Enzyme for oxidizing beta-hydroxybutyrate (BHB), test strip and sensor using enzyme

Abstract

The present disclosure relates to enzymes capable of oxidizing beta-hydroxybutyrate (BHB). In some aspects, enzymes are engineered to optimize BHB activity. Further provided are engineered enzymes comprising amino acid substitutions, truncation, and sequence deletions. The disclosure further provides uses of the enzymes described herein, such as in test strips, BHB sensors, devices and systems for detecting and measuring BHB concentrations.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 580,620, filed September 5, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list electronically submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on August 26, 2024, named 081906-1457969-253310PC_SL.xml, and has a size of 61,977 bytes. Technical Field

[0005] This disclosure relates to enzymes capable of oxidizing β-hydroxybutyrate (BHB) and methods thereof. In one aspect, this disclosure relates to the use of the enzymes described herein in various applications, such as for testing test strips and other detection platforms, BHB sensors, and systems for detecting and measuring BHB concentrations. Background Technology

[0006] The ketogenic diet is an effective approach to promoting weight loss and health. It involves minimizing carbohydrate intake while consistently increasing the consumption of fat and / or protein. This type of diet forces the body to burn fat, resulting in the production of ketone bodies (ketones) when the body breaks down fat. For optimal dietary management, monitoring ketone levels is important for individuals on a ketogenic diet. This disclosure provides a solution to this industry and medical need.

[0007] β-Hydroxybutyrate (BHB) is the conjugate base of β-hydroxybutyrate. BHB is synthesized through fatty acid metabolism, and high BHB levels indicate that the body is using fat as its primary fuel source. Therefore, there is a need for accurate and reliable measurement of BHB levels in subjects. This can be achieved through invasive, minimally invasive, or non-invasive mechanisms. This disclosure addresses this need by describing novel enzymes that aid in BHB measurement. Useful systems, apparatus, and related methods are further provided herein. Summary of the Invention

[0008] This disclosure relates to enzymes having oxidase activity. In one embodiment, the enzymes described herein are engineered and / or modified relative to natural enzymes. In the aspects described herein, the enzymes are capable of oxidizing β-hydroxybutyrate (BHB) to produce 3-oxobutyrate. In some embodiments, β-hydroxybutyrate (BHB) is (R)-β-hydroxybutyrate. In some embodiments, β-hydroxybutyrate (BHB) is (S)-β-hydroxybutyrate. In some embodiments, β-hydroxybutyrate (BHB) is a mixture of (R)-β-hydroxybutyrate and (S)-β-hydroxybutyrate.

[0009] In some embodiments, the enzyme, such as an engineered enzyme, includes (e.g., comprises) an amino acid sequence derived from, or substantially consists of, 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.

[0010] In some embodiments, the enzyme is non-naturally occurring and / or engineered, and includes (e.g., comprises) one or more amino acid substitutions, deletions, or truncations relative to a natural enzyme, and is substantially composed of or constitutes thereof.

[0011] In one implementation, the enzymes described herein cover those derived from pseudoclade algae ( Scytonema sp. The enzyme is a modified version of SEQ ID NO: 8, comprising one or more amino acid substitutions, deletions, or truncations. In one embodiment, SEQ ID NO: 8 is optimized to produce improved BHB detection activity.

[0012] In some embodiments, the enzyme comprises (e.g., includes) an amino acid sequence substantially consisting of or composed of, said amino acid sequence containing one or more mutations corresponding to N137G, Y235Q, and / or A455Y of SEQ ID NO: 8. In other embodiments, the enzymes used herein comprise SEQ ID NO: 1-43. In some aspects, the enzymes used herein comprise (e.g., include) any one of SEQ ID NO: 24 or SEQ ID NO: 40-43, fragments thereof, or modified proteins thereof, substantially consisting of or composed of.

[0013] In some embodiments, the enzyme comprises (e.g., includes) at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of any one of SEQ ID NO: 1-43, and is substantially composed of or constitutes thereof. In some embodiments, the enzyme comprises (e.g., includes) at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 40-43, fragments thereof, or modified proteins thereof, and is substantially composed of or constitutes thereof.

[0014] In another embodiment, the enzyme comprises (e.g., includes) the amino acid sequence of any one of SEQ ID NO: 1-43, substantially consisting of or consisting of therefrom, wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more amino acid substitutions. In some embodiments, the enzyme comprises the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 40-43, wherein the amino acid sequence includes (e.g., includes) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more amino acid substitutions, substantially consisting of or consisting of therefrom. In some embodiments, the amino acid substitution is a conserved substitution. In some embodiments, the substitution occurs only in the protein portion responsible for binding and / or propagation activity.

[0015] In one embodiment, this disclosure provides a protein comprising (e.g., containing) at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of any one of SEQ ID NO: 1-43, and substantially consisting of or composed of the same amino acid sequence. In some embodiments, this disclosure provides a protein, fragment thereof, or modified thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 40-43.

[0016] In other embodiments, this disclosure provides a protein comprising (e.g., including) the amino acid sequence of any one of SEQ ID NO: 1-43, substantially consisting of or consisting of therefrom, wherein said amino acid sequence includes (e.g., including) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 or more amino acid substitutions, substantially consisting of or consisting of therefrom. In some embodiments, this disclosure provides a protein having the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 40-43, wherein said amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 or more amino acid substitutions. In some embodiments, said amino acid substitutions are conservative substitutions.

[0017] In another embodiment, the engineered or modified enzymes described herein exhibit increased BHB activity relative to the corresponding wild-type enzyme. As used herein, wild-type (wild-type, wildtype, wild type) and WT are synonymous and refer to naturally occurring enzymes (i.e., in their wild or natural state). As used herein, mutant, mut, and MUT are synonymous and refer to modified and / or mutant enzymes, such as modified and / or mutant enzymes produced through enzyme design and / or enzyme engineering. Directed evolution, rational design, and semi-rational design are major computer-aided methods widely used in enzyme engineering. In one embodiment, BHB activity is measured using peroxide test strips, acetoacetic acid test strips, and / or liquid chromatography-mass spectrometry (LCMS). In one embodiment, the peroxide test strip is a Bartovation test strip, and the acetoacetic acid test strip is a Bayer Ketostix® test strip. On the one hand, when measured by LCMS, the BHB activity of the modified or engineered enzyme is about one (1), two (2), three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9), ten (10), twenty (20), thirty (30), forty (40), fifty (50), sixty (60), seventy (70), eighty (80), ninety (90), one hundred (100), two hundred (200), three hundred (300), four hundred (400), five hundred (500), six hundred (600), seven hundred (700), eight hundred (800), nine hundred (900), one thousand (1,000), ten thousand (10,000), one hundred thousand (100,000) or more.

[0018] In another embodiment, SEQ ID NO: 24 or SEQ ID NO: 40-43 and their derivatives exhibit increased BHB activity relative to SEQ ID NO: 8. In one embodiment, BHB activity is measured by a peroxide test strip, an acetoacetic acid test strip, or liquid chromatography-mass spectrometry (LCMS). In one aspect, the peroxide test strip is a Bartovation test strip, and the acetoacetic acid test strip is a Bayer Ketostix® test strip. On the one hand, when measured by LCMS, the BHB activity of SEQ ID NO: 24 or SEQ ID NO: 40-43 and their derivatives is about one (1), two (2), three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9), ten (10), twenty (20), thirty (30), forty (40), fifty (50), sixty (60), seventy (70), eighty (80), ninety (90), one hundred (100), two hundred (200), three hundred (300), four hundred (400), five hundred (500), six hundred (600), seven hundred (700), eight hundred (800), nine hundred (900), one thousand (1,000), ten thousand (10,000), one hundred thousand (100,000) or more.

[0019] In another embodiment, SEQ ID NO: 24 exhibits increased BHB activity relative to SEQ ID NO: 8. In one embodiment, BHB activity is measured by a peroxide test strip, an acetoacetic acid test strip, or liquid chromatography-mass spectrometry (LCMS). In one aspect, the peroxide test strip is a Bartovation test strip, and the acetoacetic acid test strip is a Bayer Ketostix® test strip. On the one hand, when measured by LCMS, the BHB activity of SEQ ID NO: 24 is about one (1), two (2), three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9), ten (10), twenty (20), thirty (30), forty (40), fifty (50), sixty (60), seventy (70), eighty (80), ninety (90), one hundred (100), two hundred (200), three hundred (300), four hundred (400), five hundred (500), six hundred (600), seven hundred (700), eight hundred (800), nine hundred (900), one thousand (1,000), ten thousand (10,000), one hundred thousand (100,000) or more.

[0020] In some implementations, the enzymes described herein are non-naturally occurring, engineered, isolated, and / or purified.

[0021] This disclosure further provides sensors capable of detecting and / or measuring BHB concentration, including those utilizing the enzymes described herein for detection and / or measurement, for use in non-wearable sensing systems. An example of a non-wearable sensing system is a test strip used for detecting and / or measuring BHB concentration. The test strip can be used once or multiple times.

[0022] In some embodiments, the test strip includes: a base layer; and one or more sensing reagents disposed on at least a portion of the base layer, wherein at least one of the sensing reagents includes the enzyme.

[0023] In some embodiments, at least one of the sensing reagents further comprises a cofactor, a mediator, and / or other adjuvants or excipients. 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 / methylene blue, tetrathione, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes.

[0024] In some embodiments, the substrate material includes one or more of composite materials, fibrous materials, textile fabrics, non-woven fabrics, polymers, adhesives, films, gels, polytetrafluoroethylene (PTFE), and / or silicone.

