Methods and compositions for aging and mitochondrial health

Abstract

This article provides, in particular, methods for treating, delaying, and / or reducing the severity of age-related decline in various physiological pathways, including mitochondrial health and related physical conditions, by administering compositions capable of degrading extracellular ATP (eATP), including NTPD enzymes, nucleoside enzymes such as adenosine triphosphate bisphosphatase and members of the GDA1_CD39 superfamily, phosphatases, and yeast strains that produce ATP-degrading enzymes.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 590,129, filed October 13, 2023, the disclosure of which is incorporated herein by reference in its entirety. By invoking the inclusion in the sequence list

[0002] The sequence list submitted in XML file format according to 37 CFR §§2412 is incorporated herein by reference. The XML file is named "NB42251_WO_PCT_Seq_List.xml", was created on September 30, 2024, and has a size of 150,200 bytes. Technical Field

[0003] This article provides, in particular, methods for treating, delaying, and / or reducing the severity of age-related decline associated with various physiological pathways, including mitochondrial health and related physical conditions, by administering a composition comprising ATP-degrading enzymes, including NTPD enzymes, nucleotidases such as adenosine triphosphate bisphosphatase and members of the GDA1_CD39 superfamily, acid phosphatases, and yeast strains that produce ATP-degrading enzymes. Background Technology

[0004] The biology of aging is a rapidly expanding field of biomedical research, with a growing understanding that aging is a combination of various physiological changes and their complex interactions. While classic research on aging focuses on mortality and lifespan, there is a growing interest in achieving a life free from disease and disability, or “healthy aging.”

[0005] Currently, twelve molecular, cellular, and systemic markers of aging have been identified, including cellular senescence and mitochondrial dysfunction. Therefore, it is hypothesized that restoring mitochondrial function would have a positive impact on health decline associated with unhealthy aging. While the classic role of mitochondria is oxidative phosphorylation, which generates adenosine triphosphate (ATP), essential for a wide range of thermodynamically driven biochemical reactions, extracellular adenosine triphosphate (eATP) is considered a danger-associated molecular pattern (DAMP) that is released in response to tissue damage and cellular stress and may have multiple effects on cellular processes.

[0006] However, the effects of eATP on healthy aging through the modulation of its multiple markers remain unclear. Furthermore, many areas of therapeutic approaches for achieving healthy aging, beyond classic longevity methods (including maintaining a healthy diet and exercise), remain unexplored. Therefore, there is a need for improved methods and compositions that can extend lifespan and delay the onset and morbidity of age-related diseases and disorders.

[0007] The topics disclosed in this article address these needs and offer additional benefits. Summary of the Invention

[0008] This article provides a method for treating, delaying, or reducing the severity of at least one condition selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence. The method comprises administering to a subject in need or at risk an effective amount of a) a composition containing an ATP-degrading enzyme; b) a composition containing at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

[0009] In some embodiments, the age-related immune function changes include immune function decline and increased inflammation; the age-related intestinal function decline includes age-related decreased intestinal permeability, age-related decreased nutrient sensing function, and age-related decreased nutrient absorption function; the age-related metabolic regulation function decline includes decreased insulin sensitivity, decreased systemic glucose metabolism, decreased lipid metabolism, and decreased resting energy expenditure; the age-related cognitive decline includes short-term memory loss, difficulty finding words, altered sleep patterns, insomnia, slower processing speed, and / or degenerative dementia; and / or the age-related muscle mass and function changes include muscle atrophy and sarcopenia.

[0010] In some embodiments, the ATP-degrading enzyme comprises adenosine triphosphate (ATP) diphosphatase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the ATP-degrading enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP-degrading enzyme. In some embodiments, the enzyme is a bacterial ATP-degrading enzyme. In some embodiments, the enzyme is derived from *Gallaecimonas xiamenensis*.

[0011] In some embodiments, the enzyme comprises the same as SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide. In some embodiments, the enzyme further comprises an N-terminal tripeptide containing the sequence of the amino acid AGK.

[0012] In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from *Kluyveromyces lactis*, *Cyberlindnera jadinii*, and *Saccharomyces boulardii*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is *Cyberlindnera jadinii*, and includes the strain deposited in DSMZ, numbered DSM 33763, or a strain possessing all the identifying characteristics of the *Cyberlindnera jadinii* strain deposited in DSMZ, numbered DSM 33763. In some embodiments, the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and includes strains deposited in DSMZ with the number DSM 33764 or strains having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0013] In some embodiments, the composition comprises probiotics. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral, intravenous, intramuscular, parenteral, or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or pharmaceutical preparation. In some embodiments, the at least one condition is associated with mitochondrial dysfunction. In some embodiments, the subject is a human. In some embodiments, the at least one condition affects one or more types of cells. In some embodiments, the one or more types of cells include epithelial cells, endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and / or adipocytes.

[0014] In some embodiments, the subject is a non-human animal.

[0015] This article provides a method for treating and / or preventing at least one physical condition related to mitochondrial health, the method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof. In some embodiments, the at least one physical condition is selected from the group consisting of: impaired activity of Complex I, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function, and reduced energy production.

[0016] In some embodiments, the treatment and / or prevention delays the onset of at least one condition selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence. In some embodiments, the ATP-degrading enzyme comprises adenosine triphosphate (ATP) bisphosphatase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP-degrading enzyme. In some embodiments, the enzyme is a bacterial ATP-degrading enzyme. In some embodiments, the enzyme is derived from *Gallaecimonas xiamenensis*.

[0017] In some embodiments, the enzyme comprises the same as SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide. In some embodiments, the enzyme further comprises an N-terminal tripeptide containing the sequence of the amino acid AGK.

[0018] In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from *Kluyveromyces lactis*, *Cyberlindnera jadinii*, and *Saccharomyces boulardii*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is *Cyberlindnera jadinii*, and includes the strain deposited in DSMZ, numbered DSM 33763, or a strain possessing all the identifying characteristics of the *Cyberlindnera jadinii* strain deposited in DSMZ, numbered DSM 33763. In some embodiments, the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and includes a strain deposited in DSMZ, numbered DSM 33764, or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ, numbered DSM 33764.

[0019] In some embodiments, the composition comprises probiotics. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral, intravenous, intramuscular, parenteral, or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or pharmaceutical preparation. In some embodiments, the subject is a human being.

[0020] This article provides a method for treating age-related decline in nutrient absorption, the method comprising administering to a subject in need an effective amount of a) a composition containing an ATP-degrading enzyme; b) a composition containing at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof. In some embodiments, the treatment increases nutrient absorption in a subject who has not been treated with an effective amount of the composition.

[0021] In some embodiments, the ATP-degrading enzyme comprises adenosine triphosphate (ATP) diphosphatase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP-degrading enzyme. In some embodiments, the enzyme is a bacterial ATP-degrading enzyme. In some embodiments, the enzyme is derived from *Gallaecimonas xiamenensis*.

[0022] In some embodiments, the enzyme comprises the same as SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide. In some embodiments, the enzyme further comprises an N-terminal tripeptide containing the sequence of the amino acid AGK.

[0023] In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from *Kluyveromyces lactis*, *Cyberlindnera jadinii*, and *Saccharomyces boulardii*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is *Cyberlindnera jadinii*, and includes the strain deposited in DSMZ, numbered DSM 33763, or a strain possessing all the identifying characteristics of the *Cyberlindnera jadinii* strain deposited in DSMZ, numbered DSM 33763. In some embodiments, the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and includes strains deposited in DSMZ with the number DSM 33764 or strains having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0024] In some embodiments, the composition comprises probiotics. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral, intravenous, intramuscular, parenteral, or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or pharmaceutical preparation. In some embodiments, the subject is a human being.

[0025] This document provides a method for increasing prealbumin / albumin levels in a subject, the method comprising administering to a subject in need an effective amount of a) a composition containing an ATP-degrading enzyme; b) a composition containing at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof. In some embodiments, the increase is relative to a subject not treated with an effective amount of the composition. In some embodiments, the prealbumin level is a serum prealbumin level. In some embodiments, the increase in prealbumin levels reduces inflammation in the subject relative to a subject not treated with an effective amount of the composition. In some embodiments, the inflammation is systemic inflammation.

[0026] In some embodiments, the ATP-degrading enzyme comprises adenosine triphosphate (ATP) diphosphatase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP-degrading enzyme. In some embodiments, the enzyme is a bacterial ATP-degrading enzyme. In some embodiments, the enzyme is derived from *Gallaecimonas xiamenensis*.

[0027] In some embodiments, the enzyme comprises the same as SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide. In some embodiments, the enzyme further comprises an N-terminal tripeptide containing the sequence of the amino acid AGK.

[0028] In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is selected from *Kluyveromyces lactis*, *Cyberlindnera jadinii*, and *Saccharomyces boulardii*. In some embodiments, the yeast strain producing the ATP-degrading enzyme is *Cyberlindnera jadinii*, and includes the strain deposited in DSMZ, numbered DSM 33763, or a strain possessing all the identifying characteristics of the *Cyberlindnera jadinii* strain deposited in DSMZ, numbered DSM 33763. In some embodiments, the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and includes strains deposited in DSMZ with the number DSM 33764 or strains having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0029] In some embodiments, the composition comprises probiotics. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral, intravenous, intramuscular, parenteral, or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or pharmaceutical preparation. In some embodiments, the subject is a human being.

[0030] This article provides a method for reducing the occurrence of age-related skin changes, the method comprising applying a composition topically to the skin of a subject, the composition comprising: a) an ATP-degrading enzyme; b) at least one yeast strain that produces the ATP-degrading enzyme, or c) a combination thereof, wherein the occurrence of the age-related skin changes is reduced compared to a subject in which the composition has not been applied; and wherein the age-related skin changes include fine lines, wrinkles, hyperpigmentation, melasma, age spots, skin discoloration, and / or skin inflammation.

[0031] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of the embodiment or aspect.

[0032] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure.

[0033] The headings provided herein are not intended to limit the various aspects or embodiments of this disclosure, which can be derived by referring to the specification as a whole. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter. Any defined terms are given a more complete definition when the specification is referenced as a whole.

[0034] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. For all purposes, the disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated in their full text by reference. Attached Figure Description

[0035] Figure 1 The effects of Kluyveromyces lactis and Jedings cerberlindnerella vaginalis on ATP removal from cultures were described compared with the control.

[0036] Figure 2 A schematic diagram of the experimental setup for evaluating mitochondrial function using a 3D epidermal tissue model is depicted.

[0037] Figure 3 The effects of exemplary enzymes on the activity of mitochondrial complex I in a 3D epidermal tissue model were described.

[0038] Figure 4 The effects of exemplary enzymes on IL-1β expression in a 3D epidermal tissue model were described.

[0039] Figure 5 The effect of an exemplary enzyme on ATP release in a 3D epidermal tissue model was depicted.

[0040] Figure 6 The effects of exemplary enzymes and yeast strains, Kluyveromyces lactis and J. seldomochia junceta, on mitochondrial complex I activity in a mouse colitis model were described.

[0041] Figure 7 The effects of exemplary enzymes and yeast strains, Kluyveromyces lactis and Jettselberlindnerella vaginalis, on intestinal permeability as measured as relative fluorescence units (RFU) in a mouse colitis model were described.

[0042] Figure 8 The effects of exemplary enzymes and yeast strains, Kluyveromyces lactis and J. seldomochia lindneri, on prealbumin levels in a mouse colitis model were described.

[0043] Figure 9 The effects of exemplary enzymes on the activity of mitochondrial complex I in a 3D muscle tissue model were depicted.

[0044] Figure 10The effects of exemplary enzymes on mitochondrial complex I activity in a HaCat senescent cell model were described.

[0045] Figure 11 The effect of an exemplary enzyme on GDF15 levels in a HaCat senescent cell model was depicted.

[0046] Figure 12 The effects of exemplary enzymes and yeast strain *Jedingsberlindnerella vaginalis* on body weight in a rat model of colitis were described.

[0047] Figure 13 The effects of exemplary enzymes and yeast strain *Jedin Seberlindnerella vaginalis* on cecal weight in a rat model of colitis were described.

[0048] Figure 14 The effects of exemplary enzymes and yeast strain *Gardinia sepium* on intestinal permeability as measured as relative fluorescence units (RFU) in a mouse model of colitis were depicted.

[0049] Figure 15 The effects of exemplary enzymes and yeast strain *Jedingsberlindnerella vaginalis* on mitochondrial complex I activity in a mouse colitis model were described. Detailed Implementation

[0050] Aging is a gradual pathophysiological process characterized by the decline of tissue and cellular function and a significantly increased risk of various age-related diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, and immune system diseases (Guo et al., Sig. Transduct Target Therapy, 7, 391 (2022)). While the biology of aging is not a new field of research, the growing recognition of the world’s aging population has fueled a surge in interest in the field, particularly in the possibility of achieving a lifespan free of disease and disability, or “healthy aging” (Cohen et al., A complex systems approach to agingbiology, Nat Aging, 2, 580-591 (2022)). According to the World Health Organization, by 2030, one in six people worldwide will be 60 years of age or older. The current population aged 60 and over is projected to increase from 1 billion in 2020 to 1.4 billion. By 2050, the world’s population aged 60 and over will double (to 2.1 billion).

[0051] Aging is thought to be driven by a number of cellular and molecular markers that are generally believed to contribute to the aging process and collectively determine the aging phenotype (López-Otín et al. Cell. [Cell] 2013 6 6;153(6):1194-217). As of 2023, twelve markers have been proposed, including genomic instability, telomere loss, epigenetic alterations, loss of protein homeostasis, macroautophagy dysfunction, nutrient sensing dysregulation, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis (López-Otín et al. Cell. [Cell] 2023 19 19;186(2):243-278).

[0052] Given the importance of mitochondrial dysfunction in aging, restoring mitochondrial function may potentially contribute to the development of anti-aging therapies and address health decline associated with unhealthy aging. The inventors of this disclosure have surprisingly discovered that extracellular ATP (eATP) is a major driver of mitochondrial health, in which eATP induces depolarization and thus affects complex I activity, and that the restoration of mitochondrial function can be achieved by removing eATP using NTPD enzymes of the GDA1_CD39 superfamily, acid phosphatases, yeast strains expressing ATP-degrading enzymes, or combinations thereof.

[0053] Other biomarkers of mitochondrial function include growth differentiation factor 15 (GDF15), a protein that is upregulated during aging and may play a role in aging and age-related diseases. GDF15 levels are associated with a variety of biological factors, including: GDF15 levels are positively correlated with age, and GDF15 is one of the most upregulated proteins during aging. GDF15 is associated with chronic inflammation, and elevated GDF15 levels are associated with poorer recovery after acute illness. GDF15 is elevated in patients with mitochondrial diseases and is a valuable biomarker for diagnosis (Huan Liu et al., Experimental Gerontology, Vol. 146, 2021, 111228; Montero R et al., PLoS One. 2016 Feb. 11; 11(2):e0148709).

[0054] Therefore, this disclosure relates at least in part to methods for treating, delaying or altering or reducing the severity of at least one age-related decline in the health of a subject, and for treating or preventing physical conditions related to mitochondrial health, by administering NTPD enzymes of the GDA1_CD39 superfamily or ATP-degrading enzymes of the acid phosphatase family, including yeast strains of Kluyveromyces lactis and J.C. selberlindnerella vaginalis, or combinations thereof.

[0055] I. Definition

[0056] The term "ATP-degrading enzyme" is used to refer to enzymes that can degrade ATP substrates. ATP-degrading enzymes can be found in several enzyme families, including but not limited to adenosine triphosphate diphosphatase (EC 3.6.1.5), NTPD enzyme (EC 3.6.1.5), acid phosphatase (EC 3.1.3.2), and alkaline phosphatase (EC 3.1.3.1).

[0057] The term "acid phosphatase" (EC 3.1.3.2) (also known as acid phosphomonoesterase, phosphomonoesterase, glycerol phosphatase, acid monophosphatase, acid phosphohydrolase, acid phosphomonoester hydrolase, uterine transferrin, acid nucleoside diphosphate phosphatase, acid phosphatase (class A), or orthophosphomonoester phosphohydrolase (acid-optimal)) is used to refer to an enzyme that has an optimal pH for mediating the hydrolysis of phosphate ester bonds in a substrate at a pH less than about 6.5, such as less than about 4.0. Acid phosphatases release attached phosphoryl groups from other molecules during digestion. It can be further classified as a phosphomonoesterase. Acid phosphatases are stored in lysosomes and function when the lysosome is fused with an endosome, which is acidified during function; therefore, acid phosphatases have an optimal acidic pH. This enzyme is found in many animal and plant species. In some embodiments, the acid phosphatases used in the compositions and methods disclosed herein are not derived from the genus Shigella. In some embodiments, the acid phosphatases used in the compositions and methods disclosed herein are not derived from mammals.

[0058] As used herein, “NTPD enzyme” refers to an enzyme also known as an extracellular nucleotidase, extracellular ATPase, and extracellular nucleoside triphosphate diphosphate hydrolase. As used herein, NTPD enzymes hydrolyze extracellular nucleotides. NTPD enzymes comprise four major families of extracellular nucleotidases: CD39 / NTPD enzymes (extracellular nucleotidases); extracellular nucleotidase phosphodiesterase (E-NPP); alkaline phosphatases; and extracellular 5'-nucleotidases / CD73 (see Robson et al. 2006 Purinergic Signalling 2:409-430). In some of the embodiments described herein, the NTPD enzyme is an adenosine triphosphate diphosphate phosphatase.

[0059] As used herein, the term “adenosine triphosphate diphosphatase” refers to one or more of the calcium-activating enzymes (i.e., proteins belonging to class EC.3.6.1.5) that have ATP-bisphosphate hydrolase activity and catalyze the hydrolysis of γ-phosphate from ATP and β-phosphate from ADP. As used herein, adenosine triphosphate diphosphatases can constitute ATPases, NTPases, or both. Adenosine triphosphate diphosphatases have been found in all eukaryotes and some prokaryotes, indicating that these enzymes retain their function across different species. They possess unique phosphate-hydrolyzing activities, nucleotide substrate specificity, divalent cation requirements, and sensitivity to inhibitors. (See Plesner, Int. Rev. Cytol. [International Review of Cell Science], 158:141 (1995) and Handa and Guidotti, Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications], 218(3):916 (1996)). In mammals, adenosine triphosphate (ATP) bisphosphatases are thought to function primarily as extracellular hydrolases specific for ATP and ADP, a function crucial for the inactivation of ATP molecules at the synapse after neural stimulation (see, Todorov et al., Nature, 387(6628):76 (1997)). ATP bisphosphatases in mammals are also thought to be important for inhibiting ADP-induced platelet aggregation (see, Marcus et al., J. Clin. Invest., 99(6):1351 (1997)). Recombinant potato ATP bisphosphatase is commercially available from Sigma-Aldrich. In some embodiments, the ATP bisphosphatase used in the compositions and methods disclosed herein is not derived from potatoes. In other embodiments, the ATP bisphosphatase used in the compositions and methods disclosed herein is not derived from mammals.

