KR-20260068086-A - Enzyme that oxidizes beta-hydroxybutyrate (BHB), test strip using the same, and sensor

Abstract

The present disclosure relates to an enzyme capable of oxidizing beta-hydroxybutyrate (BHB). In an embodiment, the enzyme is engineered to optimize BHB activity. Engineered enzymes comprising amino acid substitutions, cleavages, and sequence deletions are also provided. The present disclosure further provides uses of the enzyme described herein, for example, in test strips, BHB sensors, devices, and systems for detecting and measuring BHB concentrations.

Inventors

• 시겔 저스틴 비.
• 어레돈도 어거스틴

Assignees

• 더 리전츠 오브 더 유니버시티 오브 캘리포니아

Dates

Publication Date

20260513

Application Date

20240903

Priority Date

20230905

Claims (20)

1. An enzyme having oxidase activity, wherein the enzyme is engineered to exhibit improved beta-hydroxybutyrate (BHB) activity.
2. The enzyme of claim 1, wherein the enzyme is capable of oxidizing beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate.
3. The enzyme of claim 1 or 2, wherein the amino acid sequence is derived from an oxidase of the EC 1.1.3.6 family.
4. An enzyme according to any one of claims 1 to 3, wherein the oxidase of the EC 1.1.3.6 family is a cholesterol oxidase.
5. An enzyme according to any one of claims 1 to 4, wherein the enzyme is non-naturally occurring and comprises one or more amino acid substitutions, deletions, or cleavages compared to a natural or wild-type enzyme.
6. An 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. An enzyme according to any one of claims 1 to 6, wherein the amino acid sequence of the enzyme comprises an N-terminal cleavage 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 in one or more of SEQ ID NOs 8, 24, or 40 to 43.
8. An enzyme according to any one of claims 1 to 6, wherein the amino acid sequence of the enzyme comprises an N-terminal cleavage corresponding to amino acid positions 2-32 of SEQ ID NO. 8.
9. An enzyme according to any one of claims 1 to 8, comprising an amino acid sequence identical to 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 one of SEQ ID NOs 8, 24, 40, 41, 42, or 43.
10. An enzyme according to any one of claims 1 to 8, comprising one amino acid sequence of SEQ ID NOs 8, 24, 40, 41, 42, or 43, wherein the amino acid sequence comprises 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 amino acid substitutions.
11. The enzyme of claim 10, wherein the amino acid substitution is performed only on the portion of the protein that mediates BHB binding and/or BHB activity.
12. An enzyme according to either claim 10 or 11, wherein the amino acid substitution(s) are conservative substitutions.
13. An enzyme according to any one of claims 1 to 11, wherein the enzyme exhibits increased BHB activity as measured by a peroxide strip, an acetoacetic acid strip, or liquid chromatography-mass spectrometry (LCMS).
14. In claim 13, the enzyme is an enzyme that exhibits increased BHB activity as measured by liquid chromatography-mass spectrometry (LCMS).
15. The enzyme of claim 14, wherein the engineered enzyme exhibits BHB activity about 1, 2, 3, 4, 5, 10, or 20 times greater than the corresponding unmodified wild type when measured by LCMS.
16. The enzyme of claim 14, wherein the enzyme comprises a sequence that is at least 90%, at least 95%, or at least 98% identical 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. An enzyme according to any one of claims 1 to 2, wherein the beta-hydroxybutyrate is (R)-beta-hydroxybutyrate.
18. An enzyme according to any one of claims 1 to 2, wherein the beta-hydroxybutyrate is (S)-beta-hydroxybutyrate.
19. An enzyme according to any one of claims 1 to 2, wherein the beta-hydroxybutyrate is a mixture of (R)-beta-hydroxybutyrate and (S)-beta-hydroxybutyrate.
20. A non-wearable BHB sensor capable of detecting and/or measuring BHB concentration, comprising using an enzyme of any one of claims 1 to 19.