[0025] Sensors, such as non-wearable sensors, can be used to detect or measure BHB in any bodily fluid. The bodily fluid can be obtained through non-invasive, minimally invasive, or invasive methods, such as finger-prick blood collection, blood aspiration, spinal puncture as examples of invasive methods, or collection of sweat or saliva as examples of non-invasive or minimally invasive methods. Bodily fluid refers to all bodily fluids, including but not limited to whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, abdominal fluid, glandular secretions, exudate, cyst contents, and / or ascites.

[0026] This disclosure further provides sensors capable of detecting and / or measuring BHB concentration, including sensors for continuous, sustained, or on-demand detection or measurement using the enzymes described herein. Continuous means uninterrupted; without interruption; non-intermittent or sporadic; thus repeating continuously at short time intervals to form a nearly uninterrupted series. Sustained means regularly and frequently repeated in a stable and continuous state. On-demand means that detection or measurement results are readily available when the sensor is actuated by a user or when sensor data is displayed.

[0027] In some embodiments, a BHB sensor capable of sensing, detecting, and / or measuring BHB concentration includes: a sensor comprising a sensing electrode; a sensing reagent, wherein the sensing reagent composition 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 dispensed to contact the sensing electrode upon use.

[0028] In some embodiments, the sensing reagent composition further includes a cofactor and / or a mediator.

[0029] In some embodiments, the BHB sensor is configured as a wearable sensor to measure the BHB concentration of a subject and output a data stream continuously, persistently, or on demand. In some embodiments, part or all of the sensor will be located within body tissue (in vivo sensor) to measure bodily fluids in that tissue. Body tissue can include any tissue, including but not limited to the epidermis, dermis, subcutaneous tissue, muscle, and intraperitoneal space. Example embodiments of in vivo sensors include, but are not limited to, needle-based sensors, microneedle-based sensors, or continuous BHB sensors in implantable sensors. In other embodiments, the BHB sensor is configured to measure BHB in bodily fluids sampled from the body. Samples can be obtained non-invasively, minimally invasively, or invasively. Body fluids refer to all bodily fluids, including but not limited to whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudates, cyst contents, and / or ascites. Body fluids can originate from bodily fluids that come into direct contact with internal body tissues, or from bodily fluids that are sampled non-invasively or invasively from the body and sensed "on-body."

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

[0031] In some implementations, a system for detecting and measuring BHB concentration includes: a BHB sensor configured to continuously, persistently, or on-demand measure the BHB concentration of a subject and output a data stream; the sensor can use any method for BHB measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetric, iontophoresis, radiation, immunochemical, etc.; and a device connected to the BHB sensor, wherein the non-device includes: a processor configured to process the data stream from the BHB sensor; and an interface configured to display and / or transmit the measured BHB concentration value.

[0032] This disclosure further relates to a method for detecting and measuring BHB concentration using the enzymes described herein.

[0033] In some implementations, methods for detecting and measuring BHB concentration include: obtaining bodily fluid from a subject; placing the bodily fluid in a BHB sensor; determining a single-point BHB concentration in the bodily fluid; and / or displaying and / or transmitting the single-point BHB concentration on an interface.

[0034] In other embodiments, the enzymes, devices, or systems described herein are used as methods for improving the health and well-being of individuals in need. In other aspects, the enzymes, devices, or systems described herein are used as methods for weight loss. In other embodiments, the enzymes, devices, or systems described herein are used for improving mental and / or metabolic health. In yet another embodiment, the enzymes, devices, or systems described herein are used as methods for controlling or monitoring carbohydrate intake. In other embodiments, the enzymes, devices, or systems described herein are used as methods for monitoring ketone body levels, for improving cognitive function, for treating various neurological disorders (e.g., epilepsy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), traumatic brain injury, Alzheimer's disease, and dementia), for treating mental health, mental illness, mental problems, and mental disorders (e.g., depression, bipolar disorder, schizophrenia), for improving cardiometabolism, for improving responses to cancer treatment, immunotherapy, chemotherapy, and radiotherapy that modulate inflammatory pathways and immune function, for treating obesity and diabetes, for cholesterol quantification and monitoring, and for monitoring and detecting alcoholic or diabetic ketoacidosis.

[0035] In other embodiments, the enzymes, devices, or systems described herein are used in combination with other weight loss or health maintenance methods or therapies. In other embodiments, the enzymes, devices, or systems described herein are used in combination with foods or supplements formulated with low carbohydrate content or sugar alcohols.

[0036] In some embodiments, the food or supplement includes a ketone supplement or additive. In another embodiment, the food or supplement includes exogenous ketones and / or ketogenic supplements. In some embodiments, the exogenous ketones and / or ketogenic supplements include ketone bodies and / or ketone body precursors. In some embodiments, the ketone bodies and / or ketone body precursors include one or more of acetone, acetoacetic acid, β-hydroxybutyrate (BHB), β-ketovalerate, β-hydroxyvalerate, 1,3-butanediol, and medium-chain triglycerides (MCTs) containing a fatty acid with a hydrocarbon side chain 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 MCT includes one or more of the following: hexanoic acid (C6), caprylic acid (C8), decanoic acid (C10), and lauric acid (C12).

[0037] In some implementations, the apparatus, system, or method described herein is prescribed by a medical professional to an individual in need. In other implementations, the patient in need is obese or has an underlying medical condition, such as heart disease. Attached Figure Description

[0038] Figure 1 Preliminary screening of a sequence similarity network (SSN) consisting of 4,000 bacterial oxidases.

[0039] Figure 2 : Using the Alphafold model, we compared oxidase 8 (SEQ ID NO: 8) with an engineered cholesterol oxidase from Streptomyces hygrospinosus (ShCOb).

[0040] Figure 3 Visual display of an engineered enzyme (8_MUT) designed based on Alphafold prediction of wild-type oxidase 8 (8_WT).

[0041] Figure 4 The result is a detection of hydrogen peroxide (H2O2; peroxide) produced by engineered enzymes (oxidase 8_mut) containing the amino acid sequence SEQ ID NO: 24 and wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8). Commercially available Bartovation hydrogen peroxide test strips were used.

[0042] Figure 5 The test result is for acetoacetate produced by engineered enzymes (oxidase 8_mut) containing the amino acid sequence SEQ ID NO: 24 and wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8). The test strips are commercially available Bayer Ketostix® urine reagents.

[0043] Figure 6 HPLC-MS spectrum of the reaction mixture of engineered enzyme (oxidase 8_mut) containing amino acid sequence SEQ ID NO: 24.

[0044] Figure 7 Compare the HPLC-MS spectra of the reaction mixture of oxidase 8_mut (SEQ ID NO: 24) (8_MUT) with those of commercially available synthetic acetoacetate.

[0045] Figure 8Compare the HPLC-MS spectra of the reaction mixture of oxidase 8_mut (SEQ ID NO: 24) (8_MUT) with those of the reaction mixture of wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) (8_WT).

[0046] Figure 9 Compare the HPLC-MS spectra of the oxidase 8_mut (SEQ ID NO: 24) (8_MUT) reaction mixture with the HPLC-MS spectra of the substrate control.

[0047] Detailed description

[0048] This disclosure provides novel proteins and enzymes capable of exhibiting BHB activity. In one aspect, the novel proteins or enzymes can be used with the methods, systems, apparatus, and kits described herein.

[0049] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids, i.e., having carbon atoms bonded to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.

[0050] Various methods exist in the literature that allow the incorporation of non-natural amino acid derivatives or analogs into polypeptide chains in a site-specific manner; see, for example, WO 02 / 086075, the contents of which are incorporated herein by reference in their entirety.

[0051] Amino acids can be referred to in this article using either the commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Similarly, nucleotides can be referred to using their generally accepted single-letter codes.

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. All three terms apply to amino acid polymers that are artificial chemical mimics of corresponding naturally occurring amino acids, as well as both naturally occurring and non-naturally occurring amino acid polymers. As used herein, the term includes amino acid chains of any length, including full-length proteins, where amino acid residues are linked by covalent peptide bonds.

[0053] In the context of two or more nucleic acid or polypeptide sequences (e.g., the two proteases of this disclosure and the polynucleotide encoding the proteases), the term "identical" or percentage "identity" means that when compared and aligned to obtain the maximum degree of correspondence, the two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides, as measured by using one of the following sequence comparison algorithms or by visual inspection.

[0054] For sequence comparisons, typically one sequence is used as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0055] In the context of this application, a sequence “at least 80% identical to a reference sequence” is a sequence whose full length has 80% or more, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% sequence identity with the full length of the reference sequence. Proteins composed of amino acid sequences that are “at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical” to the reference sequence can include mutations, such as deletions, insertions, and / or substitutions compared to the reference sequence. In the case of substitution, a protein consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical to the reference sequence may correspond to a homologous sequence from a species other than the reference sequence.

[0056] In the context of this application, the "identity percentage" can be calculated using global alignment (i.e., comparing two sequences over their full length). Methods for comparing the identity of two or more sequences are well known in the art. For example, when considering the full length of two sequences, the "needle" procedure can be used, which utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the best alignment (including gaps) of the two sequences. The needle procedure is available, for example, on the ebi.ac.uk World Wide website and is further described in the following 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 peptides is calculated using the EMBOSS: needle (global) program and the Blosum62 matrix, wherein the "Gap Open" parameter of the EMBOSS: needle (global) program is equal to 10.0 and the "Gap Extend" parameter is equal to 0.5. Other algorithms suitable for determining percentage sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul respectively. et al. , J. Mol. Biol. 215:403-410, 1990 and Altschuel et al. , Nucleic Acids Res. 25:3389-3402, 1977. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI).

[0057] In this document, "at least one" means one or more of the specified targets, such as 1, 2, 3, 4, 5, or 6 or more of the specified targets. For example, "at least one amino acid substitution" in this document means 1, 2, 3, 4, 5, or 6 or more amino acid substitutions.