[0060] As used herein, the “GDA1_CD39 superfamily” refers to enzymes composed of nucleoside triphosphate diphosphate hydrolases that share a common motif in their protein sequences. This family is named after two proteins: yeast GDP enzyme (GDA1) and lymphocyte activation antigen CD39. In some embodiments, these proteins are cell surface enzymes that hydrolyze a range of NTPs, including extracellular ATP. Non-limiting examples include extracellular ATPases, adenosine triphosphate diphosphate phosphatases, CD39, and extracellular ATP diphosphate hydrolases (ecto-ATP / Dase) (Knowles, 2011, Purinergic Signalling, Vol. 7, pp. 21–45; Robson et al., 2006, Purinergic Signalling, 2:409–430; incorporated herein by reference).

[0061] As used in this article, “microorganism (or microbe)” refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0062] As used herein, the terms “sequence identity” or “sequence similarity” mean that two polynucleotide sequences (candidate sequence and reference sequence) are identical in length (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis). When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., vacancies) compared to the optimal alignment used for the two sequences (which does not contain additions or deletions). The optimal sequence alignment used to determine sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (such as ALIGN or Megalign (DNASTAR)) or by inspection.

[0063] As used herein, the terms “percentage (%) sequence identity” or “percentage (%) sequence similarity” with respect to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after optimal alignment of the sequences and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity.

[0064] As used herein, “corresponding to” or “corresponds to” or “corresponding to” refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue that is similar to, homologous to, or equivalent to a residue listed in a protein or peptide.

[0065] As used in this article, “preventing” and its grammatical variations refer to methods that partially or completely delay or eliminate the occurrence or recurrence of one or more of a disorder or condition and / or its accompanying symptoms, or prevent a subject from acquiring or reacquiring a disorder or condition, or reduce the risk of a subject acquiring or reacquiring one or more of a disorder or condition or its accompanying symptoms.

[0066] As used herein, the term “reduction” or “lowering” in relation to a particular trait, characteristic, feature, biological process, or phenomenon means a reduction in that trait, characteristic, feature, biological process, or phenomenon. A trait, characteristic, feature, biological process, or phenomenon may be reduced by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100%.

[0067] As used herein, the terms “subject” or “patient” refer to a mammal (e.g., a human). In some embodiments, the subject has a relevant disease, disorder, or condition, including but not limited to diseases, disorders, or conditions associated with unhealthy aging or mitochondrial dysfunction. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that are a predisposition to or risk of a disease, disorder, or condition. In some embodiments, the subject is a human. In some embodiments, the subject is a patient. In some embodiments, the subject is not a non-human animal. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human animal is a dog, cat, pig, hamster, rabbit, bird, mouse, or horse. In some embodiments, the subject is a subject who has received and / or has received diagnostic and / or therapeutic administration. In some embodiments, any of the subjects described herein are between approximately 20–30 years of age, 30–40 years of age, 40–50 years of age, 50–60 years of age, or 60–70 years of age, or 65 years of age or older.

[0068] As used herein, “administer or administering” means the act of introducing one or more strains of microorganisms, exogenous feed enzymes and / or strains and exogenous feed enzymes into a subject by feeding or tube feeding.

[0069] As used herein, "effective amount" or "therapeutic effective amount" means the amount of one or more exogenous enzymes that improve one or more indicators in a subject. Improvements in one or more indicators in a subject (such as, but not limited to, healthy aging, mitochondrial function, improved barrier integrity, reduced mortality, or reduced pathogen infection) can be measured as described herein or by other methods known in the art. Exogenous enzymes can also be administered in one or more doses.

[0070] The term "symbiotic bacteria" refers to the microbial community (normal microbiome, indigenous microbiota) composed of microorganisms that exist on the body surface covered by epithelial cells and exposed to the external environment (gastrointestinal and respiratory tracts, vagina, skin, etc.). These bacteria have been shown to influence the regulation of immune physiological functions, including but not limited to metabolism, ontogenesis, and pathogen defense. Symbiotic bacteria have also been shown to promote resistance to pathogens, which is mutually beneficial for both the host and the symbiotic microbiota (Byrd et al. 2018. Nature Reviews Microbiology, 16:143-155; Kilian et al. 2016. British Dental Journal, 221:657-666, incorporated herein by reference).

[0071] The term "probiotic" refers to a live or viable microorganism that, when administered to a subject in an effective amount, confers a health benefit to the subject. For example, probiotics contained in probiotic formulations described herein, suitable for oral or topical administration to a subject, can improve the subject's health. As used herein, the term "probiotic" encompasses both live microorganisms and viable microorganisms in a dormant state, including frozen microorganisms, dehydrated or dried microorganisms, spores, cysts, or microorganisms in various states of reduced metabolic activity that can be reconstituted upon exposure to suitable conditions. In some embodiments, the probiotic microorganism comprises or consists of one or more symbiotic bacteria. In other embodiments, the probiotic microorganism is derived from a fecal microbial source (e.g., a microbial source obtained from the feces of another healthy subject). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. In some embodiments of the methods disclosed herein, administration of a non-viable bacterial composition is also contemplated.

[0072] As used herein, the term “implantation” refers to the colonization of one or more bacterial species (such as symbiotic bacterial species provided as, for example, probiotics) in a subject or the attachment of one or more bacterial species (such as symbiotic bacterial species provided as, for example, probiotics).

[0073] Certain ranges are presented herein with the term "approximately" preceding the numerical value. The term "approximately" is used herein to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a particular stated number, the close to or approximate unstated number may be a number that is substantially equivalent to the number in the context in which the particular statement is presented. For example, with respect to numerical values, the term "approximately" refers to the range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0074] Unless the context clearly indicates otherwise, as used herein, the singular terms “a / an” and “the” include plural indicators.

[0075] It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “alone,” “only,” etc., or the use of “negative” to limit the description of the claim elements.

[0076] It should be noted that, as used herein, the term “consisting essentially of” refers to a composition in which the component following the term constitutes less than 30% by weight of the total composition in the presence of other known components, and does not affect or interfere with the function or activity of the components.

[0077] It should be further noted that, as used herein, the term “comprising” means, but is not limited to, the components following the term “comprising.” The components following the term “comprising” are essential or mandatory, but compositions comprising components may further include other non-mandatory or optional components.

[0078] It should also be noted that, as used herein, the term "composed of" means including but not limited to the components following the term "composed of". Therefore, the components following the term "composed of" are necessary or mandatory, and no other components are present in the composition.

[0079] The terms "protein" and "peptide" refer to compounds containing amino acids linked by peptide bonds and are used interchangeably. A "protein" or "peptide" comprises a polymeric sequence of amino acid residues. Throughout this disclosure, single-letter and three-letter codes for amino acids are used in accordance with the definitions of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC-IUB. The single-letter X refers to any one of the twenty amino acids. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. The position of an amino acid in a given polypeptide sequence can be named by the single-letter code of the amino acid followed by a position number. For example, glycine (G) at position 87 is represented as "G087" or "G87".

[0080] As used herein, when referring to an "amino acid sequence," it means the amino acid sequence of a protein or peptide molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. However, terms such as "peptide" or "protein" are not intended to limit the amino acid sequence to the deduced sequence, but can include post-translational modifications of the deduced amino acid sequence, such as amino acid deletions, additions, and modifications (e.g., glycosylation and the addition of lipid moieties). Additionally, unless otherwise stated, the use of non-natural amino acids, such as D-amino acids, to improve stability or pharmacokinetic behavior falls within the scope of the term "amino acid sequence."

[0081] The terms "signal sequence" and "signal peptide" refer to amino acid residue sequences that can participate in the secretion or directed transport of proteins in their mature or precursor forms. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally absent in mature proteins. Typically, after protein secretion, the signal sequence is cleaved from the protein by a signal peptidase.

[0082] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of a protein, polypeptide, or enzyme that does not have a signal peptide sequence and / or a precursor peptide sequence.

[0083] Regarding amino acid or nucleic acid sequences, the term "wild-type" indicates that the amino acid or nucleic acid sequence is natural or naturally occurring. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., protein, amino acid, or nucleic acid sequences). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant / engineered nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0084] "Biopure strain" means a strain that does not contain enough other strains to interfere with the replication of the strain or in amounts that can be detected when evaluated using techniques recognized in the art.

[0085] When used in conjunction with the organisms and cultures described herein, “isolation” includes not only biologically pure strains but also any culture of an organism grown or maintained other than those found in nature. In some embodiments, the strain is a mutant, variant, or derivative of the *Jetting-Sabrina* and / or *Kluyveromyces lactis* strains described herein, which provides benefits comparable to those provided by the preserved strains of *Jetting-Sabrina* and / or *Kluyveromyces lactis* as described herein. In some embodiments, the strain is a strain having all the identifying characteristics of the preserved strains of *Jetting-Sabrina* or *Kluyveromyces lactis* described herein. Furthermore, each individual strain (the *Jetting-Sabrina* and *Kluyveromyces lactis* strains described herein) or any combination of these strains may also provide one or more of the benefits described herein. It will also be clear that the addition of microbial strains, carriers, additives (e.g., cryoprotectants, extracts, prebiotics, postbiotics, parabiotics), enzymes, other yeasts, etc., may provide one or more benefits or improve diseases, disorders, and conditions associated with unhealthy aging or mitochondrial dysfunction in subjects, and will not constitute substantially different yeast strains.

[0086] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limit is explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limit is explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical range is explicitly stated in its entirety herein.

[0087] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] Other definitions of terms may appear throughout this specification.

[0089] II. Composition

[0090] A.Yeast

[0091] This document provides yeast strains capable of producing ATP-degrading enzymes and compositions containing yeast strains. In some embodiments, yeast strains producing ATP-degrading enzymes and compositions containing yeast strains producing ATP-degrading enzymes can be used to treat, delay, or reduce the severity of age-related conditions, treat physical conditions associated with mitochondrial dysfunction, treat age-related decline in nutrient absorption in the gut, and increase prealbumin / albumin levels in subjects. In some embodiments, yeast strains producing ATP-degrading enzymes and compositions containing yeast strains can be used to treat and / or prevent diseases, disorders, and conditions associated with age-related conditions. In some embodiments, diseases, disorders, and conditions associated with age-related conditions are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutic agents for administration to subjects experiencing physiological stress, or as part of a daily nutritional regimen to prevent disease and promote healthy aging.

[0092] In some embodiments, the compositions provided herein contain yeasts from one or more genera. For example, 2, 3, 4, 5, 6 or more different genera. In some embodiments, the compositions provided herein contain yeast species from one or more genera. For example, one or more species from 2, 3, 4, 5, 6 or more genera. In some embodiments, the compositions provided herein contain strains of yeast species from one or more genera. For example, one or more strains of species from 2, 3, 4, 5, 6 or more genera. In some embodiments, the compositions provided herein contain yeasts from a single genus. In some embodiments, the compositions provided herein contain yeast species from a single genus. For example, 2, 3, 4, 5, 6 or more yeast species from a single genus. In some embodiments, the compositions provided herein contain one or more strains of yeast species from a single genus. For example, 2, 3, 4, 5, 6 or more different strains of yeast species from a single genus. In some embodiments, the compositions provided herein contain a single strain of yeast species, such as a biopure strain. In some embodiments, the yeast genus, species, and strain are any of those described herein.

[0093] In some embodiments, the yeast that produces ATP-degrading enzymes is any yeast capable of producing ATP-degrading enzymes. In some embodiments, the strain that produces ATP-degrading enzymes belongs to the genera *C. jadinii*, *Kluyveromyces*, or *Saccharomyces*. In some embodiments, the yeast belongs to the genera *C. jadinii* or *Kluyveromyces*. In some embodiments, the yeast belongs to the genus *C. jadinii*. In some embodiments, the yeast belongs to the genus *Kluyveromyces*. In some embodiments, the yeast belongs to the genus *Saccharomyces*. In some embodiments, the yeast belongs to the species *C. jadinii*, *Kluyveromyces lactis*, or *Saccharomyces boulardii*. In some embodiments, the yeast belongs to the species *C. jadinii* or *Kluyveromyces lactis*. In some embodiments, the yeast is *C. jadinii*. In some embodiments, the yeast is *Kluyveromyces lactis*. In some embodiments, the yeast is *Saccharomyces boulardii*. In some embodiments, the yeast is a strain of *C. jadinii* or *Kluyveromyces lactis*. In some embodiments, the yeast is a strain of *C. jadinii*. In some embodiments, the yeast is a strain of *Kluyveromyces lactis*. In some embodiments, the yeast strain that produces the ATP-degrading enzyme includes one or more of Kluyveromyces lactis, Jedingsberlindnerella salina, or Blastomyces boulardii.

[0094] In some embodiments, the compositions provided herein comprise yeasts of the genera *C. jadinii*, *Kluyveromyces*, or *Saccharomyces* that produce ATP-degrading enzymes. In some embodiments, the compositions provided herein comprise yeasts of the genera *C. jadinii* or *Kluyveromyces*. In some embodiments, the compositions provided herein comprise yeasts of the genera *C. jadinii*. In some embodiments, the compositions provided herein comprise yeasts of the genera *Kluyveromyces*. In some embodiments, the compositions provided herein may comprise yeasts of the genera *C. jadinii* and *Kluyveromyces*. In some embodiments, the compositions provided herein may comprise yeasts of the species *C. jadinii* or *Kluyveromyces lactis*. In some embodiments, the composition contains *C. jadinii*. In some embodiments, the composition contains *Kluyveromyces lactis*. In some embodiments, the compositions provided herein comprise yeasts of the species *C. jadinii* and *Kluyveromyces lactis*. In some embodiments, the composition contains strains of *C. jadinii* or *Kluyveromyces lactis*. In some embodiments, the composition contains strains of *C. jadinii*. In some embodiments, the composition contains strains of *Kluyveromyces lactis*. In some embodiments, the composition contains a strain of *Jedingerberin lindnerella vaginalis* and a strain of *Kluyveromyces lactis*.

[0095] In some embodiments, the *Jerdinium sabrina* strain is the strain deposited at DSMZ [German Center for Microbiology and Cell Culture, 7B Inhofenstrasse, Braunschweig, Germany, D-38124] with the accession number DSM 33763. In some embodiments, the *Jerdinium sabrina* strain is a strain possessing all the identifying characteristics of the *Jerdinium sabrina* strain deposited at DSMZ with the accession number DSM 33763. In some embodiments, the *Jerdinium sabrina* strain possessing all the identifying characteristics of the *Jerdinium sabrina* strain deposited at DSMZ with the accession number DSM 33763 is a live strain. In some embodiments, the *Jerdinium sabrina* strain is a biopure strain of strain DSM 33763. In some embodiments, the *Jerdinium sabrina* strain is a biopure strain of *Jerdinium sabrina* possessing all the identifying characteristics of the *Jerdinium sabrina* strain DSM 33763. In some embodiments, the *J. jetting-Seberlindnerella* strain having all the identifying characteristics of the *J. jetting-Seberlindnerella* strain preserved in DSMZ, numbered DSM 33763, is a live strain.

[0096] In some embodiments, the *Kluyveromyces lactis* strain is the strain deposited at DSMZ [German Center for Microbiology and Cell Culture, 7B Inhofenstrasse, Braunschweig, Germany, D-38124] with the accession number DSM 33764. In some embodiments, the *Kluyveromyces lactis* strain is a strain possessing all the identifying characteristics of the *Kluyveromyces lactis* strain deposited at DSM with the accession number DSM 33764. In some embodiments, the *Kluyveromyces lactis* strain is a biopure strain of strain DSM 33764. In some embodiments, the *Kluyveromyces lactis* strain is a biopure strain of *Kluyveromyces lactis* possessing all the identifying characteristics of *Kluyveromyces lactis* strain DSM 33764. In some embodiments, the *Kluyveromyces lactis* strain possessing all the identifying characteristics of the *Kluyveromyces lactis* strain deposited at DSMZ with the accession number DSM 33764 is a live strain.

[0097] As used herein, identification features may be percentage genomic sequence identity and / or functional behavior. Functional behavior can be defined as: metabolic activity; functional pathways, such as signal transduction pathways; upregulated and downregulated genes, such as due to epigenetic changes; protein expression and protein secretion. In some embodiments, percentage genomic sequence identity is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0098] B. enzymes

[0099] This document provides enzymes and enzyme-containing compositions that can be used to treat, delay, or reduce the severity of age-related conditions, treat physical conditions associated with mitochondrial dysfunction, treat age-related decline in nutrient absorption in the gut, and increase prealbumin / albumin levels in subjects. In some embodiments, enzymes and enzyme-containing compositions can be used to treat and / or prevent diseases, disorders, and conditions associated with age-related conditions. In some embodiments, diseases, disorders, and conditions associated with age-related conditions are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutic agents for administration to subjects experiencing physiological stress, or as part of a daily nutritional regimen to prevent disease and promote healthy aging.

[0100] This document provides compositions comprising ATP-degrading enzymes, such as NTPD enzymes and phosphatases. In some embodiments, the phosphatase is an acid phosphatase or an alkaline phosphatase. NTPD enzymes, also known as extracellular nucleotidases, Ecto-ATPases, and extracellular nucleoside triphosphate diphosphate hydrolases, are extracellular enzymes that hydrolyze extracellular nucleotides. There are four main families of extracellular nucleotidases: CD39 / NTPD enzymes (extracellular nucleotidases); extracellular nucleotidase phosphodiesterase (E-NPP); alkaline phosphatases; and extracellular 5'-nucleotidases / CD73.

[0101] In some embodiments, any composition provided herein is capable of removing phosphatase from ATP. In some embodiments, any composition provided herein is capable of removing phosphatase from NTPs. In some embodiments, the composition is an adenosine triphosphate bisphosphatase and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an NTPD enzyme and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an acid phosphatase and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an alkaline phosphatase and is capable of removing phosphate from ATP and / or NTPs.

[0102] In some embodiments, any composition provided herein is capable of breaking the bond between sugars and purines in ATP. In some embodiments, the composition is a purine nucleoside phosphorylase (PNP) and is capable of breaking the bond between sugars and purines in ATP.

[0103] NTPD enzymes are constitutively expressed in many tissues and can differentiate based on cellular location. In some embodiments, the NTPD enzymes provided herein comprise NTPD enzymes 1, 2, 3, and 8 located on the cell surface; in some embodiments, the NTPD enzymes provided herein comprise 5 and 6, and are located intracellularly and undergo secretion following heterologous expression. In some embodiments, the NTPD enzymes provided herein comprise 4 and 7, and are entirely intracellular and face the lumen of cytoplasmic organelles. NTPD enzymes located on the cell surface require Ca2+ or Mg2+ ions to be active. These isoforms facilitate the recovery of nucleosides from salvage pathways and extracellular nucleoside phosphates.

