mut) containing the amino acid sequence of SEQ ID NO: 24. Figure 7 shows a comparison of the HPLC-MS profile of the oxidase 8_mut reaction mixture with that of a commercially available synthetic acetoacetate. Figure 8 shows a comparison of the HPLC-MS profile of the oxidase 8_mut reaction mixture with that of the wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) reaction mixture. Figure 9 compares the HPLC-MS profile of the oxidase-8_mut reaction mixture with the HPLC-MS profile of the substrate control. Figures 6-9 together show that the modified enzyme oxidase-8_mut SEQ ID NO: 24 produced acetoacetic acid.

[0143] Results of hydrogen peroxide test, acetacetate detection, and HPLC-MS analysis: BHB activity was analyzed based on the hydrogen peroxide test protocol (Bartovation hydrogen peroxide strip), the acetacetate detection protocol (Bayer Ketostix®), and the HPLC-MS protocol described herein. The results are shown in Table 4. Oxidase 8 may produce peroxides when incubated with BHB, but only in trace amounts, as shown in Figure 4. This most likely explains the positive peroxide strip activity for wild-type SEQ ID NO: 8, as described in Table 4. [Table 4]

[0144] Example 4: Further Enzyme Design Additional enzymes suitable for use in the present invention and related methods include those listed in Tables 5 and 6. [Table 5] [Table 6]

[0145] In some aspects, this disclosure provides a combination, where X 1 can be N, G, P, A, or S, and X 2 can be Y, Q, N, K, E, or D, and X 3 This could be A, Y, W, F, or H.