[0058] Amino acid substitution can be either conservative or non-conservative. On the one hand, substitution is conservative, where one amino acid is replaced by another amino acid with similar structure and / or chemical properties. On the other hand, conservative substitution produces the same or similar functional properties.

[0059] In one embodiment, conserved substitutions may include those described by Dayhoff in “The Atlas of Protein Sequence and Structure. Vol. 5”, Natl. Biomedical Research, the contents of which are incorporated herein by reference in their entirety. For example, in one aspect, amino acids belonging to any of the following groups may be interchanged to constitute a conserved 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). On the one hand, the conserved amino acid substitutions can be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G and / or T→S.

[0060] In a further embodiment, conserved amino acid substitution may include the substitution of an amino acid with another amino acid of the same class, such as (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) polar-neutral: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conserved amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

[0061] In another implementation, conservative substitutions can be made according to Table 1. Methods for predicting tolerance to protein modifications can be found, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated herein by reference in their entirety.

[0062] Table 1: Representative Conserved Amino Acid Substitutions

[0063] In another implementation, the conservative substitution may be those shown under the heading "Conservative Substitution" in Table 2. If such substitution results in a change in biological activity and, more substantially, in this case, the "Exemplary Substitutions" named in Table 2 may be introduced, and the product may be screened if necessary.

[0064] Table 2: Amino Acid Substitutions

[0065] enzymes

[0066] The enzymes or proteins described herein may be engineered, isolated, or purified. In one respect, the enzymes or proteins described herein may be non-natural. In other respects, the enzymes or proteins described herein may be modified by amino acid substitution, deletion, and / or truncation. In other respects, the enzymes described herein are optimized for the detection or analysis of BHB. In other respects, the enzymes described herein are designed for use with the systems, apparatus, kits, and methods described herein.

[0067] In some aspects, this disclosure relates to enzymes having oxidase activity, wherein said enzyme is capable of oxidizing β-hydroxybutyrate (BHB) to produce 3-oxobutyrate. In other embodiments, said enzyme comprises, consists of, or substantially consists of the following amino acid sequence, which includes one or more mutations corresponding to N137G, Y235Q, and / or A455Y of SEQ ID NO: 8. In some aspects, the enzyme described herein comprises only combinations of the N137G, Y235Q, and / or A455Y mutations of SEQ ID NO: 8. In other aspects, the enzyme described herein comprises, substantially consists of, or comprises combinations of and truncated mutations, where the combination of mutations is a combination of the N137G, Y235Q, and / or A455Y mutations of SEQ ID NO: 8, and the truncation is an N-terminal truncation at positions 2 to 32 of amino acids in SEQ ID NO: 8. In other respects, the enzyme described herein comprises, is substantially composed of, or is composed of, a combination and truncation of the following mutations, wherein the combination of mutations is a combination of N137G, Y235Q and / or A455Y mutations of SEQ ID NO: 8, and the truncation is an N-terminal truncation at the following amino acid positions selected from amino acid positions 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 30, 2 to 31 or 2 to 32 of SEQ ID NO: 8. In other respects, the enzymes described herein comprise combinations and truncations of the following mutations, consist essentially of combinations and truncations of the following mutations, or consist of combinations and truncations of the following mutations, wherein the combination of mutations is a combination of N137G, Y235Q and / or A455Y mutations of SEQ ID NO: 8, and the truncation is an N-terminal truncation of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids of SEQ ID NO: 8.

[0068] In some respects, under similar testing conditions, SEQ ID NO: 24 exhibited increased BHB activity compared to SEQ ID NO: 8. In other respects, under the same testing conditions or conditions, SEQ ID NO: 24 or SEQ ID NO: 40-43 exhibited increased BHB activity compared to SEQ ID NO: 8.

[0069] 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 derived from *Pseudococcus pseudobranchii*, *Rhodococcus rubrum*, etc. Rhodococcus erythropolis ReCO), sterol short bacilli ( Brevibacterium sterolicum , BsCO) or hygroscopic streptomyces (ShCO).

[0070] In some embodiments, the amino acid sequence described herein includes a truncation at the N-terminus and / or C-terminus. In some embodiments, the truncation has the following lengths: at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, or at least 31 amino acids. In other embodiments, the truncation has the following lengths: at most 3 amino acids, at most 5 amino acids, at most 10 amino acids, at most 15 amino acids, at most 20 amino acids, at most 25 amino acids, at most 26 amino acids, at most 27 amino acids, at most 28 amino acids, at most 29 amino acids, at most 30 amino acids, or at most 31 amino acids. In other embodiments, the truncation at the N-terminus and / or C-terminus of the enzyme or protein described herein has the following lengths: 5 to 50 amino acids, 10 to 50 amino acids, 15 to 50 amino acids, 20 to 50 amino acids, 10 to 40 amino acids, 10 to 35 amino acids, 15 to 35 amino acids, 20 to 35 amino acids, 25 to 32 amino acids, 28 to 32 amino acids, 2 to 10 amino acids, or 2 to 5 amino acids. Alternatively, the truncation described herein is in one or more of SEQ ID NO: 1-43. In another aspect, the truncation described herein is relative to SEQ ID NO: 8, SEQ ID NO: 24, or SEQ ID NO: 40-43.

[0071] In some embodiments, the enzyme comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% of the amino acid sequence identical to any one of SEQ ID NO: 1-43. In some embodiments, the enzyme comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% of the amino acid sequence identical to any one of SEQ ID NO: 24 or SEQ ID NO: 40-43. In some embodiments, the enzyme described herein comprises N137G, Y235Q and / or A455Y mutations relative to SEQ ID NO: 8, wherein the enzyme further comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% of the same amino acid sequence as SEQ ID NO: 8, SEQ ID NO: 24 or SEQ ID NO: 40-43.

[0072] In another embodiment, the enzyme comprises the amino acid sequence of any one of SEQ ID NO: 1-43, wherein the amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions. In still other embodiments, the enzyme comprises the amino acid sequence of any one of SEQ ID NO: 1-43, wherein the amino acid sequence comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 or more amino acid substitutions.

[0073] In another embodiment, the enzyme comprises the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 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 some embodiments, the amino acid substitutions are conservative substitutions. In another embodiment, the enzyme comprises the amino acid sequence of any one of SEQ ID NO: 24 or SEQ ID NO: 40-43, wherein the amino acid sequence comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 or more amino acid substitutions.

[0074] BHB sensor, and methods for detecting and measuring BHB concentration.

[0075] Typically, a biosensor is a device that measures a biological or chemical reaction by generating a signal proportional to the concentration of the analyte in 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, EssaysBiochem. 60(1):1-8, which is incorporated herein by reference in its entirety.

[0076] In other respects, this disclosure provides sensors capable of detecting and / or measuring BHB concentration, including sensors utilizing the enzymes described herein for detection and / or measurement, such as non-wearable sensors. Such sensors, for example, non-wearable sensors, may take the form of test strips or one-touch fingertip sweat sensors. In other respects, the enzyme comprises one or more of SEQ ID NO: 1-43, fragments thereof, or modified enzymes of SEQ ID NO: 1-43 as described herein. A detailed discussion of suitable applications of the enzyme in various non-wearable sensors can be found in Yin et al. (WO 2022 / 170361), the contents of which are incorporated herein by reference in their entirety.

[0077] Non-wearable sensors can be used to detect or measure BHB in body fluids, including one or more of whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudate, cyst contents, and / or ascites.

[0078] In some embodiments, the test strip includes: a base layer; and one or more sensing reagents disposed on at least a portion of the base layer, wherein at least one sensing reagent includes the enzyme.

[0079] In some embodiments, at least one of the sensing reagents further includes a cofactor, a mediator, and / or other adjuvants or excipients. In some embodiments, the cofactor includes flavin adenine dinucleotide (FAD). In some embodiments, the mediator includes one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / methylene blue, tetrathione, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes.

[0080] In other embodiments, the test strip further comprises one or more tetrazolium salts. In some embodiments, one or more tetrazolium salts are selected from 2-(p-iodophenyl)-3-(p-nitrobenzene)-5-phenyltetrazole chloride (INT) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazole bromide (MTT).

[0081] The material of the base layer may include one or more of composite materials, fibrous materials, woven fabrics, non-woven fabrics, polymers, adhesives, films, gels, PTFE, and / or silicone. This disclosure further provides a BHB sensor that senses, detects, and / or measures BHB concentration in a continuous, ongoing, or on-demand manner by utilizing the enzymes described herein. In other aspects, the enzyme comprises one or more of SEQ ID NO: 1-43, fragments thereof, or modified enzymes of SEQ ID NO: 1-43 as described herein.

[0082] In some embodiments, a BHB sensor capable of sensing, detecting, and / or measuring BHB concentration includes: a sensor comprising a sensing reagent, wherein the sensing reagent composition contains the engineered enzyme; and a reference electrode.

[0083] In some embodiments, the sensing reagent composition further includes one or more of a cofactor, mediator, adjuvant, or excipient.

[0084] In some embodiments, the wearable BHB sensor is configured to continuously, persistently, or on-demand measure the BHB concentration of a subject 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-based sensor or a microneedle-based sensor. In some embodiments, the sensor can use any method for BHB measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetric, iontophoresis, radiation, immunochemical, etc. In some embodiments, the wearable BHB sensor is configured to measure the BHB concentration in bodily fluids. In other embodiments, the body fluids include one or more of whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudate, cyst contents, and / or ascites.

[0085] This disclosure further provides a system for detecting and measuring BHB concentration using the enzyme described herein. In other aspects, the enzyme comprises one or more of SEQ ID NO: 1-43, fragments thereof, or modified enzymes of SEQ ID NO: 1-43 as described herein.