[0104] Due to their role in nucleotide metabolism, NTPD enzymes help control the availability of extracellular nucleotide agonists at P2 receptors, and thus regulate P2 receptor function. The systemic distribution of P2 receptors and the widespread propagation of purinergic signaling suggest that NTPD enzymes play an important role in a range of cellular processes. Location, substrate preference, and hydrolysis rate vary between subtypes; however, all NTPD enzymes contain five highly conserved sequence domains, known as the conserved regions of adenosine triphosphate bisphosphatase (APCR1 to APCR5).

[0105] In some of the embodiments provided herein, the enzyme exhibits phosphatase activity. A phosphatase is an enzyme that dephosphorylates its substrate. Enzymes exhibiting phosphatase activity include: adenosine triphosphate diphosphatase (also known as NTPD enzyme, ATP-bisphosphate hydrolase (EC 3.6.1.5)), inorganic pyrophosphatase (EC 3.6.1.1), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2), and exopolyphosphatase (EC 3.6.1.11). This phosphatase activity acts in the opposite direction to that of phosphorylases and kinases, which attach phosphate groups to their substrates using high-energy molecules such as ATP. In some of the embodiments, any of the NTPD enzymes is capable of hydrolyzing ATP, ADP, CTP, UTP, UDP, GTP, and / or GDP. In some embodiments, the NTPD enzyme is capable of hydrolyzing ATP. In some of the embodiments, the NTPD enzyme is soluble. In some of the embodiments, the NTPD enzyme is secreted.

[0106] In some embodiments, the ATP-degrading enzyme is not an endogenous ATP-degrading enzyme. In some embodiments, the ATP-degrading enzyme is a mammalian, bacterial, plant, or fungal ATP-degrading enzyme. In some embodiments, the ATP-degrading enzyme is not a mammalian ATP-degrading enzyme. In some embodiments, the ATP-degrading enzyme is a bacterial ATP-degrading enzyme. In some embodiments, the ATP-degrading enzyme is a GDA1_CD39 superfamily adenosine triphosphate diphosphatase. In some embodiments, the ATP-degrading enzyme is an acid phosphatase superfamily enzyme. In some embodiments, the ATP-degrading enzyme is derived from species of the genera Legionella, Beggiatoa, Galaecimonas, Betaproteobacteria, Klebsiella, Serratia, Herbaspirillum, Acinetobacters, Paraburkholderia, or Pantoeabeijingensis.

[0107] In some embodiments, the ATP-degrading enzymes are derived from Legionella pneumophila, Legionella fallonii ATCC 700992, Galaecimonas xiamenensis, Legionella santicrucis DSM23075, Legionella quateirensis, Betaproteobacteria bacterium, Klebsiella aerogenes, Serratia rubidaea, Herbaspirillum autotrophicum, Acinetobacter apis, Acinetobacter boissieri, Paraburkholderia bonniea, or Pantoea beijingensis.

[0108] The enzymes used in the compositions and methods disclosed herein are at least at a pH of about 3 to 9 (e.g., about pH 9). 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or any one of 9) or pH 3.5 to 5.5, or pH It is active at pH 3.5 to 7, or pH 5 to 7. The enzyme may be a member of the GDA1_CD39 superfamily or the acid phosphatase family. In some embodiments, the enzyme may be a member of the GDA1_CD39 superfamily, including potato-derived adenosine triphosphate (ATP) bisphosphatases. In some embodiments, the enzyme may be a member of the GDA1_CD39 superfamily, excluding potato-derived ATP bisphosphatases. In some embodiments, the enzyme is active at pH 3 to pH 9.

[0109] Other enzymes used in the compositions and methods disclosed herein are at least at pH 3 to pH 4. 9 (such as any of pH 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 9) or pH The enzyme is active at pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7, and is a member of the GDA1_CD39 superfamily or the acid phosphatase family. In some embodiments, the enzyme comprises a full-length amino acid sequence having at least 35% or at least 60% sequence identity with one or more of SEQ ID NO: 9-16, 50-56, or 77-89 (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%). The amino acid sequence of any one of the following: 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.In other embodiments, the enzyme comprises a full-length mature amino acid sequence having at least 35% sequence identity with any one of SEQ ID NO: 17-24, 57-63, 30-32, and 90-102 (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%). The amino acid sequence comprises any one of the following: 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the full-length amino acid sequence further comprises a signal sequence. In some embodiments, the amino acid sequence further comprises an N-terminal AGK tripeptide.

[0110] In some embodiments, the enzyme comprises the same as SEQ ID NO: The full-length amino acid sequence of 12 (CRC22110) has at least 35% sequence identity (such as about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity with the mature amino acid sequence of SEQ ID NO: 20 (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0111] In some embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity with the amino acid sequence of SEQ ID NO: 30, 31 or 32 (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity).

[0112] In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity with the mature amino acid sequence of SEQ ID NO: 63 (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity with the full-length amino acid sequence of SEQ ID NO: 56 (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0113] In some embodiments, the full-length amino acid sequence further includes a signal sequence. In some embodiments, the amino acid sequence further includes an N-terminal AGK tripeptide.

[0114] This document provides compositions comprising an ATP-degrading enzyme containing one or more modifications. In some embodiments, the modification comprises deletion, insertion, or substitution. In some embodiments, the modification comprises truncation. In some embodiments, the truncation is a truncation of the N-terminus. In some embodiments, the truncation is a truncation of the C-terminus. In some embodiments, the modification comprises insertion. In some embodiments, the insertion is located at the N-terminus. In some embodiments, the insertion is located at the C-terminus.

[0115] In some of the embodiments, any enzyme provided herein contains a truncated amino acid at its C-terminus having one or more of the full-length mature amino acid sequences of SEQ ID NO: 17-24, 57-63, or 90-102.

[0116] In some embodiments, any enzyme provided herein comprises the same as SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: One or more of 32 have an amino acid sequence of at least 35% (such as about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0117] In some embodiments, any enzyme described herein may be of mammalian, fungal, or bacterial origin. In some embodiments, any enzyme described herein is not a mammalian enzyme. In some embodiments, any enzyme described herein is a bacterial enzyme. In some embodiments, any enzyme described herein is an ATP-degrading enzyme, which may be of mammalian, fungal, or bacterial origin. In some embodiments, any enzyme described herein is not a mammalian ATP-degrading enzyme. In some embodiments, any enzyme described herein is a bacterial ATP-degrading enzyme.

[0118] B. carrier

[0119] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid fragment” are used interchangeably herein and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases. Isolated nucleic acid molecules in the form of DNA polymers may consist of one or more fragments of cDNA, genomic DNA, or synthetic DNA.

[0120] DNA constructs containing nucleic acids encoding the phosphatase polypeptides disclosed herein (such as nucleic acids containing any one of SEQ ID NO: 1-8 or 43-49) can be constructed to make them suitable for expression in host cells. Due to the known degeneracy in the genetic code, different polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques. It is also known that codon optimization may be required before attempting expression, depending on the desired host cell.

[0121] Polynucleotides encoding the disclosed phosphatase polypeptides can be bound to the vector. The vector can be transferred to host cells using known transformation techniques, such as those disclosed below.

[0122] Suitable vectors can be vectors that can be transformed into and / or replicated within host cells. For example, a vector containing a nucleic acid encoding a phosphatase polypeptide disclosed herein can be transformed and / or replicated in a bacterial host, fungal, or mammalian cell as a means of propagating and amplifying the vector. The vector can also be appropriately transformed into an expression host such that the encoding polynucleotide is expressed as a functional phosphatase.

[0123] Useful, non-limiting representative vectors are pTrex3gM (see, published U.S. Patent Application 20130323798), pTTT (see, published U.S. Patent Application 20110020899), and p2JM103BBI (see, Vogtentanz, Protein Expr Purif, 55:40-52, 2007), which can be inserted into the host genome. The vectors pTrex3gM, pTTT, and p2JM103BBI can be modified using conventional techniques to contain and express polynucleotides encoding the phosphatase polypeptide of the present invention.

[0124] Expression vectors typically contain control nucleotide sequences, such as promoters, operons, ribosome binding sites, translation initiation signals, and optionally repressor genes or one or more activator genes. Additionally, expression vectors may contain sequences encoding amino acids that enable the phosphatase polypeptide to target host cell organelles (such as peroxisomes) or specific host cell compartments. For expression guided by the control sequence, the nucleic acid sequence of the phosphatase polypeptide is operatively ligated to the control sequence in a manner appropriate for expression.

[0125] To express and produce a target protein (e.g., an ATP-degrading enzyme as described herein) in a cell, one or more expression vectors or expression cassettes containing one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more ATP-degrading enzymes as described herein are transformed into the cell under conditions suitable for variant expression. In some embodiments, the polynucleotide sequence encoding one or more ATP-degrading enzymes as described herein (along with other sequences contained in the vector) is integrated into the genome of the host cell; however, in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more ATP-degrading enzymes as described herein remains an autonomous extrachromosomal element within the cell. Some embodiments provide extrachromosomal nucleic acid elements and imported nucleotide sequences integrated into the host cell genome. The vectors described herein can be used to produce one or more ATP-degrading enzymes as described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variants as described herein is present on an integration vector capable of integrating the polynucleotide encoding the variant into the host chromosome and optionally amplifying it in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of the polynucleotide encoding one or more ATP-degrading enzymes as described herein is achieved via a promoter that is a wild-type promoter of the parental ATP-degrading enzyme. In some other embodiments, the promoters are heterologous to one or more ATP-degrading enzymes described herein, but are functional in the host cell. Exemplary promoters for bacterial host cells include, but are not limited to, the promoters amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, and pHpaII; the promoter of the raw maltose amylase gene from *Bacillus stearothermophilus*; the amylase gene from *Bacillus amyloliquefaciens* (BAN); the alkaline protease gene from *Bacillus subtilis*; the alkaline protease gene from *Bacillus clausii*; the xylosidase gene from *Bacillus pumilis*; cryIIIA from *Bacillus thuringiensis*; and the α-amylase gene from *Bacillus licheniformis*. Other promoters include, but are not limited to, the A4 promoter, as well as the phage λ PR or PL promoter, and the E. coli lac, trp or tac promoter, and the Bacillus rrn promoters (such as rrnI, rrnB, rrnL and rrnE ribosomal RNA promoters) and their variants.

[0126] The polynucleotide encoding the phosphatase polypeptide disclosed herein can be operatively linked to a promoter that allows transcription in a host cell. The promoter can be any DNA sequence exhibiting transcriptional activity in a selected host cell and can be derived from a gene encoding a protein homologous to or heterologous to the host cell. Examples of promoters used to direct transcription of DNA sequences encoding phosphatases, for example, in bacterial, fungal, or mammalian hosts, include the aprE promoter (SEQ ID NO:46), the promoter of the lac operon of *Escherichia coli*, the dagA or celA promoter of the *Streptomyces coelicolor* agarase gene, the promoter of the *Bacillus licheniformis* amylase gene (amyL), the promoter of the *Bacillus stearothermophilus* raw maltose amylase gene (amyM), the promoter of the *Bacillus amyloliquefaciens* amylase gene (amyQ), and the promoters of the *Bacillus subtilis* xylA and xylB genes, etc.

[0127] For transcription in fungal hosts, examples of useful promoters include those derived from genes encoding the following: *Aspergillus oryzae* TAKA amylase, *Rhizomucor miehei* aspartic protease, *Aspergillus niger* neutral α-amylase, *Aspergillus niger* acid-stable α-amylase, *Aspergillus niger* glucosylamylase, *Rhizomucor miehei* lipase, *Aspergillus oryzae* alkaline protease, *Aspergillus oryzae* triose phosphate isomerase, *Aspergillus nidulans* acetamase, etc. When genes encoding phosphatase polypeptides are expressed in bacterial species (such as *Escherichia coli*), suitable promoters can be selected from, for example, bacterial phage promoters, including the T7 promoter and the phage λ promoter. Following these lines of thought, examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of *Saccharomyces cerevisiae* and the AOX1 or AOX2 promoters of *Pichia pastoris*. Expression in filamentous fungal host cells typically involves cbh1, an endogenously inducible promoter from *T. reesei*. See Liu et al. (2008) *Acta Biochim. Biophys. Sin (Shanghai)* 40(2): 158-65.

[0128] The coding sequence can be operatively linked to the signal sequence. The DNA encoding the signal sequence can be a DNA sequence naturally associated with the gene for the target phosphatase polypeptide to be expressed, or it can originate from a different genus or species from which a portion of the phosphatase is derived. The signal sequence and promoter sequence constituting the DNA construct or vector can be introduced into the fungal host cell and can be derived from the same source. For example, the signal sequence could be the *Trichoderma reesei* cbh1 signal sequence operatively linked to the cbh1 promoter.

[0129] Expression vectors may also contain a suitable transcription terminator, and in eukaryotes, a polyadenylated sequence that is operatively linked to a DNA sequence encoding a phosphatase. The terminator and polyadenylated sequence may be appropriately derived from the same source as the promoter.

[0130] The vector may also contain selective markers, such as genes whose products compensate for defects in isolated host cells, like the dal gene from Bacillus subtilis or Bacillus licheniformis, or genes conferring antibiotic resistance (e.g., resistance to ampicillin, kanamycin, chloramphenicol, or tetracycline). Furthermore, the vector may contain Aspergillus selective markers (such as amdS, argB, niaD, and xxsC), markers inducing hygromycin resistance, or selection that can be achieved through co-transformation (as is known in the art). See, for example, published international PCT application WO 91 / 17243.

[0131] Synthetic genes encoding protein sequences corresponding to SEQ ID NO: 9-16, 50-56, and 77-89 can be generated using molecular biology methods known in the art. Synthetic genes include SEQ ID NO: 1-8, 43-49, and 64-76. These genes can be further cloned into suitable expression vectors to obtain expression plasmids containing one or more of the following: a promoter (e.g., the aprE promoter (SEQ ID NO: 25)); a signal sequence (e.g., the aprE signal sequence encoding the peptide of SEQ ID NO: 27 (SEQ ID NO. 26)); an oligonucleotide promoting the secretion of the target protein (e.g., an oligonucleotide encoding the peptide Ala-Gly-Lys); and a synthetic nucleotide sequence encoding the maturation region of the target gene (e.g., a sequence similar to SEQ ID NO: 9-16, 50-56, and 77-89). The synthetic oligonucleotides of any one of 1-8, 43-49, or 64-76 have at least 35% (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%). Synthetic oligonucleotides with any of the following sequence identity values: 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity); and / or terminators (such as the lat terminator (SEQ ID NO. 28)). In some embodiments, any oligonucleotide or encoded protein provided herein may contain a C-terminal His tag. The expression plasmid may also be further transformed into a suitable expression host cell (such as a mammalian or bacterial or fungal expression host cell).

[0132] C. host cells

[0133] Isolated cells containing a DNA construct (such as any DNA construct disclosed herein) or an expression vector (such as any expression vector disclosed herein, e.g., an expression vector containing any polynucleotide of SEQ ID NO: 1-8, 43-49, and 64-76 encoding a polypeptide of any of SEQ ID NO: 17-24, 57-63, 30-32, and 90-102) are advantageously used as host cells in the recombinant production of phosphatase polypeptides. Cells can be conveniently transformed with a DNA construct encoding the enzyme by integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered advantageous because the DNA sequence is more likely to be stably maintained in the cell. The DNA construct can be integrated into the host chromosome according to conventional methods, such as by homologous or heterologous recombination. Alternatively, cells can be transformed with an expression vector associated with a different type of host cell.

[0134] Examples of suitable bacterial host cells are Gram-positive bacterial species, such as those in the genus *Bacillaceae*, including *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus lentus*, *Bacillus brevis*, *Geobacillus stearothermophilus* (formerly *Geobacillus stearothermophilus*), *Bacillus alkalophilus*, *Bacillus amyloliquefaciens*, *Bacillus coagulans*, *Bacillus lautus*, *Bacillus megaterium*, and *Bacillus thuringiensis*; *Streptomyces* species, such as *Streptomyces murinus*; and lactic acid bacteria species, including those in the genus *Lactococcus*. *Lactococcus lactis* sp., including *Lactobacillus reuteri*, *Leuconostoc* sp., *Pediococcus* sp., and *Streptococcus* sp. Alternatively, strains of Gram-negative bacteria belonging to the family Enterobacteriaceae (including *Escherichia coli*) or the family Pseudomonadaceae can be selected as host organisms.

[0135] Suitable yeast host cells can be selected from biotechnology-related yeast species, such as, but not limited to, species of the genera *Pichia*, *Hansenula*, *Kluyveromyces*, *Yarrowinia*, *Schizosaccharomyces*, or *Saccharomyces* (including *Saccharomyces cerevisiae*), or species belonging to the genus *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*). The methyltrophic yeast strain *Pichiapastoris* can be used as a host organism. Alternatively, the host organism can be a species of the genus *Hansenula*.

[0136] Suitable host cells among filamentous fungi include species of the genus *Aspergillus*, such as *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus tubigensis*, *Aspergillus awamori*, or *Aspergillus nidus*. Alternatively, strains of the genus *Fusarium* (e.g., *Fusarium oxysporum*) or strains of the genus *Rhizomucor* (e.g., *Rhizomucor oryzae*) can serve as host organisms. Other suitable strains include species of the genera *Thermomyces* and *Mucor*. Additionally, species of the genus *Trichoderma* (e.g., *Trichoderma reesei*) can serve as hosts. Phosphatase polypeptides expressed by fungal host cells can be glycosylated, i.e., will include a glycosyl moiety. The glycosylation pattern can be the same as or different from that present in wild-type phosphatases. The type and / or degree of glycosylation may confer alterations to enzymatic and / or biochemical properties.

[0137] Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells. Other host cells may include insect cells, such as S2 cells.

[0138] It is advantageous to express a host-deleted gene, where the gene defect can be cured by the transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. Any cloned gene from a Trichoderma species or other filamentous fungal host, such as cbh1, cbh2, egl1, and egl2 genes, can be deleted. Gene deletion can be accomplished by methods known in the art by inserting the desired gene, in its desired form, into a plasmid.

[0139] Universal transformation techniques are known in the art. See, for example, Sambrook et al. (2001), ibid. Expression of heterologous proteins in *Trichoderma* is described, for example, in U.S. Patent No. 6,022,725. For transformation of *Aspergillus* strains, also refer to Cao et al. (2000) Science 9:991-1001. Genetically stable transformants can be constructed using vector systems, thereby stably integrating nucleic acids encoding phosphatases into the host cell chromosome. The transformants are then selected and purified using known techniques.

[0140] Methods for producing any enzyme disclosed herein may include culturing host cells under conditions favorable to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0141] The culture medium used to culture cells can be any conventional medium suitable for the growth of host cells and the acquisition of peptide expression. Suitable culture media and media components can be obtained from commercial suppliers or can be prepared according to published formulations (e.g., as described in the catalog of the American Type Culture Collection).