Description

Enzyme that oxidizes beta-hydroxybutyrate (BHB), test strip using the same, and sensor Cross-reference regarding related applications This application claims the benefit of U.S. Provisional Application No. 63/580,620 filed on September 5, 2023, the entirety of which is incorporated herein by reference. Sequence list The present application comprises a sequence list submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy was created on August 26, 2024, is named 081906-1457969-253310PC_SL.xml, and has a size of 61,977 bytes. Technology field The present disclosure relates to an enzyme capable of oxidizing beta-hydroxybutyrate (BHB) and a method thereof. In one embodiment, the present disclosure relates to the use of the enzymes described herein for various applications, such as use in test strips and additional detection platforms for detecting and measuring BHB concentrations, BHB sensors, and use in systems. The ketogenic diet is an effective method for promoting weight loss and wellness. The ketogenic diet involves minimizing carbohydrate intake while often simultaneously increasing the consumption of fat and/or protein. This type of diet causes the body to burn fat, which leads to the production of ketone bodies (ketones) as the body breaks down fat. For optimal dietary management, it is important for subjects on a ketogenic diet to be able to monitor their ketone levels. The present disclosure provides a solution to these industrial and medical needs. Beta-hydroxybutyrate (BHB) is the conjugate base of beta-hydroxybutyric acid. BHB is synthesized through the metabolism of fatty acids, and high levels of BHB indicate that the body is using fat as a primary fuel source. Therefore, it is necessary to accurately and reliably measure BHB levels in subjects. This can be achieved through invasive, minimally invasive, or non-invasive mechanisms. The present disclosure addresses this need by describing novel enzymes to aid in the measurement of BHB. Useful systems, devices, and related methods are also further provided herein. Brief summary of the opening The present disclosure relates to enzymes having oxidase activity. In one embodiment, the enzymes described herein are engineered and/or modified compared to native enzymes. In the embodiments described herein, the enzymes can oxidize beta-hydroxybutyrate (BHB) to produce 3-oxobutanoate. In some embodiments, beta-hydroxybutyrate (BHB) is (R)-beta-hydroxybutyrate. In some embodiments, beta-hydroxybutyrate (BHB) is (S)-beta-hydroxybutyrate. In some embodiments, beta-hydroxybutyrate (BHB) is a mixture of (R)-beta-hydroxybutyrate and (S)-beta-hydroxybutyrate. In some embodiments, the enzyme, e.g., an engineered enzyme, comprises an amino acid sequence derived from an oxidase of the EC 1.1.3.6 family (e.g., comprises, essentially comprises, or constitutes). In some embodiments, the oxidase of the EC 1.1.3.6 family is cholesterol oxidase. In some embodiments, the enzyme is non-naturally occurring and/or engineered and comprises one or more amino acid substitutions, deletions, or cleavages compared to the natural enzyme (e.g., comprises, essentially comprises, or constitutes). In one embodiment, the enzyme described herein comprises a modified oxidase 8 derived from Scytonema sp. In another embodiment, the enzyme is a modified version of SEQ ID NO. 8 comprising one or more amino acid substitutions, deletions, or cleavages. In one embodiment, SEQ ID NO. 8 is optimized to provide improved BHB detection activity. In some embodiments, the enzyme comprises an amino acid sequence comprising one or more mutations corresponding to N137G, Y235Q, and/or A455Y of SEQ ID NO. 8 (e.g., comprises, essentially comprises, or constitutes). In other embodiments, the enzymes for use herein comprise SEQ ID NOs 1-43. In some embodiments, the enzymes for use herein comprise any one of SEQ ID NO. 24 or SEQ ID NOs 40-43, or a fragment thereof, or a modified protein thereof (e.g., comprises, essentially comprises, or constitutes). In some embodiments, the enzyme comprises 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% identical to any one of SEQ ID NOs 1 to 43 (e.g., comprises, essentially comprises, or constitutes). In some embodiments, the enzyme comprises 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% identical to any one of SEQ ID NOs 24 or SEQ ID NOs 40 to 43, or a fragment thereof, or a modified protein therein (e.g., comprises, essentially comprises, or constitutes). In additional embodiments, the enzyme comprises an amino acid sequence of any one of SEQ ID NOs 1 to 43 (e.g., comprises, essentially comprises, or constitutes), and said amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more ami