[0086] In some implementations, a system for detecting and measuring BHB concentration includes: a BHB sensor configured to measure the BHB concentration of a target and output a data stream; and a device connected to the BHB sensor, wherein the device includes: a processor configured to process the data stream from the BHB sensor; and an interface configured to display and / or transmit the measured BHB concentration value.

[0087] In some implementations, BHB sensors or BHB sensing systems are created by integrating the engineered enzyme into various sensing platforms and / or devices. Platforms and / or devices that can employ the engineered enzyme include, but are not limited to, pH change sensors (e.g., Chodavarapu). et al. As described in US 7,794,584), electrochemical sensors (e.g., Simpson). et al. US 7,081,195, Lebel et al. US 6,915,147 and Jina et al. As described in US 2010 / 0049021), optical sensors (e.g., Petrich) et al. (as described in US 10,724,943), implantable sensor platforms (e.g., as described in Jain, EP 2079358), and chip-shaped blood analysis devices (e.g., Ogawa). et al. Monitoring systems (such as those described in US 7,582,259) and remotely connected control terminals (e.g., Ogawa) et al. (as described in US 7,582,259). The contents of each of these patents and applications are incorporated herein by reference in their entirety. In some embodiments according to this disclosure, the BHB sensor, BHB sensing system, BHB device, and applicable technology are non-invasive or minimally invasive, wearable biosensing and / or chemical monitoring sensors, systems, devices, and technologies.

[0088] In some embodiments of this disclosure, the electrochemical biosensor or chemical sensor for qualitative and / or quantitative measurement of BHB is a tip sensor.

[0089] In some embodiments, the BHB sensor or BHB sensing system is a one-touch fingertip sweat sensor, and the personalized data processing methods, systems, or apparatus are those described in the following patent applications, such as WIPO International Patent Application No. PCT / 2022 / 070554, filed February 7, 2022, and published on August 11, 2022, with publication number WO / 2022 / 170361; or U.S. Patent Application No. 18 / 264,755, filed August 8, 2023, and published on February 15, 2024, with publication number 2024 / 0049994 A1. The contents of each of these applications are incorporated herein by reference in their entirety.

[0090] In some embodiments, the BHB sensor or BHB sensing system utilizes reverse iontophoresis, a non-invasive or minimally invasive method for extracting biomarkers. Reverse iontophoresis is a technique that applies a small current to the skin to extract polar and nonpolar molecules onto an 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, granted July 28, 2020; U.S. Patent Application Publication No. US 2021 / 0076988, published March 18, 2021; and WIPO International Patent Application No. PCT / US2015 / 063836, filed May 31, 2017, and published June 9, 2016, under Publication No. WO / 2016 / 090189. The contents of each of these patents and applications are incorporated herein by reference in their entirety.

[0091] In some embodiments, the electrochemical biosensor or chemical sensor is a wearable epidermal electrochemical sensor device for detecting BHB in sweat. Detailed descriptions of suitable epidermal electrochemical sensor devices can be found, for example, in U.S. Patent Nos. 9,820,692 and 11,185,286. The contents of each of these patents are incorporated herein by reference in their entirety.

[0092] This disclosure further provides a method for detecting and measuring BHB concentration using the enzyme described herein. In other aspects, the enzyme comprises one or more of SEQ ID NO: 1-43, fragments thereof, or modified enzymes of SEQ ID NO: 1-43 as described herein.

[0093] In some implementations, methods for detecting and measuring BHB concentration include: obtaining bodily fluid from a subject; placing the bodily fluid in a BHB sensor; determining a single-point BHB concentration in the bodily fluid; and / or displaying and / or transmitting the single-point BHB concentration on an interface.

[0094] Test kit products

[0095] This disclosure further relates to test kit products, which are applicable to, for example, automated analyzers, systems and devices.

[0096] In some embodiments, the test kit product described herein includes reagent A and reagent B. In some embodiments, the test kit product is activated by mixing reagent A and reagent B.

[0097] In some embodiments, reagent A comprises the enzyme described herein. In some embodiments, the enzyme described herein is dissolved in a buffer solution. In some embodiments, reagent A comprises NaCl and / or KCl. In some embodiments, the concentration of NaCl and / or KCl is from 0.1 M to 5 M. In some embodiments, reagent A comprises citric acid, acetic acid, KH₂PO₄, N-cyclohexyl-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, and / or tris(hydroxymethyl)aminomethane. In some embodiments, the pH of reagent A is from 3 to 11, optionally from 4 to 10, optionally from 5 to 9, optionally from 6 to 8.

[0098] In some embodiments, reagent B comprises a cofactor, mediator, excipient, adjuvant, or carrier. In one embodiment, the enzyme-containing composition is dissolved in water. In some embodiments, the cofactor comprises one or more of flavin adenine dinucleotide (FAD), semiquinone flavin adenine dinucleotide (FADH), and / or quinone flavin adenine dinucleotide (FADH2). In some embodiments, the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / methylene blue, tetrathionyl fulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes. In some embodiments, reagent B comprises NaCl and / or KCl. In some embodiments, the concentration of NaCl and / or KCl is from 0.1 M to 5 M. In some embodiments, reagent B comprises citric acid, acetic acid, KH₂PO₄, N-cyclohexyl-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, 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 tetrazolium salts are selected from 2-(p-iodophenyl)-3-(p-nitrobenzene)-5-phenyltetrazole chloride (INT) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazole bromide (MTT).

[0099] Pharmaceutical Composition

[0100] In another embodiment, the present invention relates to the use of enzymes as a component of a composition.

[0101] In some embodiments, the composition comprises one or more active ingredients. In some embodiments, the active ingredient further comprises one or more other enzymes, such as glucose oxidase, glucosyltransferase, fructosyltransferase, catalase, amylase, lactase, lipase, and / or protease.

[0102] 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 known in the art can be derived from, for example, Margolin. et al. The contents of (US 7,718,169) are found here and are incorporated into this paper by reference.

[0103] In some embodiments, the one or more excipients comprise one or more of the following substances: microcrystalline cellulose, maltodextrin, crospovidone, colloidal silica, 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, hexanediol, methoxy polyethylene glycol, gelatin, hydroxypropyl-β-cyclohexane. Arginine, 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, dextrose, starch, glycogen), sugar alcohols (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 salts, lactates, glucuronic acid, galacturonic acid), emulsifiers or solubilizers / stabilizers (e.g., gum arabic, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polysorbate, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monolaurate, etc.) Stearic acid and other sorbitol derivatives, polyoxyethylene derivatives, waxes, polyoxyethylene derivatives, sorbitol derivatives, thickeners (e.g., agar, alginate and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives, propylene carbonate, polyethylene glycol, hexanediol, tyloxapol, or salts of such compounds). Further examples of excipients are described in the Handbook of Pharmaceutical Excipients, jointly published by the American Pharmaceutical Association and the British Medical Association.

[0104] In some embodiments, one or more adjuvants comprise one or more of water-in-oil and oil-in-water emulsions, aluminum salt adjuvants, liposomes, and / or CpG oligodeoxynucleotide species adsorbed onto aluminum salts.

[0105] In some embodiments, the one or more carriers comprise one or more polymers for encapsulating protein crystals to deliver proteins, including controlled-release biodelivery. In some embodiments, the polymers comprise biocompatible and biodegradable polymers or mixtures thereof. In some embodiments, the dissolution rate and the resulting enzyme delivery rate are determined by factors such as the specific encapsulation technology, polymer composition, polymer crosslinking, polymer thickness, polymer stability, enzyme crystal geometry, and the degree of enzyme crosslinking (if any).

[0106] In some embodiments, one or more active ingredients may be present in the composition in a form bound to a polymer carrier.

[0107] Useful polymer carriers include, for example, polymers for encapsulating protein crystals to deliver proteins, including controlled-release biodelivery. Such polymers include biocompatible and biodegradable polymers or mixtures thereof. Preferably, the polymer carrier is a biodegradable polymer. The dissolution rate and the resulting enzyme delivery rate will be determined by factors such as the specific encapsulation technology, polymer composition, polymer crosslinking, polymer thickness, polymer stability, enzyme crystal geometry, and the degree of enzyme crosslinking (if any). Other useful carriers include water.

[0108] Ketogenic diet and methods for treating diseases

[0109] This disclosure further provides a ketogenic diet and methods for treating diseases by using devices, systems, methods, or kits incorporating the enzymes described herein. In one embodiment, the ketogenic diet is used in conjunction with the devices and systems described herein. Representative descriptions of nutritional ketosis and its treatable diseases may be found, for example, in D'Agostino. et al. The content of (US2020 / 0268701) is found here and incorporated into this article in its entirety by reference.

[0110] Ketoacidosis

[0111] The terms "ketones" and / or "ketone bodies" refer to water-soluble molecules or compounds containing a ketone group derived from fatty acids. As is known in the art, a ketone group comprises one or more carbonyl groups -C(=O)-. In the context of this application, "ketones" and / or "ketone bodies" include a non-exhaustive list of compounds such as acetone (2-acetone, dimethyl ketone, or β-ketopropane), acetoacetic acid (3-oxobutyric acid, acetone carboxylic acid, or diacetic acid), acetoacetate, β-hydroxybutyric acid (3-hydroxybutyric acid), β-hydroxybutyrate (BHB, 3HB, or 3-hydroxybutyrate), β-ketovalerate (3-oxovalerate, 3-oxovalerate, or 3-ketovalerate), and β-hydroxyvalerate (3-hydroxyvalerate, 3-hydroxyvalerate, or β-hydroxyvalerate). 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 MetabRev. 15(6):412-426, which is incorporated herein by reference in its entirety.

[0112] BHB is a small anionic acid metabolite containing a hydroxyl group and is the main ketone body distributed in the human brain, serving as a primary energy source in cases of glucose deficiency. For a review of BHB, see, for example, JC Newman and E. Verdin, August 21, 2017, ꞵ-Hydroxybutyrate, Ann Rev Nutr. 37:51-76.