[0142] Any fermentation method well known in the art can be suitably used to ferment the transformed or derived fungal strains as described above. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0143] Separation and concentration techniques are known in the art, and conventional methods can be used to prepare concentrated solutions or broths containing the peptides of the present invention.

[0144] After fermentation, a fermented broth is obtained. Microbial cells and various suspended solids (including residual crude fermentation material) are removed using conventional separation techniques to obtain a solution. Commonly used methods include filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, post-centrifugation ultrafiltration, extraction, or chromatography.

[0145] Sometimes it may be necessary to concentrate the solution or broth containing the peptides to optimize recovery. Using an unconcentrated solution or broth typically increases the incubation time in order to collect the enriched or purified enzyme precipitate.

[0146] D. Enzyme composition

[0147] Any enzyme used in the methods disclosed herein can be formulated into a composition (e.g., a pharmaceutical or nutritional composition). As already mentioned, any enzyme composition described herein can be used to treat and / or prevent diseases associated with unhealthy aging and / or mitochondrial dysfunction. In one embodiment, the composition is preferably a pharmaceutical composition comprising an enzyme according to the disclosure. The pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition is not a pharmaceutical composition and is marketed to reduce the risk of disease.

[0148] In some embodiments, the composition comprises any enzyme described herein in any range between 1 nM and 100 nM or between. In some embodiments, the composition comprises any enzyme described herein in any range between 1 pM and 1 nM or between. In some embodiments, the composition comprises any enzyme described herein in any range between 100 nM and 1 µM or between. In some embodiments, any composition described herein contains any enzyme described herein in any range of about 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM or between.

[0149] In some embodiments, the composition is in the form of a liquid, solid, semi-solid, or a combination thereof. The composition may be a complete oral nutritional supplement. The composition may also be in the form of tablets, lollipops, sachets, soluble films, or combinations thereof.

[0150] In some embodiments, the composition is in the form of food, beverage, or combinations thereof. The composition may contain ingredients such as thickeners. In alternative embodiments, the composition is a topical compound.

[0151] The composition can be presented in any form, such as as a tablet, as an injectable fluid, or as an infusion fluid. Furthermore, the compositions, proteins, nucleotides, and / or carriers according to the invention can be administered via various routes, such as bronchial, topical, or oral administration.

[0152] In some embodiments, the skin care component comprises any "dermatologically acceptable" composition. In some embodiments, the dermatologically acceptable composition is suitable for use in contact with human skin tissue without excessive toxicity, incompatibility, instability, allergic reactions, etc.

[0153] In some embodiments, any skincare component or composition herein is configured for topical application to keratinized tissue. In some embodiments, any skincare component or composition may comprise dimethicone and particulate material, as well as a specific amount of water-soluble UV-blocking active ingredient, and such compositions may provide a desired feel, including non-greasy, non-sticky (indicated by mean break-up time), non-shine, refreshing, and easily spreadable (indicated by dynamic viscosity). In some embodiments, any skincare component or composition provided herein is a UV-blocking composition or a sunscreen composition.

[0154] In some embodiments, the composition may comprise at least a portion of an oral care composition or an oral care composition. Illustrative oral care compositions may include, but are not limited to, toothpaste (teeth cleaning agent), preventative paste, tooth powder, tooth polish, tooth gel (e.g., whitening gel), chewing gum, lozenges, mouthwash, oral rinses, whitening strips, patches, suspensions, emulsions, hydrogels, pastes, multiphase solutions, varnish gels, varnishes, finishes, and tubes, syringes, or dental trays comprising gels or pastes, liquids, powders, or gels or pastes applied to an application support such as dental floss or a toothbrush (e.g., a manual toothbrush, an electric toothbrush, a sonic toothbrush, a combination thereof, or an ultrasonic toothbrush). In some embodiments, the oral care composition comprises a combination of one or more oral care compositions.

[0155] The compositions of the present invention may contain a variety of optional ingredients known for use in personal care compositions, provided that one or more optional ingredients do not unduly alter the stability, aesthetics, or performance of the product. When incorporated into the composition, the optional ingredients should be suitable for contact with human keratinized tissue without undue toxicity, incompatibility, instability, allergic reactions, etc., within a reasonable judgment. The compositions herein may contain from about 0.0001% to about 50%; from about 0.001% to about 20%; or alternatively from about 0.01% to about 10% of optional ingredients by weight of the composition. Some non-limiting examples of optional ingredients include abrasives, absorbents, opacifiers, colorants (e.g., pigments, dyes, and lakes), particles, essential oils, anti-caking agents, foaming agents, defoamers, oil-controlling agents, binders, bio-additives, vitamins, minerals, peptides, glycosamines, flavonoids, antioxidants, preservatives, plant extracts, phytosterols, protease inhibitors, tyrosinase inhibitors, exfoliants, skin brighteners, non-tanning agents, anti-acne actives, anti-cellulite actives, anti-wrinkle actives, phytosterols and / or phytohormones, N-acyl amino acid compounds, antimicrobial agents, antifungal agents, moisturizers, emollients, humectants, lubricants, fragrances, anti-dandruff agents, buffers, swelling agents, chelating agents, biocides, denaturants, astringents, topical analgesics, anti-inflammatory agents, sunscreens, film-forming agents, and / or polymers, propellants, reducing agents, chelates, conditioning agents, and combinations thereof that contribute to the film-forming properties and directness of the composition.

[0156] In some embodiments, any composition provided herein may optionally contain pharmaceutically acceptable excipients, stabilizers, activators, carriers, penetrants, propellants, disinfectants, diluents, and preservatives. Suitable excipients are well known in the field of pharmaceutical formulation and can be readily identified and applied by a person skilled in the art, as referenced in, for example, Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition, 1985.

[0157] For oral administration, proteins can be administered, for example, in solid dosage forms such as capsules, tablets (e.g., with enteric coating), and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. In some embodiments, any enzymes provided herein (e.g., adenosine triphosphate bisphosphatase) can be encapsulated in gelatin capsules along with inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Examples of additional inactive ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersibility, or other known desired characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be manufactured as sustained-release products for use in the sustained release of the drug over several hours. Compressed tablets may be coated with sugar or film to mask any unpleasant taste and protect the tablets from atmospheric effects, or with enteric coating for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorings and flavorings to improve patient acceptability.

[0158] Enteric coatings prevent the release of active compounds from orally ingestible dosage forms. Depending on their composition and / or thickness, enteric coatings resist gastric acid for a required time before they begin to disintegrate and allow ATP-degrading enzymes to release slowly in the lower stomach, small intestine, or large intestine. Some examples of enteric coatings are disclosed in U.S. Patent No. 5,225,202 (incorporated by reference). Examples of enteric coatings include beeswax and glyceryl monostearate; beeswax, shellac, and cellulose, optionally comprising a neutral copolymer with polymethacrylate; copolymers of methacrylic acid and methyl methacrylate; or neutral copolymers of polymethacrylate containing metal stearates (for references on enteric coatings, see: U.S. Patent Nos. 4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560, 4,524,060, 5,536,507). Most enteric-coated polymers become soluble at pH 5.5 and higher, with maximum solubility above pH 6.5. Enteric coatings can also include a base coat and an outer coat step, as in pharmaceutical compositions designed for specific delivery in the lower GI tract, i.e., in the colon (pH 6.4 to 7.0, ileum pH 6.6), in contrast to the upper intestine, where the small intestine and duodenum have a pH range of 7.7–8 (after the addition of pancreatic juice and bile). The pH differences in the intestine can be utilized to target enteric-coated ATP-degrading enzyme compositions to specific regions of the gut. It also allows for the selection of specific ATP-degrading enzymes that are most active at specific pH levels in the intestine.

[0159] In addition to the fact that proteins according to the invention can be incorporated into pharmaceutical compositions, such ATP-degrading enzymes can also be part of nutritional compositions or nutritional products.

[0160] The proteins according to the invention can be added to nutrients (such as milk), but can also be produced within said nutrients (e.g., through molecular engineering). Furthermore, tablets and / or capsules can be prepared and subsequently added to nutrients or taken directly by humans.

[0161] In some embodiments, the compositions provided herein can be formulated for consumption by non-human animals. In some embodiments, the compositions provided herein can be formulated for consumption by dogs, cats, pigs, hamsters, rabbits, birds, mice, or horses.

[0162] In another aspect, the present invention is characterized by beverage and food products comprising an ATP-degrading enzyme that effectively addresses unhealthy aging or mitochondrial dysfunction in subjects in need. Beverage products may contain 1 unit / mL to 10,000 units / mL, for example, 1 unit / mL to 200 units / mL, 200 units / mL to 500 units / mL, 500 units / mL to 1,000 units / mL, 1,000 units / mL to 5,000 units / mL, or 5,000 units / mL to 10,000 units / mL. Food products may contain 1 unit / g to 10,000 units / g, for example, 1 unit / g to 200 units / g, 200 units / g to 500 units / g, 500 units / g to 1,000 units / g, 1,000 units / g to 5,000 units / g, or 5,000 units / g to 10,000 units / g.

[0163] In some embodiments, any composition provided herein may be prepared in various forms and may be administered via any number of routes, including but not limited to oral, intravenous, intramuscular, intra-arterial, intraperitoneal, subcutaneous, enteric, sublingual, or rectal routes. Preferably, the composition may be prepared for oral administration, such as as tablets, capsules, etc., or as an injectable preparation, such as as a liquid solution or suspension. In some embodiments, the pharmaceutical composition is an injectable preparation. Solid forms suitable for dissolution or suspension in a liquid medium prior to injection are also covered; for example, the pharmaceutical composition may be in a lyophilized form.

[0164] In some embodiments, any composition provided herein is suitable for parenteral administration, such as by injection or infusion, e.g., by bolus or continuous infusion. In some embodiments, the composition is a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulations such as suspending agents, preservatives, stabilizers, and / or dispersants. Alternatively, any composition provided herein may be in a dry form for reconstitution with a suitable sterile liquid prior to use. In some embodiments, the ATP-degrading enzyme is lyophilized. In some embodiments, the composition may be prepared as an injectable preparation, as a liquid solution or suspension. It may also be prepared in a solid form suitable for dissolution or suspension in a liquid medium prior to injection (e.g., lyophilized compositions, for example, for reconstitution with sterile water containing preservatives). For injection, such as intravenous, skin, or subcutaneous injection, or injection at the site of pain, the active ingredient may be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. As needed, preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included. For injection, the pharmaceutical composition may be provided, for example, in a pre-filled syringe.

[0165] E. Yeast preparations

[0166] The compositions disclosed herein (e.g., probiotics) typically contain at least one yeast capable of treating or preventing, or reducing, age-related or mitochondrial dysfunction-related diseases, disorders, and conditions as described herein. In some embodiments, the compositions are formulated into a freeze-dried (lyophilized) form. For example, compositions containing yeast may comprise granules or gelatin capsules, such as hard gelatin capsules, that include the yeasts disclosed herein, such as yeast strains.

[0167] In some embodiments, the yeast in the compositions described herein is dried. In some embodiments, the compositions described herein contain lyophilized yeast.

[0168] Freeze-drying of yeast is a well-established procedure in the art. In some embodiments, the compositions disclosed herein contain spray-dried yeast. Spray drying of yeast is a well-established procedure in the art. In some embodiments, the compositions disclosed herein contain drum-dried yeast. Drum drying of yeast (in some cases referred to as roll drying) is a well-established procedure in the art. Alternatively, the compositions may contain live, active yeast cultures.

[0169] In some embodiments, the compositions provided herein contain yeast in frozen, dried, freeze-dried, liquid, or solid form, in pellet or frozen pellet form, or in powder or dried powder form. In some embodiments, the compositions provided herein contain yeast in frozen form or in pellet or frozen pellet form. In some embodiments, the compositions provided herein contain yeast in dried or freeze-dried form. In some embodiments, the compositions provided herein contain yeast in powder or dried powder form. In some embodiments, the yeast is freeze-dried.

[0170] In some embodiments, any composition disclosed herein (e.g., probiotics) is encapsulated to enable yeast (such as the yeast disclosed herein) to be delivered to the intestine. Encapsulation protects the composition from degradation before delivery to the target site, degradation pathways such as rupture due to chemical or physical stimuli (e.g., stress, enzyme activity, or physical disintegration, which may be triggered by pH changes). Any suitable encapsulation method can be used. Exemplary encapsulation techniques include retention within a porous matrix, attachment or adsorption on a solid carrier surface, self-aggregation by flocculation or with a crosslinking agent, and mechanical containment by a microporous membrane or microcapsule.

[0171] The compositions disclosed herein can be administered orally and can be in the form of tablets, capsules, powders, chewable gum, or granules. In some embodiments, the tablets are effervescent tablets, chewable tablets, or lyophilized tablets. In some embodiments, the tablets, capsules, powders, or granules are formulated for extended release, such as extended-release tablets, extended-release capsules, or extended-release granules. For example, extended-release formulations can release the dose over a period of time (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours). Other ingredients (e.g., vitamin C or minerals) may be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and survival in vivo. Alternatively, the compositions disclosed herein (such as probiotic compositions) can be administered orally as food or nutritional products or as pharmaceuticals.

[0172] In some embodiments, the compositions disclosed herein are formulated as probiotics. Alternatively, in some embodiments, the compositions disclosed herein are formulated as inactive compositions, such as pasteurized or heat-treated yeast compositions.

[0173] In some embodiments, the compositions described herein are administered in the form of sachets or suspensions.

[0174] The compositions disclosed herein (e.g., probiotics) may comprise a therapeutically effective amount of the yeast disclosed herein. A therapeutically effective amount of yeast (e.g., yeast strain) is sufficient to exert a beneficial effect on a subject (e.g., a subject suffering from the disease, disorder, or condition described herein). In some embodiments, the composition contains an effective amount of *Saccharomyces cerevisiae* and / or *Kluyveromyces lactis* to treat and / or prevent any age-related condition described herein. In some embodiments, the composition contains an effective amount of *Saccharomyces cerevisiae* and / or *Kluyveromyces lactis* to treat and / or prevent age-related diseases, disorders, or conditions as described herein. A therapeutically effective amount of yeast (e.g., yeast strain) may be sufficient to result in delivery to the subject's intestine and / or partial or complete colonization of the subject's intestine. In some embodiments, a therapeutically effective amount of yeast (e.g., yeast strain) is sufficient to survive in the subject's intestine for a specific duration. In some embodiments, the duration is, at least, or approximately 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 1 month, or longer. In some embodiments, the duration is, approximately, or at least 12 days.

[0175] In some embodiments, compositions containing the yeast described herein can be applied topically. For example, in some cases, the treatment of inflammation (e.g., inflammation caused by age and / or age-related diseases, disorders, or conditions) can be accomplished through topical application. In some embodiments, inflammation can be treated or prevented by oral administration of compositions containing the yeast described herein.

[0176] This article provides a method for significantly reducing signs of skin aging, comprising applying any of the compositions described herein. In some embodiments, the method reduces the appearance of visible wrinkles when compared to skin without the application of the composition. (Li, X. et al. The Skin and Inflamm-Aging. Biology (Basel). 2023 Nov 2; 12(11):1396).

[0177] For topical application, compositions containing the yeast described herein can be in any form suitable for application to the skin surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, patches, plasters, coatings, bioadhesives, suspensions, etc., and / or can be prepared to contain liposomes, micelles, and / or microspheres. Such formulations can be used in combination with occlusive coverings so that moisture evaporating from the skin surface during and after application is retained within the formulation.

[0178] Topical formulations include those in which one or more active ingredients (e.g., yeast) are dissolved or dispersed in dermatological mediators known in the art (e.g., aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Such mediators may comprise water, aqueous buffer solutions, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycogen, or glycerin), oils (e.g., mineral oils such as liquid paraffin, natural or synthetic triglycerides such as Miglyol™), or silicone oils such as dimethicone). In some embodiments, the topical composition includes one or more pH buffers that, when dissolved in the aqueous component of the composition, provide a pH in the range of 5 to 7 (e.g., about pH 5.5).

[0179] Methods for producing topical compositions (e.g., topical pharmaceutical compositions such as creams, ointments, lotions, sprays, and sterile aqueous solutions or suspensions) are well known in the art. Suitable methods for preparing topical pharmaceutical compositions are described, for example, in WO 95 / 10999, U.S. Patent No. 6,974,585, WO 2006 / 048747, and documents cited in these references.

[0180] In some embodiments, the yeast strain can be administered via any number of routes, including but not limited to oral, intravenous, intramuscular, intra-arterial, intraperitoneal, subcutaneous, enteric, sublingual, or rectal routes. Preferably, the composition can be prepared for oral administration, such as as tablets, capsules, etc., or as an injectable preparation, such as as a liquid solution or suspension. In some embodiments, the composition is an injectable preparation. Solid forms suitable for dissolution or suspension in a liquid medium prior to injection are also covered; for example, the composition may be in a lyophilized form.

[0181] In some embodiments, the yeast strain is suitable for parenteral administration, such as by injection or infusion, e.g., by bolus or continuous infusion. In some embodiments, the yeast strain is in the form of a suspension, solution, or emulsion in an oily or aqueous medium, and may contain formulations such as suspending agents, preservatives, stabilizers, and / or dispersants. Alternatively, any yeast strain may be in a dried form for reconstitution with a suitable sterile liquid prior to use. In some embodiments, the yeast strain is lyophilized. In some embodiments, the composition may be prepared as an injectable preparation, as a liquid solution or suspension. Solid forms suitable for dissolution or suspension in a liquid medium prior to injection may also be prepared (e.g., lyophilized compositions, for example, for reconstitution with sterile water containing preservatives). For injection, such as intravenous, skin, or subcutaneous injection, or injection at the site of pain, the active ingredient may be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. As needed, preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included. For injection, the pharmaceutical composition may be provided, for example, in a pre-filled syringe.