[0146] Further numbered embodiments of this disclosure Other subjects contemplated by this disclosure are described in the following numbered embodiments. 1. An oxidase-active enzyme modified to exhibit improved beta-hydroxybutyrate (BHB) activity. 2. The enzyme according to Embodiment 1, wherein the enzyme can oxidize beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate. 3. The enzyme according to Embodiment 1 or Embodiment 2, wherein the amino acid sequence is derived from an oxidase of the EC 1.1.3.6 family. 4. An enzyme according to any one of Embodiments 1 to 3, wherein the oxidase of the EC 1.1.3.6 family is cholesterol oxidase. 5. An enzyme according to any one of Embodiments 1 to 4, wherein the enzyme is non-natural and comprises one or more amino acid substitutions, deletions, or shortenings compared to a natural enzyme or a wild-type enzyme. 6. An enzyme according to any one of Embodiments 1 to 5, wherein the amino acid sequence of the enzyme includes one or more amino acid substitutions corresponding to N137G, Y235Q and / or A455Y of SEQ ID NO: 8. 7. An enzyme according to any one of Embodiments 1 to 6, wherein the amino acid sequence of the enzyme includes an N-terminal shortening of 3 amino acids or less, 5 amino acids or less, 10 amino acids or less, 15 amino acids or less, 20 amino acids or less, 25 amino acids or less, 26 amino acids or less, 27 amino acids or less, 28 amino acids or less, 29 amino acids or less, 30 amino acids or less, or 35 amino acids or less in one or more of Sequence ID Nos. 8, 24, and 40-43. 8. An enzyme according to any one of Embodiments 1 to 6, wherein the amino acid sequence of the enzyme includes an N-terminal shortening corresponding to amino acid positions 2 to 32 of SEQ ID NO: 8. 9. An enzyme according to any one of Embodiments 1 to 8, comprising an amino acid sequence that is identical to one of SEQ ID NOs. 8, 24, 40, 41, 42, or 43 by 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% or more. 10. An enzyme according to any one of Embodiments 1 to 8, comprising one amino acid sequence from among Sequence ID No. 8, 24, 40, 41, 42, or 43, wherein the amino acid sequence includes 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, or 10 or fewer amino acid substitutions. 11. The enzyme according to Embodiment 10, wherein the amino acid substitution is performed only on the portion of the protein involved in the binding and / or propagation of BHB activity. 12. The enzyme according to Embodiment 10 or Embodiment 11, wherein one or more amino acid substitutions are conservative substitutions. 13. An enzyme according to any one of Embodiments 1 to 11, exhibiting increased BHB activity as measured by peroxide strip, acetoacetic acid strip, or liquid chromatography-mass spectrometry (LCMS). 14. The enzyme according to Embodiment 13, exhibiting increased BHB activity as measured by liquid chromatography-mass spectrometry (LCMS). 15. The enzyme according to Embodiment 14, wherein the modified enzyme exhibits BHB activity approximately 1, 2, 3, 4, 5, 10, or 20 times greater than the corresponding unmodified wild type when measured by LCMS. 16. The enzyme according to Embodiment 14, wherein the enzyme contains a sequence that is 90% or more, 95% or more, or 98% or more identical to SEQ ID NO: 24, and SEQ ID NO: 24 shows increased BHB activity compared to SEQ ID NO: 8 as measured by liquid chromatography-mass spectrometry (LCMS). 17. The enzyme according to Embodiment 1 or Embodiment 2, wherein the beta-hydroxybutyrate is (R)-beta-hydroxybutyrate. 18. The enzyme according to Embodiment 1 or Embodiment 2, wherein the beta-hydroxybutyrate is (S)-beta-hydroxybutyrate. 19. The enzyme according to Embodiment 1 or Embodiment 2, wherein the beta-hydroxybutyrate is a mixture of (R)-beta-hydroxybutyrate and (S)-beta-hydroxybutyrate. 20. A non-wearable BHB sensor capable of detecting and / or measuring BHB concentration, comprising utilizing an enzyme described in any one of Embodiments 1 to 19. 21. A non-wearable BHB sensor according to Embodiment 20, wherein the non-wearable BHB sensor is a test strip. 22. The test strip according to Embodiment 21, wherein the test strip comprises a substrate layer and one or more sensing reagents applied on at least a portion of the substrate layer, and at least one of the sensing reagents comprises an enzyme described in any one of Embodiments 1 to 19. 23. A test strip according to Embodiment 21 or Embodiment 22, wherein at least one sensing reagent comprises a cofactor, mediator, adjuvant, carrier, and / or excipient. 24. The test strip according to Embodiment 23, wherein the cofactor comprises flavin adenine dinucleotide (FAD). 25. The test strip according to Embodiment 23 or Embodiment 24, wherein the mediator comprises one or more of the following: ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complex, and / or organometallic ruthenium complex. 26. A test strip according to any one of Embodiments 22 to 25, wherein the material of the base layer comprises one or more of composite materials, fibrous materials, woven fabrics, nonwoven fabrics, polymers, adhesives, films, gels, PTFE, and / or silicones. 27. A non-wearable BHB sensor according to Embodiment 20, wherein the non-wearable sensor is a fingertip sweat sensor. 28. A non-wearable BHB sensor according to Embodiment 20, wherein the non-wearable sensor may be used to detect or measure BHB in any bodily fluid. 29. A non-wearable BHB sensor according to Embodiment 28, wherein bodily fluids can be obtained optionally by non-invasive, minimally invasive, or invasive means, such as finger puncture, blood sampling, spinal puncture, sweat and / or saliva collection. 30. A non-wearable BHB sensor according to Embodiment 29, wherein the body fluids include one or more of the following: whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites. 31. Sensors including sensing electrodes, A sensing reagent, wherein the sensing reagent composition comprises an enzyme described in any one of Embodiments 1 to 19, and Including a reference electrode, A wearable BHB sensor capable of detecting, measuring, and / or monitoring BHB concentration. 32. The wearable BHB sensor according to Embodiment 31, wherein the sensing reagent is located on or adjacent to the sensing electrode. 33. A wearable BHB sensor according to Embodiment 31, wherein a sensing reagent is applied so as to come into contact with the sensing electrode when in use. 34. The wearable BHB sensor according to Embodiment 31, wherein the wearable BHB sensor is a continuous sensor, a continual sensor, or an on-demand sensor. 35. A wearable BHB sensor according to any one of Embodiments 31 to 34, wherein the sensing reagent composition further comprises a cofactor, a mediator, an adjuvant, a carrier, and / or an excipient. 36. A wearable BHB sensor according to any one of embodiments 31 to 35, wherein the wearable BHB sensor is configured to continuously measure the target BHB concentration and output a data stream. 37. A wearable BHB sensor according to any one of Embodiments 31 to 36, wherein the wearable BHB sensor is a needle-type sensor, a microneedle-type sensor, a reverse iontophoresis sensor, a sweat-based sensor, or an implantable sensor. 38. A wearable BHB sensor according to embodiment 37, wherein the wearable BHB sensor is positioned within body tissue. 39. A wearable BHB sensor according to any one of embodiments 31 to 38, wherein the wearable BHB sensor is configured to measure the concentration of BHB in body fluids. 40. A wearable BHB sensor according to Embodiment 39, wherein the body fluids include one or more of the following: whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites. 41. A wearable BHB sensor according to any one of Embodiments 31 to 40, wherein the wearable BHB sensor is an in-body wearable BHB sensor or a body wearable BHB sensor. 42. A wearable BHB sensor according to any one of Embodiments 31 to 41, wherein the wearable BHB sensor is configured to continuously, continuously, or on demand measure the target BHB concentration and output a data stream. A device connected to a wearable BHB sensor, the device is A processor configured to process data streams from a wearable BHB sensor, and An interface configured to display and / or communicate the measured BHB concentration value. devices A system for detecting, measuring, and / or monitoring BHB concentration, including [specific components / features]. 43. Obtaining bodily fluids from the subject. The objectives are to provide a body fluid to a wearable BHB sensor described in any one of Embodiments 31 to 41, to determine the BHB concentration in the body fluid at a single time point, and Displaying and / or communicating BHB concentration at a single point in time on the interface. A method for detecting, measuring, and / or monitoring BHB concentration, including [the specified element]. 44. A method for improving health and / or wellness in a person requiring improvement, comprising using a non-wearable BHB sensor, device, or apparatus described in any one of Embodiments 20 to 30. 45. A method for improving the health and / or wellness of an object requiring improvement, comprising using a test kit, wherein the test kit utilizes an enzyme described in any one of Embodiments 1 to 19. 46. ​​A method for assisting weight loss in a person in need of assistance, comprising using a non-wearable BHB sensor, device, or apparatus described in any one of Embodiments 20 to 30. 47. The method according to any one of Embodiments 44 to 46, used in combination with a food or supplement containing at least one sugar alcohol or a low-carbohydrate composition. 48. The method according to any one of Embodiments 44 to 47, wherein the food or supplement contains less than approximately 5 grams, less than approximately 8 grams, or less than approximately 10 grams of carbohydrates per unit. 49. The method according to either Embodiment 47 or Embodiment 48, used in combination with a food or supplement, wherein the food or supplement contains an exogenous ketone supplement and / or a ketone-producing component. 50. The method according to Embodiment 49, wherein the exogenous ketone supplement comprises ketone bodies and / or precursors of ketone bodies. 51. The method according to Embodiment 50, wherein the ketone body and / or ketone body precursor comprises one or more of acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium-chain triglycerides (MCTs) containing fatty acids having hydrocarbon side chains of 6 to 12 carbon atoms. 52. The method according to Embodiment 51, wherein the ketone body and / or ketone body precursor is in the form of a salt and / or ester. 53. The method according to Embodiment 51, wherein the MCT comprises one or more of caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12).