[0113] The term "acetoacetate" refers to the conjugate base of acetoacetic acid and is used interchangeably with the term "3-oxobutyrate".

[0114] In one implementation, the enzymes, methods, apparatus, and systems described herein are associated with ketosis and can be used, for example, for nutritional or therapeutic ketosis. Nutritional or therapeutic ketosis is a physiological state characterized by elevated blood ketone body levels (typically above 0.5 mmol / L) caused by a ketogenic diet, calorie restriction, therapeutic fasting, and / or supplementation with ketogenic precursors. Ketone bodies represent alternative energy substrates for peripheral tissues and the central nervous system. The two most abundant and physiologically significant ketone bodies are acetoacetate and β-hydroxybutyrate (BHB), while a third ketone body, acetone, is produced as a byproduct of pulmonary respiration. The range of ketone body production during nutritional or therapeutic ketosis is approximately 0.3 mmol / L to 16 mmol / L. Ketone body metabolism is associated with anticonvulsant effects, enhanced brain metabolism, neuroprotection, muscle-preserving properties, and improved cognitive and physical performance. Science-based improvements in cellular metabolic efficiency through ketone supplementation management may have beneficial effects on physical, cognitive, and mental health, combatant resilience, and long-term health implications for common preventable diseases such as obesity, neurodegenerative diseases, autoimmune diseases, diabetes, and cancer.

[0115] Under normal dietary conditions, the brain relies solely on glucose metabolism for its metabolic energy. Although the brain accounts for only 2% of body weight, it consumes 25% of total glucose. During periods of limited glucose supply due to starvation / fasting, calorie restriction, or carbohydrate restriction (e.g., on a ketogenic diet), ketones can substitute glucose to supply most of the brain's metabolic energy needs (>50%). During carbohydrate deficiency, the reduced glucose supply leads to a shift in metabolism towards fatty acid β-oxidation, producing ketone bodies to maintain energy balance.

[0116] Dietary carbohydrates include simple sugars, such as sucrose found in foods like potatoes and pasta, and complex carbohydrates (starch). Over the past two centuries, carbohydrate and sugar consumption has increased dramatically in Western societies. When a person consumes sugars and carbohydrates, the pancreas secretes insulin, a hormone used to convert sugars and carbohydrates into glucose. Glucose is then used by the body as a fuel source. In most Western diets, glucose is the body's primary fuel source.

[0117] During periods of fasting, strenuous exercise, and / or low carbohydrate consumption, the body's stored glucose is rapidly used and can be depleted quickly. Because glucose stores are depleted and cannot be replenished, the body turns to another method of generating energy: ketone bodies. Ketone bodies can be used as alternative fuel by every cell in the body to meet its energy needs, including those of the brain. For example, during prolonged fasting, blood ketone levels can increase to as high as 2 or 3 mmol / L, or even higher. It is generally understood and accepted that when blood ketone levels rise above 0.5 mmol / L, the heart, brain, and surrounding tissues use ketone bodies (β-hydroxybutyrate and acetoacetate) as their primary fuel source. This condition is called ketosis or "nutritional ketosis." This is different from diabetic or alcoholic ketoacidosis, which is characterized by the uncontrolled accumulation of ketone bodies accompanied by a decrease in blood pH. Diabetic ketoacidosis is associated with insulin deficiency, which occurs in patients with type 1 diabetes. Ketoacidosis typically results in blood ketone levels exceeding 15 mmol / L, accompanied by metabolic disturbances and electrolyte imbalances.

[0118] When in ketosis, the body essentially burns fat as fuel. This is because the fat stored in the body is used to produce water-soluble ketone bodies, β-hydroxybutyrate (BHB) and acetoacetate (also known as acetylacetonate). These ketone bodies are then used by the body as its primary energy source.

[0119] When the body lacks a dietary source of glucose or sugars and its glycogen stores are depleted, it enters a state of ketosis. This typically occurs during fasting, exercise, and / or a carbohydrate-restricted ketogenic diet. Once in ketosis, the body begins breaking down fat into fatty acids and glycerol, converting the fatty acids into acetyl-CoA molecules, which are ultimately converted into ketone bodies in the liver. In other words, during ketogenic metabolism in the liver, the body uses both dietary fat and body fat as its primary energy source. Therefore, when entering ketosis, it is easy to lose body fat by reducing dietary fat intake and regulating carbohydrate intake to a sufficiently low level to maintain ketosis.

[0120] In one embodiment, the enzymes, methods, apparatus, and systems described herein relate to and may contribute to weight loss, regulating and / or monitoring weight loss, and regulating and / or monitoring carbohydrate intake. In other embodiments, the enzymes, methods, apparatus, and systems described herein relate to regulating and / or monitoring ketosis over hours, days, weeks, months, or years. In some embodiments, ketone concentrations are monitored for the purpose of detecting and preventing ketoacidosis.

[0121] Ketogenic diet and weight loss

[0122] This disclosure provides a method for treating subjects requiring weight loss using the enzymes, methods, apparatus, or systems described herein.

[0123] The ketogenic diet is a diet high in dietary fat, low in carbohydrates, and moderate in protein (approximately 1 g / kg to 2 g / kg). A classic ketogenic diet consists of a strict regimen of 4 servings of fat to 1 serving of protein daily, with less than 25 to 50 grams of carbohydrates. It has been shown that the ideal macronutrient ratio for maintaining a ketogenic diet is 65% to 85% of calories from fat, 10% to 20% from protein, and 5% from carbohydrates. In some embodiments, this disclosure provides methods for assisting subjects in monitoring and / or correcting carbohydrate intake by utilizing the enzymes, systems, and devices described herein, wherein the carbohydrate intake is approximately 10 to 30 grams, approximately 15 to 40 grams, approximately 20 to 50 grams, approximately 30 to 60 grams, or approximately 40 to 80 grams of carbohydrates per day. This dietary regimen can be used in conjunction with the devices and systems described herein.

[0124] One advantage of weight loss through a ketogenic diet is that it leads to a loss of fat storage while maintaining and protecting muscle mass. Some studies suggest that the muscle-preserving properties of a ketogenic diet improve physical performance. Athletes maintaining nutritional ketosis maintain lower insulin levels and are able to better utilize fatty acids and ketones as fuel, effectively conserving blood sugar, which optimizes and prolongs physical and mental performance. This state is called "keto-adapted." Keto-adaptation occurs when the body regulates itself to ketosis by building up the necessary fat-burning enzymes, hormone levels change to adapt to ketosis, glycogen stores in muscles and liver decrease, and the body carries less water.

[0125] Individuals following a standard US diet are expected to reach 60% to 65% of their peak oxygen consumption (VO2 maximum) during exercise; higher exercise levels deplete glycogen stores. Keto-adapted individuals draw a greater proportion of substrate from fats and ketones (conserving glycogen) and are able to shift peaks to higher VO2 levels, thus sustaining exercise for extended periods. Transitioning to ketosis (blood ketones >0.5 mmol / L) typically takes 1 to 2 weeks, accompanied by strict carbohydrate restriction (<25 g / day) and moderate protein restriction (1 g / kg / day), balancing macronutrients in fats. A sustained physiological decline in glucose and insulin is necessary for sustained hepatic ketogenesis, which is very difficult for most people.

[0126] Vlahakos (US 6,613,356) (the contents of which are incorporated herein by reference in their entirety) provides a weight-loss composition using the butyrate ion from potassium butyrate or related compounds. Butyrate stimulates receptors in the stomach, creating a feeling of fullness and retaining food in the stomach. Therefore, ingesting a butyrate precursor before meals can reduce food consumption. Tests have shown that this composition improves patients' tolerance to strenuous exercise, improves hypercholesterolemia and hypertriglyceridemia, and reduces fatigue.

[0127] Another advantage of using a ketogenic diet for weight loss is that being in ketosis can reduce feelings of hunger. In fact, hunger is a frequently cited major obstacle to maintaining a traditional calorie-restricted diet.

[0128] Because the presence of blood ketones can be easily measured using one of the many ketone test strips or devices available on the commercial market, those hoping to achieve ketosis can easily track their progress. Just as those on a conventional diet can get positive feedback by weighing themselves and measuring weight loss, those pursuing ketosis can also get encouragement by measuring their blood ketone levels. However, when transitioning to ketosis, any measurable increase in blood ketone levels may take several days to two weeks or longer to show up in a urine test.

[0129] Methods of implementing a ketogenic diet and monitoring ketosis using a BHB sensor

[0130] In some embodiments, a ketogenic diet comprises approximately 5% to 10% carbohydrates; approximately 55% to 85% fat; and approximately 10% to 40% protein. In some embodiments, a ketogenic diet comprises 15 g to 50 g carbohydrates; 60 g to 400 g fat; and 40 g to 100 g protein. In some embodiments, a ketogenic diet provides 1500 kcal / day to 3000 kcal / day. In some embodiments, a ketogenic diet includes intermittent or prolonged fasting. In some aspects, these parameters can be adjusted and / or monitored using the methods, systems, and apparatus described herein.

[0131] In some implementations, the duration of daily consumption of the ketogenic diet is at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.

[0132] In some implementation schemes, 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.

[0133] In some embodiments, ketosis monitoring is performed by detecting BHB concentration using a β-hydroxybutyrate (BHB) sensor. In some embodiments, the BHB concentration is the blood BHB concentration or the interstitial fluid (ISF) BHB concentration. In some embodiments, ketosis monitoring is performed at least once daily, at least twice daily, at least three times daily, at least four times daily, at least five times daily, 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 implementations, ketosis monitoring is conducted at 7:00 AM, 8:00 AM, 9:00 AM, 10:00 AM, 11:00 AM, 12:00 PM, 1:00 PM, 2:00 PM, 3:00 PM, 4:00 PM, 5:00 PM, 6:00 PM, 7:00 PM, 8:00 PM, 9:00 PM, and / or 10:00 PM. In some implementations, ketosis monitoring is conducted 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 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 after dinner, 1 hour after dinner, 2 hours after dinner, and 3 hours after dinner.