[0182] In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently at least or at least about 10. 5 10 6 10 7 10 8 10 9 10 10 Or 10 11 One colony-forming unit (CFU). In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently at least or at least about 10 9 10 10 Or 10 11 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently at least or at least about 10. 10 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently about 1 x 10⁻⁶. 5 To approximately 1 x 10 11 CFU; for example, approximately 1 x 10 6 To approximately 1 x 10 11 CFU, approximately 1 x 10 7 To approximately 1 x 10 11 CFU, approximately 1 x 10 8 To approximately 1 x 1011 CFU, or approximately 1 x 10 9 To approximately 1 x 10 11 CFU, or approximately 1 x 10 10 To approximately 1 x 10 11 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently 1 x 10⁻⁶. 8 To approximately 1 x 10 11 CFU, 1 x 10 9 To approximately 1 x 10 11 CFU, 1 x 10 9 To approximately 1 x 10 10 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently 0.5 x 10⁻⁶. 10 To approximately 5 x 10 10 CFU, approximately 1 x 10 10 To approximately 5 x 10 10 CFU, approximately 1.5 x 10 10 To approximately 5 x 10 10 CFU, approximately 2 x 10 10 To approximately 5 x 10 10 CFU, approximately 2.5 x 10 10 To approximately 5 x 10 10 CFU, approximately 3 x 10 10 To approximately 5 x 10 10 CFU, approximately 3.5 x 10 10 To approximately 5 x 10 10 CFU, approximately 4 x 10 10 To approximately 5 x 10 10 CFU, or approximately 4.5 x 10 10 To approximately 5 x 10 10 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently 1 x 10⁻⁶. 10 1.5 x 10 10 2 x 10 10 Or 2.5 x 10 10 CFU. In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently 2.5 x 10⁻⁶. 10CFU. In some embodiments, the CFU may be a CFU / weight (e.g., gram) composition. Thus, in some of any embodiments, the CFU is a CFU / weight composition. In some embodiments, the CFU described herein is a CFU / gram composition (CFU / g). In some embodiments, the CFU described herein is a CFU / kilogram composition (CFU / kg). In some embodiments, the composition contains one or more yeasts as described herein, the amount of which is independently 2.5 x 10⁻⁶. 13 CFU / kg.

[0183] In some embodiments, the weight of the composition administered to a subject (e.g., a human) is 50 mg to 3000 mg, 100 mg to 2500 mg, 150 mg to 2000 mg, 200 mg to 1500 mg, 250 mg to 1000 mg, 300 mg to 950 mg, 400 mg to 900 mg, 450 mg to 850 mg, 500 mg to 800 mg, 550 mg to 750 mg, or 600 mg to 700 mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered in doses of 200 mg to 3000 mg, 200 mg to 2500 mg, 200 mg to 2000 mg, 200 mg to 1500 mg, or 200 mg to 1000 mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at or about the following doses: 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, 2500 mg, or 3000 mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at or about the following doses: 500 mg, 1000 mg, 1500 mg, 2000 mg, 2500 mg, or 3000 mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at or about the following doses: 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg.

[0184] In some embodiments, the composition contains a therapeutically effective amount of *G. gerbinafine* and / or *Kluyveromyces lactis* (e.g., as described herein) to treat, delay, or reduce diseases, disorders, and conditions associated with them as described herein. For example, the composition may contain a therapeutically effective amount of strains of *G. gerbinafine* and / or *Kluyveromyces lactis* as described herein to effectively treat, delay, or reduce the severity of at least one condition associated with aging or mitochondrial health. In some embodiments, the therapeutically effective amount is at least about 1 x 10⁻⁶. 10 CFU / g up to at least about 5 x 10 10 CFU / g composition. In some embodiments, the therapeutically effective amount is or is about 2.5 x 10⁻⁶. 10 CFU / g composition.

[0185] In some embodiments, the amount of yeast (e.g., as described herein) in the composition is a suitable daily dose for a subject (e.g., a human subject). In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a human child. The compositions provided herein can be repeatedly administered to the subject.

[0186] In some embodiments, the amount of yeast (e.g., as described herein) in the composition is a suitable daily dose for a subject (e.g., a non-human animal). Typically, the probiotic is optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are typically indigestible carbohydrates, such as oligosaccharides or polysaccharides or sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and trans-galacto-oligosaccharides.

[0187] In some embodiments, the probiotic compositions described herein are formulated to include a prebiotic compound in an amount of about 1% to about 30% by weight (e.g., 5% to 20% by weight) relative to the total weight of the composition. The carbohydrates may be selected from the group consisting of: fructooligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomaltooligosaccharides, pectin, xylooligosaccharides (or XOS), chitosan oligosaccharides (or COS), human milk oligosaccharides, β-glucan, gum arabic modified starch and resistant starch, polydextrose, D-tagatose, gum arabic fiber, carob, oat and citrus fiber. In one aspect, the prebiotic is a short-chain fructooligosaccharide (hereinafter referred to as FOSs-cc for simplicity); said FOSs-cc is not a digestible carbohydrate, is typically obtained by the conversion of beet sugar and comprises a sucrose molecule bound with three glucose molecules.

[0188] The yeast-containing compositions described herein may further contain pharmaceutically acceptable excipients or carriers. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include, but are not limited to, ethanol, glycerin, and water. The choice of drug carrier, excipient, or diluent may be based on the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain, in addition to, any suitable binder, lubricant, suspending agent, coating agent, or solubilizer. Examples of suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring agents can be provided in pharmaceutical compositions. Examples of preservatives include, but are not limited to, esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0189] In some embodiments, such as when the composition described herein is formulated as a probiotic, the composition contains one or more excipients. In some embodiments, one or more excipients are fructose, lactose monohydrate, and / or colloidal anhydrous silica.

[0190] The compositions disclosed herein can be formulated as food products. For example, in addition to the therapeutic effects of the invention, food products can also provide nutritional benefits, such as in nutritional supplements. Similarly, food products can be formulated to enhance the flavor of the compositions of the invention, or to make the compositions more consumer-appealing by making them more similar to ordinary food rather than pharmaceutical compositions. In some embodiments, food products are fruit juices; bars; cheeses; fresh fermented products; kimchi; Korean kimchi; miso; kombucha; kefir or other fermented milk; Indonesian fermented soybeans; indigenous fermented foods; sauerkraut and other fermented vegetables; coffee; cocoa and other yeast-containing fermented foods; sourdough bread; beer; cereals; milk; milk powder; infant formula; compositions suitable for athletes, such as energy drinks, protein solutions / powders; specialized nutrition, such as for the elderly or infants; hospital nutrition; and medical foods.

[0191] In some embodiments, the compositions disclosed herein contain a single yeast species or strain and contain no other yeast species or strains. Such compositions may contain only trace amounts or biologically irrelevant amounts of other yeast or bacterial strains or species. Such compositions may be cultures that are substantially free of other biological species.

[0192] The compositions used according to the methods disclosed herein may or may not require market authorization.

[0193] In some cases, freeze-dried yeast (e.g., yeast strains) is reconstituted before application. In other cases, reconstitution is performed using the diluents described herein.

[0194] Any yeast-containing composition disclosed herein may contain pharmaceutically acceptable excipients, diluents, or carriers.

[0195] In some embodiments, this document provides a pharmaceutical composition comprising: one or more yeasts or yeast strains as described herein; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the yeast is present in an effective amount for treating, delaying, or reducing the severity of at least one disease, disorder, or condition (including its symptoms) related to age or mitochondrial health as described herein.

[0196] In some embodiments, the present invention provides the above-described pharmaceutical composition comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

[0197] In some embodiments, the present invention provides the above-described pharmaceutical composition comprising a diluent selected from the group consisting of ethanol, glycerol, and water.

[0198] In some embodiments, the present invention provides the above-described pharmaceutical composition comprising an excipient selected from the group consisting of: starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweetener, gum arabic, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0199] In some embodiments, the present invention provides the above-described pharmaceutical composition, which further comprises at least one of a preservative, an antioxidant, and a stabilizer.

[0200] In some embodiments, the present invention provides the above-described pharmaceutical composition comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.

[0201] In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the yeast (as described herein) is lyophilized.

[0202] In some embodiments, the above-described pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere with 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of yeast, as measured in colony-forming units, persists for a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years.

[0203] The yeast species and strains disclosed herein can be cultured using standard microbiological techniques, such as those described herein.

[0204] III. Methods

[0205] This document provides enzymes, yeasts, and compositions containing enzymes and / or yeasts that can be used to treat, delay, or reduce the severity of age-related conditions, treat physical conditions associated with mitochondrial dysfunction, treat age-related nutrient absorption in the gut, and increase prealbumin / albumin levels in subjects. In some embodiments, these enzymes, yeasts, and compositions containing enzymes and / or yeasts can be used to treat and / or prevent diseases, disorders, and conditions associated with age-related conditions. In some embodiments, diseases, disorders, and conditions associated with age-related conditions are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutic agents for administration to subjects experiencing physiological stress, or as part of a daily nutritional regimen to prevent disease and promote healthy aging.

[0206] In some of the embodiments, the enzymes, yeasts, and compositions containing enzymes and / or yeasts provided herein are capable of reducing the severity of age-related conditions. In some embodiments, the enzymes, yeasts, and compositions containing enzymes and / or yeasts provided herein are capable of reducing the severity of age-related conditions by at least 1%, at least 5%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a reference. In some of the embodiments, the reference is a subject not treated with an effective amount of the enzymes, yeasts, and compositions containing enzymes and / or yeasts provided herein.

[0207] The time-dependent accumulation of aging or cellular damage, and the progressive loss of tissue and organ function over time (Childs et al. Nat. Med. [Nature Medicine] Dec. 2015; 21(12):1424-1435), are processes that occur over decades of life. Not wishing to be bound by theory, this paper provides methods for treating age-related signs and symptoms by reversing them. In some embodiments, any composition provided herein can be used in methods for reversing cellular damage in cells to a level commensurate with the cell's time age by one, two, three, or four or more decades. In some embodiments, the methods provided herein are methods for reducing or reversing the biological age of cells relative to their time age. In some embodiments, the reduction or reversal is a reduction or reversal of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or more relative to the cell's biological age. In some embodiments, the reduction is a reduction of one, two, three, or four or more decades relative to the cell's biological age. Metabolic changes affect all cellular activities and can contribute to the onset and progression of many neurodegenerative disorders that become more prevalent with aging (Trigo et al., FEBS Letters, 2022, 1095-1110). Metabolic changes can include decreased insulin sensitivity, systemic glucose metabolism, lipid metabolism, and changes in resting energy expenditure (Palmer et al., J. Clin Invest, 2022 Aug 15; 132(16):e158451). Furthermore, increasing age increases the risk of age-related diseases, including Alzheimer's disease. Cellular senescence (which has a major impact on maintaining normal tissue homeostasis and pathological conditions) is a major pathogenic factor in the aging process of an organism and promotes aging and age-related diseases. Cellular senescence is the gradual decline in the cell's ability to proliferate and differentiate, as well as its physiological functions, over time. In 1956, Denham Harman proposed the free radical theory of aging, arguing that degenerative changes during aging are mediated by the harmful effects of free radicals generated during normal cellular metabolism (Guo et al., Sig. Transduct Target Ther [Signal Transduction and Targeted Therapy] 7, 391 (2022)). Furthermore, aging is accompanied by a significant decrease in muscle mass (known as sarcopenia) and a significant decrease in muscle strength (known as dynapenia) (Law et al., 2016; Annu Rev Gerontol Geriatr. [Annals of Gerontology and Geriatrics] 36(1):205-228).

[0208] During aging, mitochondrial function, particularly mitochondrial membrane potential (MMP) and respiratory capacity per mitochondria, declines, often accompanied by increased production of reactive oxygen species (ROS). ROS are byproducts of oxidative phosphorylation and important signaling molecules. Mitochondrial dysfunction and reduced antioxidant detoxification lead to oxidative stress, lipid peroxidation, and mtDNA damage. Furthermore, the persistent loss of MMPs during aging may reflect a decrease in electron transport chain capacity, including reduced activity of Complex I in the brain, liver, skeletal muscle, and immune system. Decreased Complex I activity is a driver of mitochondrial dysfunction and has been shown to be associated with numerous health-related conditions, including age-related health decline (Enders et al., Int. J. Mol. Sci [International Journal of Molecular Sciences] 2023, 24, 10818). Mitochondrial function also plays a role in the gastrointestinal epithelium by maintaining gut health. Emerging research suggests the involvement of mitochondrial dysfunction in inflammatory bowel disease (IBD) and may serve as a starting point for investigating mitochondrial-targeted interventions for IBD therapy. (Ho et al. Annu. Rev. Physiol [Annals of Physiology] 202284:435-59).

[0209] During aging, the decline in metabolic health can lead to mitochondrial dysfunction, which promotes metabolic inflammation. This metabolic inflammation can then further impair mitochondrial function (Yuan et al., Oxidative Medicine and Cellular Longevity, Vol. 2022, Article ID 8803404). The aging process also has additional effects on the immune system, including the ability to maintain a healthy symbiotic microbiome. Normally, the immune system identifies microbes by utilizing specialized pattern recognition receptors (PRRs) to detect evolutionarily conserved structures known as microbe-associated molecular patterns (MAMPs). Activation of these PRRs triggers the production of pro-inflammatory cytokines, thereby coordinating a powerful immune response aimed at eradicating pathogens. However, this process can simultaneously lead to collateral damage to the host's own tissues, potentially resulting in life-threatening outcomes. Cell wall glycoconjugates of symbiotic bacteria, such as Firmicutes, can act as widely distributed signaling molecules throughout the body, and the chelation of cell wall glycoconjugates by plasma albumin can help inhibit PRR activation. Several studies have shown that older adults exhibit altered gut microbiota composition and that impaired albumin chelate glycoconjugates may lead to increased systemic inflammation (Tang and Wu, 2023 Cell Host & Microbe 31:1422-1425).

[0210] Age-related changes in nutrient intake may also lead to age-related gut microbiota dysbiosis. Diet is one of the major contributors to gut health, and advanced age is associated with deterioration in all aspects of nutrient intake and absorption, including dentition, salivary function, digestion, and intestinal transit time. In addition, gut microbiota dysbiosis (generally defined as a disturbance or change in the density and / or composition of the gut microbiota) has been associated with a variety of diseases and health conditions, including cystic fibrosis, inflammatory bowel diseases (irritable bowel syndrome, Crohn's disease, and colon cancer), neurological disorders (Parkinson's disease, Alzheimer's disease, and multiple sclerosis) and musculoskeletal disorders (including frailty, osteoporosis, rheumatoid arthritis, and gout) (Buford, Microbiome 2017; 5:80).

[0211] Unbound by theory, it is believed that the application of an ATP-degrading enzyme (such as adenosine triphosphate diphosphatase, including adenosine triphosphate diphosphatases and acid phosphatases of the GDA1_CD39 superfamily, including amino acid sequences identical to any one of SEQ ID NO: 17-24, 57-63, and 30-32) is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical; or identical to SEQ ID No: The composition of an amino acid sequence of 17-24, 57-63, 30-32, and 90-102 is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical) can a) treat, delay, or reduce the severity of at least one age-related condition by removing eATP; b) treat and / or prevent at least one physical condition related to mitochondrial health by removing eATP; c) increase age-related nutrient absorption in the gut by removing eATP; and / or d) increase prealbumin / albumin levels in a subject by removing eATP.

[0212] Unbound by theory, it is believed that administration of a composition comprising at least one of Kluyveromyces lactis and J.C. selberlindnerella vaginalis can a) treat, delay, or reduce the severity of at least one age-related condition by removing eATP; b) treat and / or prevent at least one physical condition related to mitochondrial health by removing eATP; c) increase age-related nutrient absorption in the gut by removing eATP; and / or d) increase prealbumin / albumin levels in subjects by removing eATP.

[0213] Unbound by theory, it is believed that application of a composition comprising at least one of *Kluyveromyces lactis* and *Jedinus sabrina* and an ATP-degrading enzyme (such as adenosine triphosphate diphosphatase, including adenosine triphosphate diphosphatases of the GDA1_CD39 superfamily or an ATP-degrading enzyme of the acid phosphatase family, including amino acid sequences of at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to any of SEQ ID NO: 17-24, 57-63, 30-32, and 90-102) can a) treat, delay, or reduce the severity of at least one age-related condition by removing eATP, b) a) Treating and / or preventing at least one physical condition related to mitochondrial health by removing eATP; c) Increasing age-related nutrient absorption in the gut by removing eATP; and / or d) Increasing prealbumin / albumin levels in subjects by removing eATP.

[0214] The source of the ATP-degrading enzyme can be any ATP-degrading enzyme, or any composition containing an ATP-degrading enzyme, and any tool capable of producing a functional ATP-degrading enzyme in the context of this invention, such as DNA or RNA nucleic acids encoding the ATP-degrading enzyme. The nucleic acid encoding the ATP-degrading enzyme can be embedded in a suitable vector (e.g., plasmids, phage particles, bacteriophages, (retro)viruses, transposons, gene therapy vectors, and other vectors capable of inducing or conferring the production of the ATP-degrading enzyme). Additionally, natural or recombinant microorganisms, such as bacteria, fungi, protozoa, and yeast, can be used as a source of the ATP-degrading enzyme in the context of this invention. In some embodiments, the source of the ATP-degrading enzyme in the context of this invention is not a mammalian ATP-degrading enzyme. In some embodiments, the source of the ATP-degrading enzyme in the context of this invention is a bacterial ATP-degrading enzyme.

[0215] Polynucleotides encoding ATP-degrading enzymes can be optimized for their expression. For example, codon optimization of the nucleotide sequence can be used to improve the translation efficiency of expression systems used to produce ATP-degrading enzymes. Therefore, polynucleotides encoding ATP-degrading enzymes can be codon-optimized. Those skilled in the art are familiar with various tools for codon optimization, such as those described in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Vol. 30, No. 15, August 1, 2014, pp. 2210-2212; or those described in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. July 1, 2005; 33 (Web server issue): W526-31). Nucleic acid molecules may contain heterologous elements (i.e., elements that are not inherently present on nucleic acid molecules that have the same coding sequence as ATP-degrading enzymes), for example, for the expression of ATP-degrading enzymes (e.g., heterologous expression). For example, nucleic acid molecules may contain heterologous promoters, heterologous enhancers, heterologous UTRs (e.g., for optimal translation / expression), heterologous poly-A tails, etc. In some embodiments, nucleic acid molecules may contain elements that confer resistance to antibiotics. In other embodiments, nucleic acid molecules do not contain elements that confer resistance to antibiotics.

[0216] In some embodiments, the polynucleotide encoding an ATP-degrading enzyme can be manipulated to insert, delete, or alter certain nucleic acid sequences. In some embodiments, the polynucleotide encoding an ATP-degrading enzyme is manipulated to introduce restriction sites, correct codon usage, add or optimize transcriptional and / or translational regulatory sequences, etc. In some embodiments, the polynucleotide encoding an ATP-degrading enzyme is altered to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) substitutions, deletions, and / or insertions into the amino acid sequence of the ATP-degrading enzyme. In some embodiments, the polynucleotide encoding an ATP-degrading enzyme may contain mutations that modify stability, post-translational modifications, or mutations that increase expression yield. In some embodiments, the polynucleotide encoding an ATP-degrading enzyme may contain amino acids for attaching covalent groups or introducing tags (including histidine (His) tags). In some embodiments, mutations may be "silent," i.e., not reflected in the amino acid sequence due to redundancy of the genetic code as described above. In some embodiments, the nucleic acid encoding an ATP-degrading enzyme may be randomly or directionally mutated to introduce different properties in the encoded amino acids.