[0147] Built-in by reference All references, articles, publications, patents, patent gazettes, and patent applications cited herein and / or below in the text above are incorporated by reference in their entirety for all purposes. All documents and similar materials cited in this application, including patents, patent applications, articles, books, professional works, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. If the definitions of terms in the incorporated references appear to differ from the definitions provided in this application, the definitions provided in this application shall prevail.

[0148] However, any references to references, articles, publications, patents, patent gazettes, and patent applications cited herein are not, and should not be construed as, an endorsement or suggestion in any form that they constitute valid prior art or form part of common general knowledge in any country of the world.

Claims

1. An oxidase-active enzyme modified to exhibit improved beta-hydroxybutyrate (BHB) activity.

2. The enzyme according to claim 1, wherein the enzyme can oxidize beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate.

3. The enzyme according to claim 1 or claim 2, wherein the amino acid sequence is derived from an oxidase of the EC 1.1.3.6 family.

4. The enzyme according to any one of claims 1 to 3, wherein the oxidase of the EC 1.1.3.6 family is cholesterol oxidase.

5. The enzyme according to any one of claims 1 to 4, wherein the enzyme is non-natural and comprises one or more amino acid substitutions, deletions, or shortenings compared to a natural enzyme or a wild-type enzyme.

6. The enzyme according to any one of claims 1 to 5, wherein the amino acid sequence of the enzyme includes one or more amino acid substitutions corresponding to N137G, Y235Q, and / or A455Y of SEQ ID NO:

8.