[0134] Effects of ketosis on cognition and physical performance

[0135] Performance studies have shown that ketone supplementation improves motor function, endurance, and cognitive function. In rats given ketone supplementation, cardiopulmonary and neurological function recovery was achieved under extreme oxidative stress (hyperxia). Many people on a ketogenic diet report greater mental clarity, improved multitasking ability, and better mood and balance.

[0136] Other benefits of the ketogenic diet include anti-aging and mood-stabilizing effects. Other studies have shown that elevated blood ketone levels are associated with superior performance in endurance time, oxygen consumption, heart rate, blood lactate levels, and power output.

[0137] Therefore, in one implementation, the enzymes, methods, apparatus, and systems described herein are related to, and may contribute to, methods for improving endurance, cognitive function, and mood.

[0138] Example

[0139] The following examples illustrate specific aspects of this disclosure and are not intended to limit this disclosure in any way.

[0140] Example 1: Enzyme Design

[0141] (R)-β-hydroxybutyrate (R-BHB) can be converted to 3-oxobutyrate by oxidation of secondary alcohols, as shown below.

[0142]

[0143] Naturally occurring alcohol oxidases can promote the oxidation of secondary alcohols to ketones. For example, cholesterol oxidases belonging to the redox enzyme family EC1.1.3.6 have the ability to convert cholesterol to cholesterol-4-en-3-one. The reaction for converting cholesterol to cholesterol-4-en-3-one is shown below. This ability makes cholesterol oxidases potential candidates for developing optimized engineered enzymes capable of converting (R)-β-hydroxybutyrate to 3-oxobutyrate.

[0144]

[0145] According to this disclosure, any suitable assay can be used to determine the presence and / or amount of hydrogen peroxide (H2O2) produced by an enzymatic reaction. For example, the obtained hydrogen peroxide can be detected using a sensitive and stable fluorescent probe. See, for example, Allain et al., Clin Chem 1974, 20:470-475; Amundson et al., J BiochemBiophys Meth 1999, 38:43-52; and dos Santos Ferreira et al., Clin Chim Acta 2015, 446:263-266. The contents of each of these applications are incorporated herein by reference in their entirety.

[0146] To identify suitable candidate oxidases for further engineering, a sequence similarity network (SSN) consisting of 4000 bacterial oxidases was screened. The SSN method is described in Atkinson. et al. , PLoS ONE In February 2009, 4(2), e4345, it was incorporated into this paper by reference. Preliminary screening results are as follows: Figure 1 As shown in Table 3, 39 amino acid sequences were identified as potential candidate sequences.

[0147] The cholesterol oxidase (oxidase 8) of Uniprot ID: A0A1Z4IKQ1, derived from *Pseudococcus pseudobranchii*, was identified using the Alphafold protein structure database for structure prediction. The Alphafold prediction method is described in detail in Varadi. et al. , Nucleic Acids Res. , Vol. 50, Issue D1, 7 January 2022, pagesD439–D444 and Jumper et al. , Nature Each of the above-mentioned references is incorporated herein by reference in Vol. 596, pages 583–589 (2021). The Alphafold model was then visualized using PyMol (http: / / citebay.com / how-to-cite / pymol / ). The Alphafold model was used to compare oxidase 8 with engineered cholesterol oxidase (ShCOb, SEQ ID NO: 21, from *Streptomyces hygroscopicus*, see Heath). et al. , Chembiochem. 2022Apr 5;23(7):e202200075) Figure 2 As shown.

[0148] The computer-aided design of the engineered enzyme was then analyzed. The mutant N137G was introduced to create space for BHB at the enzyme's active site, and the mutants Y235Q and A455Y were introduced to form hydrogen bonds with the carboxylic acid of wild-type BHB (reference sequence = SEQ ID NO: 8). A 31-amino acid N-terminal truncation was introduced (amino acid positions 2 to 32 of SEQ ID NO: 8 [oxidase 8 wt]). Figure 3 A visual representation of the engineered enzyme design predicted by Alphafold is shown. The amino acid sequences of the engineered enzyme are also provided in Table 3. As shown in Table 3, when compared with SEQ ID NO: 8 (also referred to herein as 8_WT), SEQ ID NO: 24 (also referred to herein as 8_MUT or oxidase 8_mut) contains all three amino acid substitutions and truncations at positions 2 to 32. Considering the 31 truncated amino acids, these three mutations correspond to N106G, Y204Q, and / or A424Y in the resulting SEQ ID NO: 24. Alternatively, if the “M” at amino acid position number 1 (i.e., methionine or Met) is ignored and the 31 truncated amino acids are considered, these three mutations correspond to N105G, Y203Q, and / or A423Y in the resulting SEQ ID NO: 24.

[0149] Table 3: Amino acid sequences of wild-type and mutant oxidases

[0150] Example 2: Enzyme Expression and Purification

[0151] Methods for expressing and purifying engineered enzymes are described in Yoshikuni. et al. In US Patent Publication No. 2022 / 0348970 A1, the contents of each of its articles are incorporated herein by reference in their entirety.

[0152] Overnight culture of BLR cells suspended in 2 mL was transformed with the pet29b+ plasmid (encoding a target polypeptide with a C-terminal His tag) and grown in Terrific Broth containing 50 μg / mL kanamycin. The culture was diluted 1:1000 with 500 mL Terrific Broth containing 1 mM MgSO4, 1% glucose, and 50 μg / mL antibiotic and incubated at 37°C for 24 h. The culture was then settled at 5000G for 10 min and resuspended in self-induction medium (TB broth, 1 mM MgSO4, 1×NPS, and 1×5052) and induced at 18°C ​​for 24 h. 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) and 1 mM benzyl sulfonyl fluoride. The cell lysate suspension was sonicated for 2 minutes and then centrifuged at 5,000 G. The supernatant was loaded into a gravity flow column with 500 μL cobalt beads and washed five times with 15 mL of lysis buffer. Proteins were eluted with 1 mL of elution buffer (1×PBS, pH 7.5, 200 mM imidazole). Protein concentrations were determined using a Synergy H1 spectrophotometer (Biotek) by measuring absorbance at 280 nm using the calculated extinction coefficient. Enzymes at concentrations of at least 0.1 mg / mL were then determined.

[0153] Example 3: Enzyme Activity

[0154] Enzyme activity is evaluated by measuring the hydrogen peroxide and acetoacetic acid produced. Commercial test strips are used to detect the presence of hydrogen peroxide and acetoacetic acid.

[0155] To prepare the substrate, 100 mM BHB was dissolved in 400 mM sodium phosphate (pH 7) and 100 mM NaCl. In a 96-well plate, 100 µL of 0.5 mg / mL enzyme and 100 µL of the dissolved substrate were combined. The reaction was allowed to proceed at 21°C for 24 hours. After the incubation period, 10 µL of each reactant was placed in a separate test strip for colorimetric analysis using hydrogen peroxide and acetoacetic acid test strips.

[0156] Detection of hydrogen peroxide: Use Bartovation hydrogen peroxide test strips (https: / / bartovation.com) to detect the presence of hydrogen peroxide. These test strips are calibrated at 0 ppm, 1 ppm, 3 ppm, 10 ppm, 50 ppm, and 100 ppm. If hydrogen peroxide is present, the test strip turns blue, with a bluer color indicating a higher concentration of hydrogen peroxide.

[0157] like Figure 4 As shown, the engineered enzyme (oxidase 8_mut) containing the amino acid sequence SEQ ID NO: 24 produces hydrogen peroxide in the presence of 2-pentanol, (R)-2-pentanol, BHB, or (R)-β-hydroxybutyrate (R-BHB), as the test strip turns blue upon contact with the reaction mixture of these reactants. In contrast, wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) exhibits less activity in oxidizing BHB, as indicated by a paler blue color; and does not oxidize R-BHB, as the test strip does not turn blue. The mutant enzyme of SEQ ID NO: 24 produced positive test results, while none of SEQ ID NO: 1-23 and SEQ ID NO: 25-39 produced positive results.

[0158] Detection of acetoacetic acid: The Bayer Ketostix® Urine Reagent Test Strips are used to detect the presence of acetoacetic acid in urine. These strips are calibrated at 0 mg / dL, 5 mg / dL, 15 mg / dL, 40 mg / dL, 80 mg / dL, and 100 mg / dL. The test strip turns red if acetoacetic acid is present.

[0159] like Figure 5As shown, when R-BHB is used as a reactant, the engineered enzyme (oxidase 8_mut) containing the amino acid sequence SEQ ID NO: 24 produces acetoacetic acid, as the test strip turns red. In contrast, when R-BHB is used as a reactant, wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) does not produce acetoacetic acid, as the test strip does not turn red.

[0160] HPLC-MS analysis of acetoacetate: The method for detecting acetoacetate using high-performance liquid chromatography-mass spectrometry (HPLC-MS) is described in detail in U. et al. , Nat. Prod. Chem. Res. The content of 2019 7(2):364 is incorporated into this paper by reference.

[0161] 2,4-Dinitrophenylhydrazine (2,4-DNP) is commonly used for the detection of ketones and aldehydes because it readily reacts with the C=O carbonyl group to form hydrazone compounds. The reaction of R-BHB with 2,4-DNP is shown below.