[0217] In some embodiments, the ATP-degrading enzyme is encoded by a nucleic acid that is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleic acid sequence of any one of SEQ ID No: 1-8, 43-49, and 64-76. The nucleic acid sequences of 1-8, 43-49, and 64-76 are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical.

[0218] In some embodiments, the composition comprising an ATP-degrading enzyme contains a nucleic acid whose amino acid sequence is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to that of any one of SEQ ID No: The amino acid sequences of 1-8, 43-49, and 64-76 are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical.

[0219] In some embodiments, age-related disorders are selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related bowel dysfunction, age-related cognitive decline, and cellular senescence. In some embodiments, an age-related disorder is age-related metabolic dysfunction. In some embodiments, an age-related disorder is age-related immune function changes. In some embodiments, an age-related disorder is age-related muscle mass and function changes. In some embodiments, an age-related disorder is age-related bowel dysfunction. In some embodiments, an age-related disorder is age-related cognitive decline. In some embodiments, an age-related disorder is cellular senescence. In some embodiments, an age-related disorder is associated with mitochondrial dysfunction.

[0220] In some embodiments, the age-related decline in metabolic function includes decreased insulin sensitivity, decreased systemic glucose metabolism, decreased lipid metabolism, and decreased resting energy expenditure. In some embodiments, the age-related decline in metabolic function includes conditions associated with decreased insulin sensitivity, decreased systemic glucose metabolism, decreased lipid metabolism, and decreased resting energy expenditure.

[0221] In some embodiments, age-related conditions are age-related changes in immune function. In some embodiments, these age-related changes in immune function include immune decline and increased inflammation. In some embodiments, the inflammation does not include inflammation of the skin or oral mucosa. In some embodiments, these age-related changes in immune function do not include increased inflammation. In some embodiments, these age-related changes in immune function include conditions associated with immune decline and increased inflammation.

[0222] In some embodiments, age-related conditions are age-related changes in muscle mass and function. In some embodiments, these age-related changes in muscle mass and function include muscle atrophy and sarcopenia. In some embodiments, these age-related changes in muscle mass and function include conditions associated with muscle atrophy and sarcopenia.

[0223] In some embodiments, age-related disorders are age-related intestinal dysfunction. In some embodiments, age-related disorders are age-related intestinal dysfunction, including decreased intestinal permeability, age-related nutrient sensing impairment, and age-related malabsorption. In some embodiments, age-related disorders are age-related intestinal dysfunction, including disorders associated with decreased intestinal permeability, age-related nutrient sensing impairment, and malabsorption.

[0224] In some embodiments, age-related symptoms are age-related cognitive decline. In some embodiments, this age-related cognitive decline includes changes in synaptic structure and function. In some embodiments, this age-related cognitive decline includes short-term memory loss, difficulty finding words, altered sleep patterns, insomnia, slower processing speed, and / or degenerative dementia. In some embodiments, this degenerative dementia is Alzheimer's disease, vascular dementia, or Lewy body dementia. In some embodiments, age-related symptoms are cellular senescence.

[0225] In some embodiments, this document provides a method for treating, delaying, or reducing the severity of at least one condition selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence, wherein the method comprises administering to an individual in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one of Kluyveromyces lactis and J.C. selberlindnerella vaginalis; or c) a combination thereof.

[0226] In some embodiments, the subject has a relevant disease, disorder, or condition, including but not limited to diseases, disorders, or conditions associated with unhealthy aging or mitochondrial dysfunction. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that are predisposing to or at risk of the disease, disorder, or condition. In some embodiments, the subject is a human being. In some embodiments, the subject is not a non-human animal. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject who has received and / or has received diagnostic and / or therapeutic administration. In some embodiments, any subject described herein is between approximately 20–30 years of age, 30–40 years of age, 40–50 years of age, 50–60 years of age, or 60–70 years of age, or 65 years of age or older.

[0227] This article provides a method for administering an effective amount of any composition provided herein to a subject, thereby reducing the severity of the condition so that the subject appears to be an individual without the condition.

[0228] This article provides a method for administering an effective amount of any composition provided herein to a subject to reduce the severity of the condition so that the subject appears to be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years younger.

[0229] In some embodiments, this document provides a method of administering an effective amount of any composition provided herein to a subject to reduce the severity of the condition so that the subject appears to be an individual at least about 1–10 years, 10–20 years, 30–40 years, 40–50 years, or 50–60 years younger than the subject.

[0230] In some embodiments, this document provides a method of administering an effective amount of any composition provided herein to a subject to reduce the severity of the condition so that the subject appears to be an individual at least about 1–10 years, 10–20 years, 30–40 years, 40–50 years, or 50–60 years younger than the subject.

[0231] In some embodiments, this document provides a method of administering an effective amount of any composition provided herein to a subject, thereby reducing the subject's cellular age to appear as cells that are at least about 1–10 years, 10–20 years, 30–40 years, 40–50 years, or 50–60 years younger than the subject's age.

[0232] In some embodiments, the individual is healthy. In some embodiments, the individual does not exhibit any of the conditions described herein. In some embodiments, the individual does not exhibit any of the diseases or disorders described herein. In some embodiments, the individual is a healthy young adult. In some embodiments, the individual is between 10 and 20 years of age. In some embodiments, the individual is at least about 1 to 10 years, 10 to 20 years, 30 to 40 years, 40 to 50 years, or 50 to 60 years younger than the subject. In some embodiments, the individual is between 20 and 30 years of age. In some embodiments, the individual is between 30 and 40 years of age. In some embodiments, the individual is between 40 and 50 years of age. In some embodiments, the individual is between 50 and 60 years of age.

[0233] In some embodiments, the individual is of similar age to the subject. In some embodiments, the individual is healthy and of similar age to the subject. In some embodiments, the individual does not exhibit any of the conditions described herein and is of similar age to the subject. In some embodiments, the individual does not exhibit any of the diseases or disorders described herein and is of similar age to the subject. In some embodiments, any composition provided herein may substantially purify or enrich the target NTPD enzyme, yeast strain, or combination such that at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more of the sample is the desired ATP-degrading enzyme, yeast strain, or combination. In some embodiments, about 40%, 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the composition comprises another yeast strain, enzyme, bacterial strain, probiotic, prebiotic, or minor component. In some embodiments, the minor component is active. In some embodiments, the minor component is inactive.

[0234] In some embodiments, any composition provided herein may be applied for at least 1 week. In some embodiments, any composition provided herein may be applied for between 1 and 2 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 3 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 4 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 5 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 6 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 7 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 8 weeks. In some embodiments, any composition provided herein may be applied for more than 8 weeks.

[0235] In some embodiments, any composition provided herein may be applied for at least one month. In some embodiments, any composition provided herein may be applied for between one and two months. In some embodiments, any composition provided herein may be applied for between one and three months. In some embodiments, any composition provided herein may be applied for between one and four months. In some embodiments, any composition provided herein may be applied for between one and five months. In some embodiments, any composition provided herein may be applied for between one and six months. In some embodiments, any composition provided herein may be applied for between one and seven months. In some embodiments, any composition provided herein may be applied for between one and eight months. In some embodiments, any composition provided herein may be applied for between one and nine months. In some embodiments, any composition provided herein may be applied for between one and ten months. In some embodiments, any composition provided herein may be applied for between one and eleven months. In some embodiments, any composition provided herein may be applied for between one and twelve months. In some embodiments, any composition provided herein may be applied for more than twelve months.

[0236] In some embodiments, any condition described herein is associated with or may be associated with mitochondrial dysfunction. In some embodiments, any condition described herein may affect one or more cells. In some embodiments, the one or more cells include epithelial cells, endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and / or adipocytes. Cells to be treated according to this disclosure include epithelial cells, endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and / or adipocytes.

[0237] In some embodiments, this document provides a method for treating and / or preventing at least one physical condition associated with mitochondrial health. In some embodiments, the at least one physical condition associated with mitochondrial health is selected from impaired complex I activity, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function, and reduced energy production. In some embodiments, the at least one physical condition associated with mitochondrial health is impaired complex I activity. In some embodiments, the at least one physical condition associated with mitochondrial health is impaired mitochondrial respiration. In some embodiments, the at least one physical condition associated with mitochondrial health is oxidative stress. In some embodiments, the at least one physical condition associated with mitochondrial health is DNA damage. In some embodiments, the at least one physical condition associated with mitochondrial health is impaired cellular function. In some embodiments, the at least one physical condition associated with mitochondrial health is reduced energy production.

[0238] In some embodiments, the treatment and / or prevention of at least one physical condition associated with mitochondrial health delays the onset of at least one condition selected from age-related decline in metabolic regulation, age-related changes in immune function, age-related changes in muscle mass and function, age-related decline in intestinal function, age-related cognitive decline, and cellular aging.

[0239] In some embodiments, this document provides a method for treating and / or preventing at least one physical condition related to mitochondrial health, the method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one of Kluyveromyces lactis and J.C. selberlindnerella vaginalis; or c) a combination thereof.

[0240] This article also provides methods for treating age-related nutrient absorption in the gut. In some embodiments, any of the methods provided herein can increase age-related nutrient absorption by altering dentition, salivary function, digestion, and intestinal transit time. In some embodiments, these alterations include increases in salivary function, digestion, and intestinal transit time.

[0241] This article provides a method for treating age-related decline in nutrient absorption in the gut, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one of *Kluyveromyces lactis* and *Jett's sabrina lindneri*; or c) a combination thereof. In some embodiments, the increase is relative to a subject who has not been treated with an effective amount of the composition.

[0242] This document also provides methods for increasing prealbumin / albumin levels in subjects. In some embodiments, the method includes increasing prealbumin / albumin levels using any composition provided herein, which increases chelation to glycoconjugates. Any glycoconjugates described herein may be of eukaryotic origin. Any glycoconjugates described herein may be of prokaryotic origin. In some embodiments, the glycoconjugates may be of mammalian, non-mammal, fungal, bacterial, or plant origin. In some embodiments, the glycoconjugates are of bacterial origin. In some embodiments, the bacterial origin is from symbiotic bacteria.

[0243] This document provides a method for increasing prealbumin / albumin levels in a subject, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one of *Kluyveromyces lactis* and *Jettson's sabrina lindneri*, or c) a combination thereof. In some embodiments, the increase is relative to a subject not treated with an effective amount of the composition. In some embodiments, prealbumin levels are present in serum. In some embodiments, the increase in prealbumin levels reduces inflammation in the subject relative to a subject not treated with an effective amount of the composition. In some embodiments, the inflammation is systemic inflammation.

[0244] This disclosure also provides compositions containing ATP-degrading enzyme sources, including pharmaceutical and nutritional compositions containing ATP-degrading enzyme sources. The compositions may optionally contain pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeabilizers, propellants, disinfectants, diluents, and preservatives. Suitable excipients are well known in the field of pharmaceutical formulation and can be readily identified and applied by those skilled in the art, as referenced in, for example, Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition, 1985.

[0245] In another embodiment, compositions containing an ATP-degrading enzyme source or compositions containing *Kluyveromyces lactis* and / or *Jett's Seberlindnerella vaginalis* are suitable for oral administration and contain an enteric coating to protect the ATP-degrading enzyme or yeast strain from the adverse effects of gastric juice and low pH. Enteric coatings and controlled-release formulations are well known in the art (references are provided above). Enteric-coated compositions in the art may comprise a solution of a water-soluble enteric-coating polymer mixed with one or more active ingredients (such as ATP-degrading enzymes or yeast strains) and other excipients, dispersed in an aqueous solution and subsequently dried and / or granulated. The resulting enteric coating resists the attack of atmospheric moisture and oxygen during storage, as well as the attack of gastric juice and low pH on the ATP-degrading enzyme after ingestion, while readily decomposing under the alkaline conditions present in the lower intestine.

[0246] This article also provides a method for reducing the occurrence of age-related skin changes, the method comprising applying a composition topically to the skin of a subject, the composition comprising: a) an ATP-degrading enzyme; b) at least one yeast strain that produces the ATP-degrading enzyme, or c) a combination thereof, wherein the occurrence of the age-related skin changes is reduced compared to a subject in which the composition has not been applied; and wherein the age-related skin changes include fine lines, wrinkles, hyperpigmentation, melasma, age spots, skin discoloration, and / or skin inflammation.

[0247] Exemplary embodiments

[0248] 1. A method for treating, delaying, or reducing the severity of at least one condition, said at least one condition being selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence, said method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

[0249] 2. The method as described in Example 1, wherein the age-related immune function changes include immune function decline and increased inflammation; the age-related intestinal function decline includes age-related decreased intestinal permeability, age-related decreased nutrient sensing function, and age-related decreased nutrient absorption function; the age-related metabolic regulation function decline includes decreased insulin sensitivity, decreased systemic glucose metabolism, decreased lipid metabolism, and decreased resting energy expenditure; the age-related cognitive decline includes short-term memory loss, difficulty finding words, altered sleep patterns, insomnia, slower processing speed, and / or degenerative dementia; and / or the age-related muscle mass and function changes include muscle atrophy and sarcopenia.

[0250] 3. The method as described in Example 1 or Example 2, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

[0251] 4. The method as described in any one of Examples 1-3, wherein the enzyme is not a mammalian ATP-degrading enzyme.

[0252] 5. The method as described in any one of Examples 1-4, wherein the enzyme is a bacterial ATP-degrading enzyme.

[0253] 6. The method as described in any one of Examples 1-5, wherein the enzyme is derived from Galicia xiamenensis.

[0254] 7. The method as described in any one of Examples 1-6, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

[0255] 8. The method as described in Example 7, wherein the enzyme further comprises an N-terminal tripeptide comprising the sequence of the amino acid AGK.

[0256] 9. The method of any one of Examples 1-8, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*.

[0257] 10. The method of any one of Examples 1-9, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

[0258] 11. The method of any one of Examples 1-10, wherein the yeast strain that produces the enzyme is *Gerdinium sabinatum*, and comprises a strain deposited in DSMZ with the number DSM 33763 or a strain having all the identifying characteristics of the *Gerdinium sabinatum* strain deposited in DSMZ with the number DSM 33763.

[0259] 12. The method of any one of Examples 1-11, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and includes a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0260] 13. The method of any one of Examples 1-12, wherein the composition comprises probiotics.

[0261] 14. The method of any one of Examples 1-13, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

[0262] 15. The method of any one of Examples 1-14, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration, or topical administration.

[0263] 16. The method of any one of Examples 1-15, wherein the composition comprises a food product, a food ingredient, a dietary supplement, or a pharmaceutical agent.

[0264] 17. The method as described in any one of Examples 1-16, wherein the at least one condition is associated with mitochondrial dysfunction.

[0265] 18. The method as described in any one of Examples 1-17, wherein the subject is a human.

[0266] 19. The method as described in any one of Examples 1-17, wherein the subject is a non-human animal.

[0267] 20. The method as described in any one of Examples 1-19, wherein the at least one condition affects one or more cells.

[0268] 21. The method as described in Example 20, wherein the one or more cells comprise epithelial cells, endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and / or adipocytes.

[0269] 22. A method for treating and / or preventing at least one physical condition related to mitochondrial health, the method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

[0270] 23. The method as described in Example 22, wherein the at least one physical state is selected from the group consisting of: impaired activity of complex I, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function, and reduced energy production.

[0271] 24. The method as described in Example 22 or Example 23, wherein the treatment and / or prevention delays the onset of at least one condition, said at least one condition being selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence.

[0272] 25. The method of any one of Examples 22-25, wherein the enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

[0273] 26. The method of any one of Examples 22-25, wherein the enzyme is not a mammalian ATP-degrading enzyme.

[0274] 27. The method of any one of Examples 22-26, wherein the enzyme is a bacterial ATP-degrading enzyme.

[0275] 28. The method as described in any one of Examples 22-27, wherein the enzyme is derived from Galicia maculata.

[0276] 29. The method as described in any one of Examples 22-28, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

[0277] 30. The method as described in Example 29, wherein the enzyme further comprises an N-terminal tripeptide comprising the sequence of the amino acid AGK.

[0278] 31. The method of any one of Examples 22-30, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cerberlindnerella*, *Kluyveromyces*, and *Saccharomyces*.

[0279] 32. The method of any one of Examples 22-31, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

[0280] 33. The method of any one of Examples 22-32, wherein the yeast strain that produces the ATP-degrading enzyme is *Gardinia sepium* Lindnerella vaginalis, and comprises a strain deposited in DSMZ with the number DSM 33763 or a strain having all the identifying characteristics of the *Gardinia sepium* strain deposited in DSMZ with the number DSM 33763.

[0281] 34. The method of any one of Examples 22-33, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis and includes a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0282] 35. The method of any one of Examples 22-34, wherein the composition comprises probiotics.

[0283] 36. The method of any one of Examples 22-35, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

[0284] 37. The method of any one of Examples 22-36, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration, or topical administration.

[0285] 38. The method of any one of Examples 22-37, wherein the composition comprises a food product, a food ingredient, a dietary supplement, or a pharmaceutical agent.

[0286] 39. The method as described in any one of Examples 22-38, wherein the subject is a human.

[0287] 40. The method of any one of Examples 22-38, wherein the subject is a non-human animal.

[0288] 41. A method for treating age-related decline in nutrient absorption, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

[0289] 42. The method as described in Example 41, wherein the treatment increases the nutrient absorption of the subject relative to a subject not treated with an effective amount of the composition.

[0290] 43. The method as described in Example 41 or Example 42, wherein the enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

[0291] 44. The method as described in any one of Examples 41-43, wherein the enzyme is not a mammalian ATP-degrading enzyme.

[0292] 45. The method of any one of Examples 41-44, wherein the enzyme is a bacterial ATP-degrading enzyme.

[0293] 46. ​​The method as described in any one of Examples 41-45, wherein the enzyme is derived from Galicia maculata.

[0294] 47. The method as described in any one of Examples 41-46, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

[0295] 48. The method of Example 47, wherein the enzyme further comprises an N-terminal tripeptide comprising the sequence of the amino acid AGK.

[0296] 49. The method of any one of Examples 41-48, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cerberlindnerella*, *Kluyveromyces*, and *Saccharomyces*.

[0297] 50. The method of any one of Examples 41-49, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

[0298] 51. The method of any one of Examples 41-50, wherein the yeast strain that produces the ATP-degrading enzyme is *Gardinia sepium*, and comprises a strain deposited in DSMZ with the number DSM 33763 or a strain having all the identifying characteristics of the *Gardinia sepium* strain deposited in DSMZ with the number DSM 33763.

[0299] 52. The method of any one of Examples 41-51, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and comprises a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0300] 53. The method of any one of Examples 41-52, wherein the composition comprises probiotics.

[0301] 54. The method of any one of Examples 41-53, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

[0302] 55. The method of any one of Examples 41-54, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration, or topical administration.

[0303] 56. The method of any one of Examples 41-55, wherein the composition comprises a food product, a food ingredient, a dietary supplement, or a pharmaceutical agent.