7. The enzyme according to any one of claims 1 to 6, wherein the amino acid sequence of the enzyme includes an N-terminal shortening of 3 amino acids or less, 5 amino acids or less, 10 amino acids or less, 15 amino acids or less, 20 amino acids or less, 25 amino acids or less, 26 amino acids or less, 27 amino acids or less, 28 amino acids or less, 29 amino acids or less, 30 amino acids or less, or 35 amino acids or less in one or more of sequence numbers 8, 24, and 40 to 43.

8. The enzyme according to any one of claims 1 to 6, wherein the amino acid sequence of the enzyme includes an N-terminal shortening corresponding to amino acid positions 2 to 32 of SEQ ID NO:

8.

9. An enzyme according to any one of claims 1 to 8, comprising an amino acid sequence that is identical to one of sequence numbers 8, 24, 40, 41, 42, or 43 by 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% or more.

10. An enzyme according to any one of claims 1 to 8, comprising one amino acid sequence from sequence numbers 8, 24, 40, 41, 42, or 43, wherein the amino acid sequence comprises one, two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, or ten or fewer amino acid substitutions.

11. The enzyme according to claim 10, wherein the amino acid substitution is performed only on the portion of the protein involved in the binding and / or propagation of BHB activity.

12. The enzyme according to claim 10 or claim 11, wherein one or more amino acid substitutions are conservative substitutions.

13. The enzyme according to any one of claims 1 to 11, wherein the enzyme exhibits increased BHB activity as measured by peroxide strip, acetoacetate strip, or liquid chromatography-mass spectrometry (LCMS).

14. The enzyme according to claim 13, wherein the enzyme exhibits increased BHB activity as measured by liquid chromatography-mass spectrometry (LCMS).

15. The enzyme according to claim 14, wherein the modified enzyme exhibits BHB activity approximately 1, 2, 3, 4, 5, 10, or 20 times greater than the corresponding unmodified wild type when measured by LCMS.

16. The enzyme according to claim 14, wherein the enzyme contains a sequence that is 90% or more, 95% or more, or 98% or more identical to SEQ ID NO: 24, and SEQ ID NO: 24 shows increased BHB activity compared to SEQ ID NO: 8 as measured by liquid chromatography-mass spectrometry (LCMS).

17. The enzyme according to claim 1 or claim 2, wherein the beta-hydroxybutyrate is (R)-beta-hydroxybutyrate.

18. The enzyme according to claim 1 or claim 2, wherein the beta-hydroxybutyrate is (S)-beta-hydroxybutyrate.

19. The enzyme according to claim 1 or claim 2, wherein the beta-hydroxybutyrate is a mixture of (R)-beta-hydroxybutyrate and (S)-beta-hydroxybutyrate.

20. A non-wearable BHB sensor capable of detecting and / or measuring BHB concentration, comprising utilizing an enzyme according to any one of claims 1 to 19.

21. The non-wearable BHB sensor according to claim 20, wherein the non-wearable BHB sensor is a test strip.

22. The aforementioned test strip, Substrate layer, and It comprises one or more sensing reagents applied to at least a portion of the substrate layer, At least one of the sensing reagents comprises the enzyme described in any one of claims 1 to 19. The test strip according to claim 21.

23. The test strip according to claim 21 or 22, wherein the at least one sensing reagent comprises a cofactor, mediator, adjuvant, carrier, and / or excipient.

24. The test strip according to claim 23, wherein the cofactor comprises flavin adenine dinucleotide (FAD).

25. The test strip according to claim 23 or claim 24, wherein the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complex, and / or organometallic ruthenium complex.

26. The test strip according to any one of claims 22 to 25, wherein the material of the base layer comprises one or more of composite materials, fibrous materials, woven fabrics, nonwoven fabrics, polymers, adhesives, films, gels, PTFE, and / or silicones.

27. The non-wearable BHB sensor according to claim 20, wherein the non-wearable sensor is a fingertip sweat sensor.

28. The non-wearable BHB sensor according to claim 20, wherein the non-wearable sensor may be used to detect or measure BHB in any bodily fluid.

29. The non-wearable BHB sensor according to claim 28, wherein the bodily fluid can be obtained optionally by non-invasive, minimally invasive, or invasive means, such as by finger puncture, blood sampling, spinal puncture, sweat and / or saliva collection.