[0162]

[0163] The reaction described above 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, a sample was prepared for HPLC-MS analysis using 200 µL of the reaction mixture, 100 µL of 4 M acetate, and 700 µL of the derivatization mixture. The reaction was allowed to proceed at 60°C for 1 hour. The reaction mixture was then centrifuged at 5000 G for 10 minutes. The supernatant was collected for HPLC-MS analysis.

[0164] An Agilent Eclipse Plus C18 RRHD 1.8µm column was used for separation. Analysis was performed with mobile phase A (H₂O, 0.1% formic acid) and mobile phase B (100% ACN, 0.1% formic acid) at a flow rate of 0.3 mL / min. A gradient process was used, starting with 80% MPA / 20% MPB and transitioning to 0% MPA / 100% MPB over a 10-minute process. For the detection of acetoacetic acid, single-ion mode was used in MS to search for derivatized products. Figure 6 The HPLC-MS spectrum of the reaction mixture containing the engineered enzyme (oxidase 8_mut) with amino acid sequence SEQ ID NO: 24 is shown. Figure 7 The HPLC-MS spectra of the oxidase 8_mut reaction mixture were compared with those of commercially available synthetic acetoacetate. Figure 8The HPLC-MS spectra of the oxidase 8_mut reaction mixture were compared with those of the wild-type oxidase 8 (oxidase 8_WT, SEQ ID NO: 8) reaction mixture; Figure 9 The HPLC-MS spectra of the oxidase 8_mut reaction mixture were compared with those of the substrate control. Figures 6 to 9 Together, it was shown that the engineered enzyme oxidase 8_mut SEQ ID NO: 24 did indeed produce acetoacetic acid.

[0165] Results of hydrogen peroxide test, acetoacetate detection and HPLC-MS analysis: BHB activity was analyzed using the hydrogen peroxide assay (Bartovation hydrogen peroxide test strips), the acetoacetate assay (Bayer Ketostix®), and the HPLC-MS protocol described herein. Results are presented in Table 4. When incubated with BHB, oxidase 8 sometimes produces peroxides, but at trace levels, the amounts are extremely small, such as... Figure 4 As indicated. This most likely explains the positive activity of the peroxide test strip for wild-type SEQ ID NO: 8 in Table 4.

[0166] Table 4: Overview of Representative Activities

[0167] Example 4: Further Enzyme Design

[0168] Other enzymes applicable to the present invention and related methods include those listed in Tables 5 and 6.

[0169] Table 5: Amino acid sequences of representative enzymes according to this disclosure

[0170] Table 6: Amino acid sequences of representative enzymes according to this disclosure

[0171] On one hand, this disclosure provides a combination in which X 1 It can be N, G, P, A, or S; X 2 It can be Y, Q, N, K, E, or D; and X 3 It can be A, Y, W, F, or H.

[0172] This disclosure further numbered the implementation plan

[0173] Other topics considered in this disclosure are set forth in the embodiments numbered below: 1. An enzyme having oxidase activity, wherein the enzyme is engineered to exhibit improved β-hydroxybutyrate (BHB) activity.

[0174] 2. The enzyme according to embodiment 1, wherein the enzyme is capable of oxidizing β-hydroxybutyrate (BHB) to produce 3-oxobutyrate.

[0175] 3. The enzyme according to any one of embodiments 1 or 2, wherein the amino acid sequence is derived from an oxidase of the EC 1.1.3.6 family.

[0176] 4. The enzyme according to any one of embodiments 1 to 3, wherein the EC 1.1.3.6 family oxidase is a cholesterol oxidase.

[0177] 5. The enzyme according to any one of embodiments 1 to 4, wherein the enzyme is not naturally occurring and comprises one or more amino acid substitutions, deletions, or truncations relative to a natural or wild-type enzyme.

[0178] 6. The enzyme according to any one of embodiments 1 to 5, wherein the amino acid sequence of the enzyme comprises one or more amino acid substitutions corresponding to N137G, Y235Q and / or A455Y of SEQ ID NO: 8.

[0179] 7. The enzyme according to any one of embodiments 1 to 6, wherein the amino acid sequence of the enzyme comprises an N-terminal truncation of up to 3 amino acids, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, or up to 35 amino acids from one or more of SEQ ID NO: 8, SEQ ID NO: 24, or SEQ ID NO: 40-43.

[0180] 8. The enzyme according to any one of embodiments 1 to 6, wherein the amino acid sequence of the enzyme comprises an N-terminal truncation at positions 2 to 32 of the amino acid corresponding to SEQ ID NO: 8.

[0181] 9. The enzyme according to any one of embodiments 1 to 8, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% of the same amino acid sequence as one of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

[0182] 10. The enzyme according to any one of embodiments 1 to 8, comprising an amino acid sequence of one of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43, wherein the amino acid sequence comprises 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions.

[0183] 11. The enzyme according to embodiment 10, wherein the amino acid substitution occurs only in the protein portion responsible for binding and / or propagating BHB activity.

[0184] 12. The enzyme according to any one of embodiments 10 or 11, wherein the amino acid substitution is a conserved substitution.

[0185] 13. The enzyme according to any one of embodiments 1 to 11, wherein the enzyme exhibits increased BHB activity when measured by a peroxide test strip, an acetoacetic acid test strip, or liquid chromatography-mass spectrometry (LCMS).

[0186] 14. The enzyme according to embodiment 13, wherein the enzyme exhibits increased BHB activity when measured by liquid chromatography-mass spectrometry (LCMS).

[0187] 15. The enzyme according to embodiment 14, wherein, when measured by LCMS, the engineered enzyme exhibits approximately 1, 2, 3, 4, 5, 10, or 20 times higher BHB activity than the corresponding unmodified wild-type enzyme.

[0188] 16. The enzyme according to embodiment 14, wherein the enzyme comprises at least 90%, at least 95%, or at least 98% identical sequence to SEQ ID NO: 24, and wherein SEQ ID NO: 24 exhibits increased BHB activity compared to SEQ ID NO: 8 when measured by liquid chromatography-mass spectrometry (LCMS).

[0189] 17. The enzyme according to any one of embodiments 1 to 2, wherein the β-hydroxybutyrate is (R)-β-hydroxybutyrate.

[0190] 18. The enzyme according to any one of embodiments 1 to 2, wherein the β-hydroxybutyrate is (S)-β-hydroxybutyrate.

[0191] 19. The enzyme according to any one of embodiments 1 to 2, wherein the β-hydroxybutyrate is a mixture of (R)-β-hydroxybutyrate and (S)-β-hydroxybutyrate.

[0192] 20. A non-wearable BHB sensor capable of detecting and / or measuring BHB concentration, said non-wearable BHB sensor comprising an enzyme utilizing any one of embodiments 1 to 19.

[0193] 21. The non-wearable BHB sensor according to embodiment 20, wherein the non-wearable BHB sensor is a test strip.

[0194] 22. The test strip according to embodiment 21, wherein the test strip comprises: basal layer; and One or more sensing reagents are disposed on at least a portion of the substrate layer, wherein at least one of the sensing reagents comprises an enzyme as described in any one of embodiments 1 to 19.

[0195] 23. The test strip according to embodiment 21 or 22, wherein the at least one sensing reagent comprises a cofactor, mediator, adjuvant, carrier and / or excipient.

[0196] 24. The test strip according to embodiment 23, wherein the cofactor comprises flavin adenine dinucleotide (FAD).

[0197] 25. The test strip according to any one of embodiments 23 to 24, wherein the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / methylene blue, tetrathione, quinone derivatives, ferrocene, organometallic osmium complexes and / or organometallic ruthenium complexes.

[0198] 26. The 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, textiles, non-woven fabrics, polymers, adhesives, films, gels, PTFE and / or silicone.

[0199] 27. The non-wearable BHB sensor according to embodiment 20, wherein the non-wearable BHB sensor is a fingertip sweat sensor.

[0200] 28. The non-wearable BHB sensor according to embodiment 20, wherein the non-wearable BHB sensor can be used to detect or measure BHB in any bodily fluid.

[0201] 29. The non-wearable BHB sensor according to embodiment 28, wherein the bodily fluid can be obtained by non-invasive, minimally invasive or invasive means, optionally by finger-prick blood collection, blood sampling, spinal puncture, sweat and / or saliva collection.

[0202] 30. The non-wearable BHB sensor according to embodiment 29, wherein the body fluid includes one or more of whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudate, cyst contents, and / or ascites.

[0203] 31. A wearable BHB sensor capable of detecting, measuring, and / or monitoring BHB concentration, said wearable BHB sensor comprising: a sensor including a sensing electrode; a sensing reagent, wherein the sensing reagent composition comprises the enzyme described in any one of embodiments 1 to 19; and a reference electrode.

[0204] 32. The wearable BHB sensor according to embodiment 31, wherein the sensing reagent is on or adjacent to the sensing electrode.

[0205] 33. The wearable BHB sensor according to embodiment 31, wherein the sensing reagent is dispensed to contact the sensing electrode during use.

[0206] 34. The wearable BHB sensor according to embodiment 31, wherein the wearable BHB sensor is a continuous sensor, a persistent sensor, or an on-demand sensor.

[0207] 35. The wearable BHB sensor according to any one of embodiments 31 to 34, wherein the sensing reagent composition further comprises a cofactor, mediator, adjuvant, carrier and / or excipient.

[0208] 36. The wearable BHB sensor according to any one of embodiments 31 to 35, wherein the wearable BHB sensor is configured to continuously measure the BHB concentration of an object and output a data stream.

[0209] 37. The wearable BHB sensor according to any one of embodiments 31 to 36, wherein the wearable BHB sensor is a needle-based sensor, a microneedle-based sensor, a reverse iontophoresis sensor, a sweat-based sensor, or an implantable sensor.

[0210] 38. The wearable BHB sensor according to embodiment 37, wherein the wearable BHB sensor is located in body tissue.