[0304] 57. The method as described in any one of Examples 41-56, wherein the subject is a human.

[0305] 58. The method as described in any one of Examples 41-56, wherein the subject is a non-human animal.

[0306] 59. A method for increasing prealbumin / albumin levels in a subject, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

[0307] 60. The method as described in Example 59, wherein the increase is relative to a subject who has not been treated with an effective amount of the composition.

[0308] 61. The method as described in Example 59 or Example 60, wherein the prealbumin level is the serum prealbumin level.

[0309] 62. The method as described in any one of Examples 59-61, wherein the increase in prealbumin levels reduces inflammation in the subject relative to a subject not treated with an effective amount of the composition.

[0310] 63. The method as described in Example 62, wherein the inflammation is systemic inflammation.

[0311] 64. The method of any one of Examples 59-63, wherein the enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

[0312] 65. The method of any one of Examples 59-64, wherein the enzyme is not a mammalian ATP-degrading enzyme.

[0313] 66. The method as described in any one of Examples 59-65, wherein the enzyme is a bacterial ATP-degrading enzyme.

[0314] 67. The method as described in any one of Examples 59-66, wherein the enzyme is derived from Galicia maculata.

[0315] 68. The method as described in any one of Examples 59-67, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

[0316] 69. The method of Example 68, wherein the enzyme further comprises an N-terminal tripeptide comprising the sequence of the amino acid AGK.

[0317] 70. The method of any one of Examples 59-69, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cerberlindnerella*, *Kluyveromyces*, and *Saccharomyces*.

[0318] 71. The method of any one of Examples 59-70, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

[0319] 72. The method of any one of Examples 59-71, wherein the yeast strain that produces the ATP-degrading enzyme is *Gardinia sepium*, and comprises a strain deposited in DSMZ with the number DSM 33763 or a strain having all the identifying characteristics of the *Gardinia sepium* strain deposited in DSMZ with the number DSM 33763.

[0320] 73. The method of any one of Examples 59-72, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis and comprises a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0321] 74. The method of any one of Examples 59-73, wherein the composition comprises probiotics.

[0322] 75. The method of any one of Examples 59-74, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

[0323] 76. The method of any one of Examples 59-75, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration, or topical administration.

[0324] 77. The method of any one of Examples 59-76, wherein the composition comprises a food product, a food ingredient, a dietary supplement, or a pharmaceutical agent.

[0325] 78. The method as described in any one of Examples 1-77, wherein the subject is a human.

[0326] 79. The method as described in any one of Examples 1-77, wherein the subject is a non-human animal.

[0327] 80. A method for reducing the occurrence of age-related skin changes, the method comprising applying a composition topically to the skin of a subject, the composition comprising:

[0328] a) an ATP-degrading enzyme; b) at least one yeast strain that produces an ATP-degrading enzyme, or c) a combination thereof.

[0329] The occurrence of age-related skin changes was reduced in subjects who did not receive the composition; and

[0330] The age-related skin changes mentioned above include fine lines, wrinkles, hyperpigmentation, melasma, age spots, skin discoloration, and / or skin inflammation.

[0331] 81. The method of Example 80, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

[0332] 82. The method as described in any one of Examples 80-81, wherein the enzyme is not a mammalian ATP-degrading enzyme.

[0333] 83. The method of any one of Examples 80-82, wherein the enzyme is a bacterial ATP-degrading enzyme.

[0334] 84. The method as described in any one of Examples 80-83, wherein the enzyme is derived from Galicia maculata.

[0335] 85. The method as described in any one of Examples 80-84, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

[0336] 86. The method of Example 85, wherein the enzyme further comprises an N-terminal tripeptide comprising the sequence of the amino acid AGK.

[0337] 87. The method of any one of Examples 80-86, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cerberlindnerella*, *Kluyveromyces*, and *Saccharomyces*.

[0338] 88. The method of any one of Examples 80-87, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

[0339] 89. The method of any one of Examples 80-88, wherein the yeast strain that produces the ATP-degrading enzyme is *Gardinia sepium*, and comprises a strain deposited in DSMZ, numbered DSM 33763, or a strain having all the identifying characteristics of the *Gardinia sepium* strain deposited in DSMZ, numbered DSM 33763.

[0340] 90. The method of any one of Examples 80-89, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis and comprises a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

[0341] 91. The method of any one of Examples 80-91, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

[0342] 92. The method of any one of Examples 80-91, wherein the composition is formulated for topical application.

[0343] 93. The method as described in any one of Examples 80-92, wherein the subject is a human.

[0344] 94. The method as described in any one of Examples 80-93, wherein the subject is a non-human animal. Example

[0345] Example 1. Preparation and characterization of enzyme candidates

[0346] Phylogenetically diverse sequences from the GDA1 / CD39 enzyme family and acid phosphatases, known to have ATP hydrolytic activity, were identified from NCBI or internal databases. Codon optimization of the target gene sequences was performed based on Bacillus subtilis codon preferences, and synthetic genes encoding various enzyme sequences were generated using standard techniques (Generay Biotech (Shanghai) Co., Ltd). The sequences of codon-optimized genes (SEQ ID NO: 1-8, 43-49), native full-length polypeptide sequences (SEQ ID NO: 9-16, 50-56), and predicted mature enzymes (SEQ ID NO: 17-24, 57-63) are listed in Table 1. The genes were cloned into the expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif. [Protein Expression and Purification] 55:40-52, 2007). Expression of codon-optimized nucleotide sequences was performed using the nucleotide sequence SEQ ID NO: 26, which encodes an aprE signal peptide (SEQ ID NO: 27) with an additional 3 amino acids (Ala-Gly-Lys) between the signal sequence and the predicted mature sequence of the target gene (to promote the secretion of the target protein in Bacillus subtilis). This sequence encodes the predicted mature polypeptide driven by the aprE promoter (SEQ ID NO: 25) and terminated by the lat terminator (SEQ ID NO: 28). Competent Bacillus subtilis cells were transformed with each corresponding expression vector and plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies were inoculated into flasks containing LB medium and grown at 37°C with shaking at 200 rpm for 5–6 h. Seed cultures were inoculated into production media containing minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, calcium chloride, citric acid, etc.), one or more carbon sources (e.g., glucose), complex nitrogen sources (e.g., hydrolyzed soybean peptone, yeast extract), and buffer components (e.g., MOPS, Tris, etc.). Cultures were grown at 32°C with shaking at 250 rpm for 24–40 h.

[0347] The general purification procedure is as follows: Collect the culture broth by centrifugation, concentrate and replenish with ammonium sulfate to a final concentration of 1 M, then apply it to a hydrophobic interaction column (e.g., phenyl FF, butyl FF), pre-equilibrate with 50 mM Tris pH 7.5 containing 1 M ammonium sulfate. Elute the column in stepwise mode (0.75 M, 0.5 M, 0.25 M, 0.02 M ammonium sulfate, H2O, 20% ethanol), and combine the fractions containing the target active enzyme, exchanging the buffer to 20 mM Tris pH 7.5. Load the combined sample onto anion exchange chromatography (e.g., QFF) and elute in gradient mode (0–0.5 M NaCl). Identify the fractions containing the purified enzyme based on SDS-PAGE analysis and activity assays, and combine them. Store the purified sample at -20°C in a buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 40% glycerol until use.

[0348]

[0349] In separate experiments, samples were assayed using a standard malachite green phosphate assay kit (Sigma-Aldrich, catalog number MAK307) to determine their ability to hydrolyze ATP or ADP at pH 3 to 9. Reference enzymes, purchased from Sigma-Aldrich, included potato adenosine triphosphate bisphosphatase (catalog number A6535), recombinant human CD39 His tag (catalog number SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog number P0114).

[0350] The enzyme reaction was performed in 96-well plates with a total volume of 150 µL containing 250 µM substrate, 50 mM buffer, 5 mM CaCl2, and varying concentrations of enzyme. The substrate was either ATP or ADP. The buffer was glycine (pH 3), acetate (pH 4, 4.5, 5), bis-Tris (pH 5.5, 6, 6.5), or Tris (pH 7, 8, 9). The enzyme concentration varied from approximately 1 µg / mL to 1 ng / mL and was adjusted based on enzyme activity to ensure that the amount of phosphate released during the assay conformed to the dynamic range of the assay (5–100 µM phosphate). The enzyme concentration was determined by absorbance at 280 nm and using the predicted molecular weight and molar extinction coefficient to calculate the concentration. The enzyme reaction was initiated by adding 15 µL of enzyme and 135 µL of other reaction components to achieve the final reaction composition as previously described. After 5 min, the reaction was stopped by mixing 80 µL of the reaction mixture with 20 µL of malachite green working reagent. According to the manufacturer's instructions, the malachite green working reagent was prepared by mixing 100 parts of reagent A with 1 part of reagent B. A phosphate standard solution (0–80 µM) and an enzyme-free negative control were also mixed with the malachite green working reagent. All samples were tested in duplicate. The malachite green reaction mixture was allowed to stand at room temperature for 45–60 min, and the absorbance at 620 nm was measured. Finally, the absorbance readings of each sample were converted to phosphate concentration using the absorbance of the phosphate standard, and the negative control was subtracted from each reaction. The number of micromoles of phosphate released per milligram of protein per minute of reaction time (µmol / mg / min) using ATP or ADP as substrates is shown in Tables 2 and 3.

[0351]

[0352]

[0353] As shown in Tables 2 and 3, CRC22110 is the most active enzyme, exhibiting activity over a wide pH range, including pH 3 to pH 9. The tested enzymes have near-neutral pH optimum values ​​(pH 6–8) and a wide range of activity between pH 5 and 7, with some enzymes even maintaining activity at pH 9.

[0354] Example 2. Expression and characterization of truncated forms of target enzymes

[0355] The CRC22110 *Adenosine triphosphate diphosphatase* molecule with an N-terminal AGK peptide expressed in *Bacillus subtilis* as described in Example 1 was selected for further evaluation, including truncation of the predicted sequence (SEQ ID NO: 29). Various C-terminal truncations of the CRC22110 ATP diphosphatase were designed to remove C-terminal residues to a final cysteine ​​residue involving one of the seven disulfide bonds in the mature enzyme: CRC22110-V1 (SED IQ NO: 30), CRC22110-V2 (SEQ ID NO: 31), and CRC22110-V3 (SEQ ID NO: 32). The expression methods of the truncated forms of CRC22110 are described below, wherein in some cases, an additional three AGK residues are included at the predicted N-terminus of the mature polypeptide sequence. A first DNA fragment containing the flanking region of the (5') skfA gene (5' skfA gene FR, SEQ ID NO: 33) is operatively linked to a polynucleotide construct (e.g., an expression cassette) containing an upstream (5') Bacillus subtilis rrnI-p2 promoter region DNA sequence (SEQ ID NO: 34), a DNA sequence operatively linked to a DNA sequence of the 5' untranslated region (5' UTR) of Bacillus subtilis aprE (SEQ ID NO: 35), a DNA sequence operatively linked to DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO: 26), a DNA sequence operatively linked to a DNA sequence encoding a desired C-terminal truncated Adenosine triphosphate diphosphatase of *Galium spp.* (polypeptide sequence SEQ ID NO: 36, 37, or 38) with added nucleotide sequences encoding the N-terminal tripeptide AGK, and a DNA sequence operatively linked to the *Bacillus amyloliquefaciens* BPN terminator (SEQ ID NO: 36, 37, or 38). 39), the terminator is operatively linked to the flanking region of the (3')skfA gene (3'skfA gene FR) (SEQ ID NO: 40).A second DNA fragment containing the (5') amyE gene flanking region (5' amyE gene FR) (SEQ ID NO: 41) is operatively linked to a polynucleotide construct (e.g., an expression cassette) containing an upstream (5') Bacillus subtilis rrnI-p2 (SEQ ID NO: 34) promoter region DNA sequence, which is operatively linked to a DNA sequence of the Bacillus subtilis aprE 5' untranslated region (5' UTR) (SEQ ID NO: 35), which is operatively linked to DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO: 26), which is operatively linked to a DNA sequence encoding the desired C-terminal truncated Adenosine triphosphate diphosphatase of *Galium spp.* (polypeptide sequence SEQ ID NO: 36, 37, or 38) with added nucleotide sequence encoding the N-terminal tripeptide AGK, and which is operatively linked to the *Bacillus amyloliquefaciens* BPN terminator (SEQ ID NO: 36, 37, or 38). 39), the terminator is operatively linked to the (3') amyE gene flanking region (3' amyE gene FR) (SEQ ID NO: 42). More specifically, these DNA fragments were assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes for producing two-copy strains. Using standard molecular biology techniques, suitable Bacillus subtilis strains containing nine protease deletions were used to integrate the above-described first and second linear DNA expression cassette fragments into the genome.

[0356] As described below, the relative enzyme activity of the truncated form of CRC22110 adenosine triphosphate diphosphatase against ATP substrates was then determined.

[0357] Obtain the culture supernatant from Bacillus subtilis fermentation by filtration, and add ammonium sulfate and 1 M Tris pH 8 to a final concentration of 1 M ammonium sulfate and 20 mM Tris pH 8. Centrifuge and filter the sample. Load the filtrate onto a 300 mL phenyl agarose column equilibrated with 20 mM Tris and 1 M ammonium sulfate pH 8. Run a linear gradient from 0% to 100% 20 mM Tris pH 8 at 10 mL / min over 300 min. Collect the elution fractions and monitor ATPase activity using an ATPase activity assay. Combine, concentrate, and buffer-exchange the active fractions to 20 mM Tris pH 8. Load this fraction onto a 25 mL Q agarose column equilibrated with 20 mM Tris pH 8. It was then eluted with a step gradient (pH 8) of 50 mM NaCl in 20 mM Tris, followed by 100 mM NaCl, 200 mM NaCl, 400 mM and 500 mM NaCl in 20 mM Tris. The active fraction and the pure fraction (>95% as checked by SDS-PAGE) were combined to form the purified protein, which was then quantified.

[0358] Protein quantification by UPLC: Protein concentration was determined by UPLC (ultra-high performance liquid chromatography) and OD280 density determination. For UPLC determination, purified enzymes were diluted in 20 mM Tris pH 8, and protein fractions were separated using a Zorbax 300 SB-C3 column (Agilent Technologies). A linear gradient was run between 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B), and detection was performed at 220 nm on UHPLC to determine the concentration. 10 μL of sample was loaded onto the column, and the peak area of ​​the diluted sample was determined. The enzyme concentration of the sample was calculated using a standard curve of a purified reference enzyme (e.g., full-length CRC22110). Protein concentration was also determined by OD280 measurement. Purified enzymes were diluted in 20 mM Tris pH 8, and their OD280 was measured in quartz cuvettes. Protein concentration was calculated based on their respective extinction coefficients. The final concentration was calculated based on the average value determined by the UPLC method and OD280.

[0359] ATPase activity comparison: ATPase activity was measured using the previously described ATPase assay, with samples diluted to 0.003 ppm or 0.0015 ppm in assay buffer (50 mM Tris pH 8, 5 mM CaCl2, 0.01% Tween 80). 10 μL of the diluted enzyme sample was added to 0.25 mM ATP in the assay buffer to initiate the reaction. The reaction mixture was incubated at 25°C for 10 min, then 50 μL of the reaction mixture was added to 100 μL of QuantiChrom™ Malachite Green reagent (VWR catalog number 75878), and the reaction mixture was incubated at 25°C for 20 min before measuring OD620. ATPase activity (without enzyme control) was compared after subtracting the blank. Relative ATPase activity was calculated relative to the full-length CRC22110 based on OD after background subtraction. The results are shown in Table 4 below.

[0360] As shown in Table 4, the relative ATPase activities of CRC22110 and C-terminal truncated ATPases are highly comparable, thus confirming that this region of the protein sequence can be truncated without affecting enzyme function.

[0361]

[0362] Example 3. Preparation and characterization of yeast candidates

[0363] This example describes the evaluation of the ability of various yeast strains to remove ATP when ATP is added to the growth medium.

[0364] The following deposit was previously made in accordance with the Budapest Convention on the International Recognition of the Deposit of Microorganisms for Patent Proceeding Purposes.

[0365] The strain of *Saccharomyces cerevisiae* (DGCC3540) was deposited on January 19, 2021, at DSMZ [German Center for Microbiology and Cell Culture, 7B Inhofenstrasse, Braunschweig, Germany, D-38124, Germany] with accession number DSM 33763.

[0366] Kluyveromyces lactis (DGCC3550) was deposited on January 19, 2021, at DSMZ [German Center for Microbiology and Cell Culture, 7B Inhofenstrasse, Braunschweig, Germany, D-38124, Germany] with accession number DSM 33764.

[0367] *Pichia membranifaciens*, *Kluyveromyces lactis* (DGCC3550) (previously deposited under the Budapest Treaty on the International Recognition of the Preservation of Microorganisms with accession number DSM 33764), and *C. jadinii* (DGCC3540) (previously deposited under the Budapest Treaty on the International Recognition of the Preservation of Microorganisms with accession number DSM 33763) were grown overnight in YPD medium at 27°C. The cultures were used as inoculum and added at 1% v / v to fresh medium containing 1 mM ATP. Cells were then grown anaerobically at 37°C to simulate the temperature and oxygen availability of colonic conditions.

[0368] Supernatant was collected at 0 and 4 hours, and ATP was measured according to the manufacturer's instructions using the ATP Determination Kit (Thermo Fisher Scientific catalog number A22066). In short, supernatant was collected at 0 and 4 hours and filtered through a 0.2-micron filter to remove cells. ATP levels were measured using the A22066 kit. Results were normalized relative to a culture medium-only control.

[0369] like Figure 1 As shown, compared with the control, *Kluyveromyces lactis* and *Jettsonia sebifera* were able to remove ATP. These results indicate that both yeast strains have the ability to degrade eATP.

[0370] Example 4. Evaluation of mitochondrial function in an epidermal tissue model

[0371] This example illustrates the use of an exemplary enzyme in a 3D epidermal tissue model.

[0372] In short, the 3D epidermal tissue model (catalog number EPI 200) was obtained from MatTek Inc., Massachusetts, USA. The tissue was maintained and used in experiments according to the manufacturer's instructions (https: / / www.mattek.com / mattekproduct / epiderm / ).

[0373] To determine the role of ATP in mitochondrial function, tissues were incubated with 10 mM ATP or GTP or AMP or CRC22110-V1 enzyme (100 nM) or ATP + CRC22110-V1 enzyme at 37°C for 3 hours. The test reagent was added to the top of the air-exposed tissue, and the procedure was performed as shown in the experimental setup. Figure 2Tissue and supernatant were collected after 3 hours, and complex I activity was measured in the tissues according to the manufacturer's protocol to evaluate mitochondrial activity under different conditions. Briefly, tissues were homogenized in PBS to prepare protein extracts, and complex I activity was measured using 500 μg of total protein using a colorimetric complex I activity assay kit (Abcam, catalog number ab109721). Samples were normalized relative to cell controls only. Figure 3 As shown, the presence of ATP reduced the activity of complex I compared to the cell control alone, while the addition of AMP showed the least effect on the activity of complex 1. The addition of the exemplary enzyme CRC22110-V1 restored the activity of complex 1 compared to the cell control alone.