30. The non-wearable BHB sensor according to claim 29, wherein the bodily fluids include one or more of the following: whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites.

31. Sensor including sensing electrodes, A sensing reagent, wherein the sensing reagent composition comprises the enzyme described in any one of claims 1 to 19, and Including a reference electrode, A wearable BHB sensor capable of detecting, measuring, and / or monitoring BHB concentration.

32. The wearable BHB sensor according to claim 31, wherein the sensing reagent is located on or adjacent to the sensing electrode.

33. The wearable BHB sensor according to claim 31, wherein the sensing reagent is applied so as to come into contact with the sensing electrode when in use.

34. The wearable BHB sensor according to claim 31, wherein the wearable BHB sensor is a continuous sensor, a continuous sensor, or an on-demand sensor.

35. The wearable BHB sensor according to any one of claims 31 to 34, wherein the sensing reagent composition further comprises a cofactor, a mediator, an adjuvant, a carrier, and / or an excipient.

36. The wearable BHB sensor according to any one of claims 31 to 35, wherein the wearable BHB sensor is configured to continuously measure the target BHB concentration and output a data stream.

37. The wearable BHB sensor according to any one of claims 31 to 36, wherein the wearable BHB sensor is a needle-type sensor, a microneedle-type sensor, a reverse iontophoresis sensor, a sweat-based sensor, or an implantable sensor.

38. The wearable BHB sensor according to claim 37, wherein the wearable BHB sensor is placed within body tissue.

39. The wearable BHB sensor according to any one of claims 31 to 38, wherein the wearable BHB sensor is configured to measure the BHB concentration in a body fluid.

40. The wearable BHB sensor according to claim 39, wherein the bodily fluids include one or more of the following: whole blood, serum, plasma, blood fractions other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, milk, mucus, erosion, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, cystic contents, and / or ascites.

41. The wearable BHB sensor according to any one of claims 31 to 40, wherein the wearable BHB sensor is an in-body wearable BHB sensor or a body wearable BHB sensor.

42. A wearable BHB sensor according to any one of claims 31 to 41, wherein the wearable BHB sensor is configured to continuously, continuously, or on demand measure the target BHB concentration and output a data stream. A device connected to the wearable BHB sensor, wherein the device is A processor configured to process the data stream from the wearable BHB sensor, and An interface configured to display and / or communicate the measured BHB concentration value. devices A system for detecting, measuring, and / or monitoring BHB concentration, including [specific components / features].

43. To obtain bodily fluids from the subject, To provide bodily fluids to the wearable BHB sensor according to any one of claims 31 to 41, To determine the BHB concentration in the aforementioned body fluid at a single time point, and Displaying and / or communicating the BHB concentration at a single point in time on the interface. A method for detecting, measuring, and / or monitoring BHB concentration, including [the specified element].

44. A method for improving health and / or wellness in a person requiring improvement, comprising using a non-wearable BHB sensor, device, or apparatus as described in any one of claims 20 to 30.

45. A method for improving health and / or wellness in a subject requiring improvement, comprising using a test kit, wherein the test kit utilizes an enzyme described in any one of claims 1 to 19.

46. A method for assisting weight loss in a person in need of assistance, comprising using a non-wearable BHB sensor, device, or apparatus as described in any one of claims 20 to 30.

47. The method according to any one of claims 44 to 46, used in combination with a food or supplement containing at least one sugar alcohol or a low-carbohydrate composition.

48. The method according to any one of claims 44 to 47, wherein the food or supplement contains less than approximately 5 grams, less than approximately 8 grams, or less than approximately 10 grams of carbohydrates per unit.

49. The method according to claims 47 and 48, used in combination with a food or supplement, wherein the food or supplement contains an exogenous ketone supplement and / or a ketone-producing component.

50. The method according to claim 49, wherein the exogenous ketone supplement comprises ketone bodies and / or precursors of ketone bodies.

51. The method according to claim 50, wherein the ketone body and / or precursor of the ketone body comprises one or more of acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium-chain triglycerides (MCTs) having hydrocarbon side chains of 6 to 12 carbon atoms.

52. The method according to claim 51, wherein the ketone body and / or ketone body precursor is in the form of a salt and / or ester.

53. The method according to claim 51, wherein the MCT comprises one or more of caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12).

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