[0211] 39. The 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 bodily fluids.

[0212] 40. The wearable BHB sensor according to embodiment 39, wherein the body fluid includes one or more of whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudate, cyst contents, and / or ascites.

[0213] 41. The wearable BHB sensor according to any one of embodiments 31 to 40, wherein the wearable BHB sensor is an in vivo wearable BHB sensor or a surface wearable BHB sensor.

[0214] 42. A system for detecting, measuring, and / or monitoring BHB concentration, the system comprising: a wearable BHB sensor according to any one of embodiments 31 to 41, wherein the wearable BHB sensor is configured to continuously, persistently, or on-demand measure the BHB concentration of a subject and output a data stream; and a device connected to the wearable BHB sensor, wherein the device comprises: a processor configured to process the data stream from the wearable BHB sensor; and an interface configured to display and / or transmit the measured BHB concentration values.

[0215] 43. A method for detecting, measuring, and / or monitoring BHB concentration, comprising: obtaining a body fluid from a subject; placing the body fluid in a wearable BHB sensor according to any one of embodiments 31 to 41; determining a single-point BHB concentration in the body fluid; and displaying and / or transmitting the single-point BHB concentration on an interface.

[0216] 44. A method for improving the health and / or well-being of individuals in need, comprising using a non-wearable BHB sensor, device, or apparatus as described in any one of embodiments 20 to 30.

[0217] 45. A method for improving the health and / or well-being of individuals in need, comprising using a test kit product, wherein the test kit product utilizes an enzyme as described in any one of embodiments 1 to 19.

[0218] 46. ​​A method for assisting individuals in need to lose weight, comprising using a non-wearable BHB sensor, device, or apparatus as described in any one of embodiments 20 to 30.

[0219] 47. The method according to any one of embodiments 44 to 46, used in combination with a food or supplement comprising at least one sugar alcohol or low-carbohydrate composition.

[0220] 48. The method according to any one of embodiments 44 to 47, wherein each unit of the food or supplement contains less than about 5 grams of carbohydrates, less than about 8 grams of carbohydrates, or less than about 10 grams of carbohydrates.

[0221] 49. The method according to any one of embodiments 47 and 48, used in combination with a food or supplement, wherein the food or supplement contains an exogenous ketone supplement and / or a ketogenic ingredient.

[0222] 50. The method according to embodiment 49, wherein the exogenous ketone supplement comprises ketone bodies and / or ketone body precursors.

[0223] 51. The method according to embodiment 50, wherein the ketone body and / or the precursor of the ketone body comprises one or more of acetone, acetoacetic acid, β-hydroxybutyrate (BHB), β-ketovalerate, β-hydroxyvalerate, 1,3-butanediol, and medium-chain triglycerides (MCT), wherein the medium-chain triglycerides (MCT) contain a fatty acid with a hydrocarbon side chain of 6 to 12 carbon atoms in length.

[0224] 52. The method according to embodiment 51, wherein the ketone body and / or the precursor of the ketone body is in the form of a salt and / or an ester.

[0225] 53. The method according to embodiment 51, wherein the MCT comprises one or more of the following: hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), and lauric acid (C12).

[0226] By incorporating via reference

[0227] All references, articles, publications, patents, patent publications, and patent applications cited above and / or below are incorporated herein by reference in their entirety for all purposes. All literature and similar materials cited in this application, including patents, patent applications, articles, books, papers, and internet web pages, are expressly incorporated herein by reference in their entirety for all purposes. Where the definitions of terms in the incorporated references differ from those provided in this application, the definitions provided in this application shall prevail.

[0228] However, any references, articles, publications, patents, patent publications and patent applications mentioned herein are not, and should not be, considered as an admission or in any way implied to constitute valid prior art or as part of common general knowledge in any country of the world.

Claims

1. An enzyme having oxidase activity, wherein the enzyme is engineered to exhibit improved β-hydroxybutyrate (BHB) activity.

2. The enzyme according to claim 1, wherein the enzyme is capable of oxidizing β-hydroxybutyrate (BHB) to produce 3-oxobutyrate.

3. The enzyme according to any one of claims 1 or 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 EC 1.1.3.6 family oxidase is a cholesterol oxidase.

5. The enzyme according to any one of claims 1 to 4, wherein the enzyme is not naturally occurring and comprises one or more amino acid substitutions, deletions, or truncations relative to a natural or wild-type enzyme.

6. The enzyme according to any one of claims 1 to 5, wherein the amino acid sequence of the enzyme comprises 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 comprises an N-terminal truncation of up to 3 amino acids, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, or up to 35 amino acids from one or more of SEQ ID NO: 8, SEQ ID NO: 24, or SEQ ID NO: 40-43.

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

8.

9. The enzyme according to any one of claims 1 to 8, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% of the same amino acid sequence as one of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO:

43.

10. The enzyme according to any one of claims 1 to 8, comprising an amino acid sequence of one of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43, wherein the amino acid sequence comprises 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions.

11. The enzyme of claim 10, wherein the amino acid substitution occurs only in the protein portion responsible for binding and / or propagating BHB activity.

12. The enzyme according to any one of claims 10 or 11, wherein the amino acid substitution is a conserved substitution.

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

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

15. The enzyme of claim 14, wherein, when measured by LCMS, the engineered enzyme exhibits about 1, about 2, about 3, about 4, about 5, about 10, or about 20 times higher BHB activity than the corresponding unmodified wild-type enzyme.

16. The enzyme of claim 14, wherein the enzyme comprises at least 90%, at least 95%, or at least 98% identical sequence to SEQ ID NO: 24, and wherein SEQ ID NO: 24 exhibits increased BHB activity compared to SEQ ID NO: 8 when measured by liquid chromatography-mass spectrometry (LCMS).

17. The enzyme according to any one of claims 1 to 2, wherein the β-hydroxybutyrate is (R)-β-hydroxybutyrate.

18. The enzyme according to any one of claims 1 to 2, wherein the β-hydroxybutyrate is (S)-β-hydroxybutyrate.

19. The enzyme according to any one of claims 1 to 2, wherein the β-hydroxybutyrate is a mixture of (R)-β-hydroxybutyrate and (S)-β-hydroxybutyrate.

20. A non-wearable BHB sensor capable of detecting and / or measuring BHB concentration, said non-wearable BHB sensor comprising an enzyme utilizing 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 test strip according to claim 21, wherein the test strip comprises: basal layer; and One or more sensing reagents are disposed on at least a portion of the substrate layer, wherein at least one of the sensing reagents comprises the enzyme according to any one of claims 1 to 19.

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 of claim 23, wherein the cofactor comprises flavin adenine dinucleotide (FAD).

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

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, textiles, non-woven fabrics, polymers, adhesives, films, gels, PTFE and / or silicone.

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

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

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

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

31. A wearable BHB sensor capable of detecting, measuring, and / or monitoring BHB concentration, said wearable BHB sensor comprising: A sensor, which includes sensing electrodes; A sensing reagent, wherein the sensing reagent composition comprises the enzyme according to any one of claims 1 to 19; and Reference electrode.

32. The wearable BHB sensor of claim 31, wherein the sensing agent is on or adjacent to the sensing electrode.

33. The wearable BHB sensor of claim 31, wherein the sensing reagent is dispensed to contact the sensing electrode during use.

34. The wearable BHB sensor according to claim 31, wherein the wearable BHB sensor is a continuous sensor, a persistent 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, mediator, adjuvant, carrier, and / or 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 BHB concentration of an object 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-based sensor, a microneedle-based sensor, a reverse iontophoresis sensor, a sweat-based sensor, or an implantable sensor.

38. The wearable BHB sensor of claim 37, wherein the wearable BHB sensor is located in 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 concentration of BHB in bodily fluids.

40. The wearable BHB sensor of claim 39, wherein the body fluid comprises one or more of whole blood, serum, plasma, blood components other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal secretions, breast milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneal fluid, glandular secretions, exudate, cyst 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 vivo wearable BHB sensor or a surface wearable BHB sensor.

42. A system for detecting, measuring, and / or monitoring BHB concentration, said system comprising: The wearable BHB sensor according to any one of claims 31 to 41, wherein the wearable BHB sensor is configured to continuously, persistently or on demand measure the BHB concentration of an object and output a data stream; and A device, which is connected to the wearable BHB sensor, wherein the device includes: A processor configured to process data streams from the wearable BHB sensor; and An interface configured to display and / or transmit measured BHB concentration values.

43. Methods for detecting, measuring, and / or monitoring BHB concentration, including: Obtain bodily fluids from an object; The bodily fluid is placed in the wearable BHB sensor according to any one of claims 31 to 41; Determine the concentration of BHB at a single point in the body fluid; and Display and / or transmit the single-point BHB concentration on the interface.

44. A method for improving the health and / or well-being of individuals in need, 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 the health and / or well-being of a subject in need, comprising using a test kit product, wherein the test kit product utilizes the enzyme of any one of claims 1 to 19.

46. ​​A method for assisting individuals in need of weight loss, 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 comprising at least one sugar alcohol or low-carbohydrate composition.

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

49. The method according to any one of claims 47 and 48, used in combination with a food or supplement, wherein the food or supplement comprises an exogenous ketone supplement and / or a ketogenic ingredient.

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

51. The method of claim 50, wherein the ketone body and / or the precursor of the ketone body comprises one or more of acetone, acetoacetic acid, β-hydroxybutyrate (BHB), β-ketovalerate, β-hydroxyvalerate, 1,3-butanediol, and medium-chain triglycerides (MCT), wherein the medium-chain triglycerides (MCT) contain a fatty acid with a hydrocarbon side chain of 6 to 12 carbon atoms in length.

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

53. The method according to claim 51, wherein the MCT comprises one or more of the following: hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), and lauric acid (C12).

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