[0374] In separate experiments, cytokines were measured using supernatant collected from 3-hour time points via LEGENDplex multianalyte flow assay (BioLegend, catalog number 740808). Briefly, cytokines were measured using 100 μL of supernatant following the manufacturer's instructions. Cytokine levels are expressed as mean fluorescence units (MFI). Figure 4 As shown, ATP can induce IL-1β expression in 3D epidermal tissue, while the addition of the exemplary enzyme CRC22110-V1 can reduce the effect of ATP on tissue.

[0375] The ATP release from the collected supernatant was further evaluated using an ATP determination kit (catalog number A22066) similar to that described in Example 3. Figure 5 As shown, ATP accelerates the release of ATP from tissues, while the addition of the exemplary enzyme CRC22110-V1 reduces the ATP release effect.

[0376] In summary, the results indicate that exogenous ATP (eATP) can induce a decrease in the activity of complex I in skin epithelial cells, and the addition of an exemplary ATP-degrading enzyme can remove the exogenous ATP pool and restore the activity of mitochondrial complex I, which is associated with mitochondrial health and involved in the aging process.

[0377] Example 5. Exemplary enzymes and yeast strains for evaluating mitochondrial activity and related gut health.

[0378] This example describes the use of the exemplary enzymes and yeast strains outlined in the examples above for assessing mitochondrial function and its effects on gut health in a mouse model of antibiotic-induced colitis.

[0379] Six- to seven-week-old Apc / minJ mice with a mutant *Escherichia coli* gene for adenomatous polyposis and a predisposition to colitis and tumors were given a mixture of vancomycin, ampicillin, and neomycin (1 mg / mL) in their drinking water for 10 days. Wild-type mice and Apc / minJ mice not receiving the antibiotic mixture were used as controls. After 10 days, the mice were divided into six groups as outlined in Table 5, and as follows: Group 1 (WT mice) and Group 2 (mutant Apc / MinJ mice) were untreated and served as controls, drinking only distilled water; Group 3 (mutant Apc / MinJ) received the mediator control; Groups 4 and 5 (mutant Apc / MinJ) received the yeast strains *Kluyveromyces lactis* and *Jettia sebifera* once daily via oral feeding (PO) from day 1 to day 13; and Group 6 received the exemplary enzyme CRC22110-V3 twice daily via feeding (PO) at 6-hour intervals from day 1 to day 13.

[0380]

[0381] Animals were administered antibiotics via drinking water for 10 days, except for groups 1 and 2. Animals were given a tube feeding medium (group 3), ATP-degrading enzyme, or yeast daily. On day 13, after isoflurane anesthesia, animals were euthanized by exsanguination through the abdominal aorta. Blood and colonic tissue were collected. Colonic tissue was stored at -80°C and blood was processed, while serum was stored at -20°C for further analysis.

[0382] In the first experiment, the effect of the treatment group on mitochondrial complex I activity was evaluated. Colon tissue collected from mice was washed in cold PBS and then homogenized. Protein concentration was determined by nanodrop. 500 μg of total protein was used to determine complex I activity using the AB287839 kit, according to the manufacturer's instructions. Figure 6 As shown, compared with the untreated mutant Apc / MinJ mouse control (MT), the antibiotic-treated mutant Apc / MinJ mice (MT+Abx) exhibited reduced complex I activity in colon tissue. Compared with the MT control, the addition of *Saccharomyces cerevisiae* and CRC22110-V3 (MT+Abx+*Saccharomyces cerevisiae* and MT+Abx+CRC22110-V3, respectively) to antibiotic-treated Apc / MinJ mice resulted in the restoration of complex I activity.

[0383] In the second experiment, the effect of the treatment group on intestinal permeability was assessed. On day 13, mice were fasted for 4 hours, then administered 150 μl of 80 mg / ml FITC dextran (4 kDa) via gavage. Four hours later, the mice were sacrificed, and blood was collected and processed. Fluorescence was measured at 530 nm and excited at 485 nm. Figure 7 As shown, antibiotic-treated mutant Apc / MinJ mice (MT+Abx) exhibited increased intestinal permeability, as measured by relative fluorescence units (RFU). Compared to the WT untreated control (WT), untreated mutant Apc / MinJ mice (MT) and mutant Apc / MinJ mice treated with antibiotics and Kluyveromyces lactis showed a smaller increase in RFU, while mice treated with antibiotics and Giardia lamblia (MT+Abx+Giardia lamblia) or CRC22110-V3 (MT+Abx+CRC22110-V3) did not show an increase in RFU and showed similar and / or improved levels relative to the WT control.

[0384] In the third experiment, since albumin levels had already been shown to be positively correlated with reduced inflammation, the effect of the treatment group on prealbumin levels was evaluated. Serum prealbumin was measured using a prealbumin ELISA kit (catalog number KA2070). Serum samples (100 μL / well) were added to the ELISA plate and incubated at room temperature for 30 min. After incubation, the wells were washed, and the bound prealbumin was quantified using an enzyme-antibody conjugate and TMB as substrates. Measurements were taken at OD450 nm, and values ​​were reported after background subtraction. Figure 7 In the middle. For example Figure 8 As shown, antibiotic-treated mutant Apc / MinJ mice (MT+Abx) exhibited decreased prealbumin levels, while the addition of Giddings cerevisiae (MT+Abx+Giddings cerevisiae) and CRC22110-V3 (MT+Abx+CRC22110-V3) restored prealbumin levels to levels similar to those of wild-type (WT) and untreated mutant Apc / MinJ (MT) controls.

[0385] In summary, antibiotic treatment induces intestinal inflammation, and eATP plays a key role in the manifestation of this inflammation. These results suggest that removing eATP using ATPases or yeast can counteract the effects of inflammation by improving the intestinal barrier and albumin levels, both of which are associated with the aging process.

[0386] Example 6. Biochemical characterization of other ATP-degrading enzyme candidates

[0387] Additional samples of the phylogenetic diversification enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma-Aldrich and included potato adenosine triphosphate bisphosphatase (catalog number A6535), recombinant human CD39 His tag (catalog number SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog number P0114).

[0388] Similar to measuring the ability of candidate enzymes to degrade ATP as described in Example 1.

[0389] Using ATP as a substrate, the number of micromoles of phosphate released per milligram of protein per minute of reaction time (µmol / mg / min) is shown in Tables 6 and 7.

[0390]

[0391]

[0392] As shown in Tables 6 and 7, CRC34822 is the most active enzyme at pH 5 among the enzymes tested in this experiment.

[0393] Example 7. Evaluating mitochondrial function in a 3D muscle tissue model

[0394] This example illustrates the use of an exemplary enzyme in a 3D bovine muscle tissue model.

[0395] In short, the bovine muscle cells (catalog number B354-05) were obtained from Sigma-Aldrich. According to the manufacturer's protocol, the cells were grown in 3D matrix gel (Corning, catalog number 356237) to provide a 3D tissue model.

[0396] To determine the role of ATP in mitochondrial function, muscle tissue grown in matrix gel was incubated for 3 days at 37°C with either 10 mM ATP, or either CRC22110-V1 (10 nM), CRC34822 (10 nM and 100 nM), CRC33930 (10 nM), CRC33849 (10 nM), or CRC22105 (10 nM).

[0397] Tissue was collected at 3 hours and complex I was measured to evaluate mitochondrial function under different conditions according to the manufacturer's protocol and similar to those described in Example 4. Briefly, tissue was homogenized in PBS to prepare a protein extract, and complex I activity was measured using 500 μg of total protein using a complex I activity assay kit (ab109721). Samples were normalized relative to cell-only controls. Figure 9As shown, the presence of ATP reduced the activity of complex I compared to the cell-only control, while the addition of the exemplary enzyme restored the activity of complex 1.

[0398] Example 8. Evaluating mitochondrial function in a senescent cell model

[0399] This example illustrates the use of an exemplary enzyme in a senescent HaCat cell model.

[0400] To test the effects of ATP on mitochondrial health during aging, HaCat cells were obtained from Acellerate and grown for 20 passages. In this experiment, cells were passaged every three days, and ATP or an ATP-degrading enzyme was added to the cells 24 hours after passage. Cells were incubated with 5 mM ATP + LPS (1 μg / mL), or CRC22110-V1 (10 nM), or CRC34822 (100 nM), or alkaline phosphatase (10 nM), or potato ATP-dipase (10 nM).

[0401] Cells were collected at passages 1, 10, and 20, and complex I measurements were performed according to the manufacturer's protocol to evaluate mitochondrial activity under different conditions. Briefly, cells were homogenized in PBS to prepare a protein extract, and complex I activity was measured using 500 μg of total protein using a complex I activity assay kit (Abogen AB109721) similar to that described in Examples 4 and 7.

[0402] like Figure 10 As shown, the activity of complex I decreased in cells from passage 1 to passage 20, and the addition of extracellular ATP accelerated this decline. The exemplary ATP-degrading enzymes tested were able to reverse the decrease in complex I activity, indicating that they play a role in improving cell health.

[0403] In another experiment, the levels of growth differentiation factor 15 (GDF15), a protein that is upregulated during aging and may play a role in aging and age-related diseases, were assessed under similar conditions.

[0404] To test GDF15 levels, HaCat cells were incubated for 24 hours with LPS (1 μg / mL), LPS + CRC22110-V1 (10 nM), CRC34822 (100 nM), or potato ATP bisphosphatase (10 nM). After 24 hours, the supernatant was collected, and GDF15 levels were measured using a human GDF15 ELISA kit (catalog number BMS2258, Thermo Fisher Scientific).

[0405] like Figure 11As shown, LPS induces stress in cells, thereby increasing GDF15 levels, while the exemplary ATP-degrading enzymes tested were able to reduce GDF15 levels compared to stressed cells, indicating that they have a positive effect on cell health and function.

[0406] Example 9. Additional exemplary enzymes and yeast strains for evaluating mitochondrial activity and related gut health.

[0407] This example describes the use of additional exemplary enzymes and yeast strains outlined in the examples above for assessing mitochondrial function and its effects on gut health in a mouse model of antibiotic-induced inflammation.

[0408] Similar to that described in Example 5, 6-7 week old AJ mice were administered a mixture of vancomycin, ampicillin, and neomycin (1 mg / mL) in drinking water for 10 days and were sacrificed three days after the antibiotics were discontinued. AJ mice that did not receive the antibiotic mixture served as controls. Groups 2 through 6 received the yeast strain *G. jeddingensis* (live or dead) once daily via oral feeding (PO) from day 1 to day 13, and group 7 received the exemplary enzyme CRC34822 once daily via feeding (PO) from day 1 to day 13.

[0409]

[0410] Animals were weighed at different time points, including at the start of antibiotic administration, throughout the course of antibiotic administration, and after antibiotic administration was discontinued. Figure 12 As shown, mice given antibiotics showed weight loss compared to control mice (natural; no antibiotics), and intervention with Jadin Cyberlindnerella vaginalis and the CRC34822 enzyme was able to counteract the weight loss associated with antibiotic administration.

[0411] On day 13, after isoflurane anesthesia, the animals were euthanized by exsanguination from the abdominal aorta. Blood and colonic tissue were collected. The colonic tissue was stored at -80°C and the blood was processed, while the serum was stored at -20°C for further analysis.

[0412] In the first experiment, the effect of the treatment group on cecal weight was assessed. Colonic tissue collected from mice was weighed. Figure 13 As shown, mice given antibiotics showed a significant increase in cecal weight compared to control mice (without antibiotics), and intervention with Jadins cerebral lindnerella vaginalis and CRC34822 was able to counteract the negative effects of antibiotics on cecal weight.

[0413] In the second experiment, the effect of the treatment group on intestinal permeability was assessed. On day 13, mice were fasted for 4 hours, then administered 150 μl of 80 mg / ml FITC dextran (4 kDa) via gavage. Four hours later, the mice were sacrificed, and blood was collected and processed. Fluorescence was measured at 530 nm and excited at 485 nm. Figure 14 As shown, antibiotic administration affects intestinal permeability, as evidenced by the high FITC glucan profile in the serum of mice treated with antibiotics (Abx), while intervention with Giardin Seberlindnerella vaginalis and CRC34822 can counteract the negative effects of antibiotics on intestinal permeability.

[0414] In the third experiment, the effects of the treatment group on mitochondrial complex I activity were evaluated. Ileum was collected from animals and used to prepare protein lysis buffers using lysis buffer (Invitrogen™ Cell Extraction Buffer (Catalogue No. FNN0011) supplemented with 0.5 M EDTA and a mixture of Thermo Scientific™ Halt™ protease inhibitors (100X) (Catalogue No. 78430)).

[0415] According to the manufacturer's instructions, 500 μg of total protein was used to determine complex I activity using the ab109721 kit. Figure 15 As shown, mice treated with antibiotics (Abx) exhibited reduced complex I activity in their ileal tissue compared to the untreated control (control). Adding *Gerdinium saccharidum* and CRC34822 to antibiotic-treated mice resulted in a recovery of complex I activity compared to the MT control.

[0416] In summary, antibiotic treatment induces intestinal inflammation, and eATP plays a crucial role in the manifestation of this inflammation. These results suggest that removing eATP using ATP-degrading enzymes or yeast that produces ATP-degrading enzymes can counteract the effects of inflammation by improving the intestinal barrier and mitochondrial activity, both of which are associated with the aging process.

Claims

1. A method for treating, delaying, or reducing the severity of at least one condition, said at least one condition being selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence, said method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

2. The method of claim 1, wherein the age-related immune function changes include immune function decline and increased inflammation; the age-related intestinal function decline includes age-related decreased intestinal permeability, age-related decreased nutrient sensing function, and age-related decreased nutrient absorption function; the age-related metabolic regulation function decline includes decreased insulin sensitivity, decreased systemic glucose metabolism, decreased lipid metabolism, and decreased resting energy expenditure; the age-related cognitive decline includes short-term memory loss, difficulty finding words, altered sleep patterns, insomnia, slower processing speed, and / or degenerative dementia; and / or the age-related muscle mass and function changes include muscle atrophy and sarcopenia.

3. The method of claim 1 or claim 2, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

4. The method according to any one of claims 1-3, wherein the enzyme is not a mammalian ATP-degrading enzyme.

5. The method according to any one of claims 1-4, wherein the enzyme is a bacterial ATP-degrading enzyme.

6. The method according to any one of claims 1-5, wherein the enzyme comprises the enzyme with SEQ ID NO: The amino acid sequence shown in any one of 17-24, 57-63, 30-32, and 90-102 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide.

7. The method according to any one of claims 1-6, wherein the yeast strain that produces the ATP-degrading enzyme is selected from the genera *Cyberlindnera*, *Kluyveromyces*, and *Saccharomyces*.

8. The method according to any one of claims 1-7, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

9. The method of any one of claims 1-8, wherein the yeast strain that produces the ATP-degrading enzyme is *Gardinia sepium*, and comprises a strain deposited in DSMZ with the number DSM 33763 or a strain having all the identifying characteristics of the *Gardinia sepium* strain deposited in DSMZ with the number DSM 33763.

10. The method of any one of claims 1-9, wherein the yeast strain that produces the ATP-degrading enzyme is Kluyveromyces lactis, and comprises a strain deposited in DSMZ with the number DSM 33764 or a strain having all the identifying characteristics of the Kluyveromyces lactis strain deposited in DSMZ with the number DSM 33764.

11. The method of any one of claims 1-10, wherein the composition comprises probiotics.

12. The method of any one of claims 1-11, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

13. The method of any one of claims 1-12, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration, or topical administration.

14. The method of any one of claims 1-13, wherein the composition comprises a food product, a food ingredient, a dietary supplement, or a pharmaceutical agent.

15. The method of any one of claims 1-14, wherein the at least one condition is associated with mitochondrial dysfunction.

16. The method of any one of claims 1-15, wherein the subject is a human being.

17. The method of any one of claims 1-16, wherein the subject is a non-human animal.

18. The method of any one of claims 1-17, wherein the at least one condition affects one or more cells.

19. The method of claim 18, wherein the one or more cells comprise epithelial cells, endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and / or adipocytes.

20. A method for treating and / or preventing at least one physical condition related to mitochondrial health, the method comprising administering to a subject in need or at risk an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

21. The method of claim 20, wherein the at least one physical state is selected from the group consisting of: impaired activity of complex I, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function, and reduced energy production.

22. The method of claim 20 or claim 21, wherein the treatment and / or prevention delays the onset of at least one condition, said at least one condition being selected from age-related metabolic dysfunction, age-related immune function changes, age-related muscle mass and function changes, age-related intestinal dysfunction, age-related cognitive decline, and cellular senescence.

23. The method of any one of claims 20-22, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

24. The method of any one of claims 20-23, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

25. A method for treating age-related decline in nutrient absorption, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

26. The method of claim 25, wherein the treatment increases the nutrient absorption of the subject relative to a subject not treated with an effective amount of the composition.

27. The method of claim 25 or claim 26, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

28. The method of any one of claims 25-27, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

29. A method for increasing prealbumin / albumin levels in a subject, the method comprising administering to a subject in need an effective amount of a) a composition comprising an ATP-degrading enzyme; b) a composition comprising at least one yeast strain that produces an ATP-degrading enzyme; or c) a combination thereof.

30. The method of claim 29, wherein the increase is relative to a subject who has not been treated with an effective amount of the composition.

31. The method of claim 29 or claim 30, wherein the prealbumin level is a serum prealbumin level.

32. The method of any one of claims 29-31, wherein the increase in prealbumin levels reduces inflammation in the subject relative to a subject not treated with an effective amount of the composition.

33. The method of claim 32, wherein the inflammation is systemic inflammation.

34. The method of any one of claims 29-33, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

35. The method of any one of claims 29-34, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

36. A method for reducing the occurrence of age-related skin changes, the method comprising applying a composition topically to the skin of a subject, the composition comprising: a) an ATP-degrading enzyme; b) at least one yeast strain that produces an ATP-degrading enzyme, or c) a combination thereof. The occurrence of age-related skin changes was reduced in subjects who did not receive the composition; and The age-related skin changes mentioned above include fine lines, wrinkles, hyperpigmentation, melasma, age spots, skin discoloration, and / or skin inflammation.

37. The method of claim 36, wherein the ATP-degrading enzyme comprises adenosine triphosphate diphosphatase, a member of the GDA1_CD39 superfamily, or a member of the acid phosphatase family.

38. The method of claim 36 or claim 37, wherein the yeast strain that produces the ATP-degrading enzyme is selected from Kluyveromyces lactis, Jettselberlindnerella vaginalis, and Blastomyces boulardii.

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