Stabilization of oxidase by glycine

Abstract

This invention provides methods and compositions related to the stabilization of oxidase enzymes. [Solution] A method for increasing the thermal stability of glucose oxidase in a composition, comprising mixing the glucose oxidase with the free amino acid glycine in a ratio of 1.6 to 3.2 grams of glycine per gram of glucose oxidase, wherein the mixed glucose oxidase exhibits increased activity in the composition compared to glucose oxidase in a composition that is otherwise identical except for the lack of glycine.

Description

Technical Field

[0001] The compositions and methods of the present invention relate to the stabilization of oxidase enzymes using the amino acid glycine. The compositions and methods allow for heat drying of an oxidase-containing composition having an improved amount of enzyme activity and, optionally, long-term storage of the dried oxidase-containing composition without the need for refrigeration.

Background Art

[0002] Oxidase (EC 1.1.3) is an enzyme that catalyzes oxidation-reduction reactions typically involving dioxygen (O2) as an electron acceptor. Examples of oxidases are hexose oxidase, glucose oxidase, monoamine oxidase, xanthine oxidase, L-gulonolactone oxidase, lysyl oxidase, NADPH oxidase, polyphenol oxidase, cytochrome P450 oxidase, and laccase. A number of additional enzymes are listed herein.

[0003] Glucose oxidase (GOx; EC 1.1.3.4) is an oxidase that can be overexpressed in heterologous hosts for large-scale production and has particularly wide-ranging commercial applications. GOx (sometimes referred to as GOD) is widely used, for example, in wine production to control microbial contamination and in biochemical analyses and biosensors for measuring free glucose, for example, in blood and urine. GOx is also used in baking to produce stronger dough, in food packaging to remove oxygen, and to prevent browning of certain foods such as egg white.

[0004] GOx is a heat-sensitive enzyme, and commercial production is costly and inefficient due to significant loss of activity during production and storage. Processes such as spray drying and pelletizing for animal feed production are particularly destructive to GOx. Given the countless applications of GOx and other oxidases, there is a need for means to increase stabilization in a cost-effective manner.

[0005] Hexose oxidase (HOx: EC 1.1.3.5) catalyzes the oxidation of monosaccharides and disaccharides to their corresponding lactones, accompanied by the reduction of molecular oxygen to hydrogen peroxide. This enzyme is commercially produced by overexpression in certain methylotrope yeasts. Hexose oxidase can oxidize a wide range of substrates, including D-glucose, D-galactose, maltose, cellobiose, and lactose. This broad substrate specificity distinguishes this enzyme from GOx, which is highly specific to D-glucose. HOx is also used in baking to produce stronger dough.

[0006] GOx and HOx are sensitive enzymes, and their commercial production is costly and inefficient due to significant loss of activity during production and storage. There is a need for methods to increase their stabilization in a cost-effective manner. [Overview of the project] [Means for solving the problem]

[0007] The present invention relates to the stabilization of oxidase using the amino acid glycine. This composition and method allows for the heat drying of an oxidase-containing composition with improved enzyme activity, and enables long-term storage of the dried oxidase-containing composition with reduced need for refrigeration. Aspects and embodiments of this composition and method are described in the following independently numbered paragraphs. 1. In one embodiment, a method is provided for increasing the stability of an oxidase enzyme in a composition, comprising mixing an oxidase enzyme with the free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase enzyme, wherein the mixed oxidase enzyme has increased stability in the composition compared to an oxidase enzyme in a composition that is otherwise identical but lacks glycine. 2. In some embodiments of the method of paragraph 1, the ratio is at least 6.4 grams of glycine per gram of oxidase enzyme. 3. In some embodiments of the methods of paragraph 1 or 2, glycine is the primary stabilizer of the oxidase enzyme in the preparation. 4. In some embodiments of any of the methods described in paragraphs 1-3, glycine is present in the substantial absence of free acidic amino acids. 5. In some embodiments of any of the methods described in paragraphs 1-4, glycine is the only free amino acid present. 6. In some embodiments of any of the methods described in paragraphs 1-5, the oxidase enzyme is glucose oxidase or hexose oxidase. 7. In some embodiments of any of the methods described in paragraphs 1 to 6, the oxidase enzyme and glycine are mixed in an aqueous solution or suspension. 8. In some embodiments of the method of paragraph 7, the mixed oxidase enzyme and glycine are then dried. 9. In another embodiment, a composition is provided comprising an oxidase enzyme and the free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase enzyme, wherein the oxidase enzyme has increased stability in the composition compared to an oxidase enzyme in a composition that lacks glycine but is otherwise identical. 10. In some embodiments of the composition of paragraph 9, the proportion is at least 6.4 grams of glycine per gram of oxidase enzyme. 11. In some embodiments of the compositions of paragraph 9 or 10, glycine is present in the substantial absence of acidic amino acid residues. 12. In some embodiments of any of the compositions in paragraphs 9-11, glycine is the only free amino acid present. 13. In some embodiments of any of the compositions in paragraphs 9-12, the oxidase enzyme is glucose oxidase or hexose oxidase. 14. In some embodiments of any of the methods or compositions described in paragraphs 9-13, the oxidase enzyme and glycine are incorporated into granules, films, pads, gels, or other solid and / or liquid compositions.

[0008] These aspects and embodiments of the composition and method, as well as other aspects and embodiments, will be apparent from the description of the present invention. [Brief explanation of the drawing]

[0009] [Figure 1] This graph shows the ratio of bound FAD to free FAD in the presence of a large amount of glycine per gram of active GOx. [Modes for carrying out the invention]

[0010] I. Introduction The inventors have discovered that the incorporation of the amino acid glycine, rather than any other amino acid, improves the heat-drying yield in the production of enzyme compositions from formulated oxidase enzyme concentrates. While the stabilization of oxidases such as glucose oxidase (GOx) using amino acids has been described (see, for example, U.S. Patent No. 4,543,326), these studies have consistently shown that other amino acid residues, such as acidic amino acid residues (i.e., aspartic acid and glutamic acid, and their salts), are most preferred for stabilization. It has been identified as a suitable amino acid. The present invention, in particular, relates to the use of glycine, a neutral amino acid, as a preferred stabilizer for oxidase.

[0011] II. Definitions and Abbreviations Before describing in detail the compositions and methods of the present invention, the following terms are defined for clarity. Terms not defined shall be given their common meaning as used in the relevant art.

[0012] As used herein, the term "granule" refers to small particles of a substance. A particle contains a nucleus optionally accompanied by one or more coating layers.

[0013] As used herein, “weight percentage,” “weight fraction,” “mass fraction,” or simply “fraction” refers to a relative amount of mass on a % wt / wt or fractional wt / wt basis, for example, the relative amount of the mass of an ingredient to the total mass of the granules.

[0014] As used herein, the terms “pellets” and “pelletization” refer to solid, round, spherical, and cylindrical tablets or pellets, and the processes for forming such solid shapes, in particular feed pellets and solid, extruded animal feed.

[0015] As used herein, the term “recovered activity” or “activity recovery” means the ratio of (i) the activity of the enzyme after treatment including one or more of the following stressors: heating, increased pressure, increased pH, decreased pH, storage, drying, exposure to surfactants, exposure to solvents, and mechanical stress, to (ii) the activity of the enzyme before treatment. The recovered activity may be expressed as a percentage. The recovered activity (percent) is calculated as follows:

number

[0016] As used herein, the term "approximately" refers to ±15% of the value mentioned.

[0017] For ease of reference, the elements of the compositions and methods of the present invention can be arranged under one or more headings. It should be noted that the compositions and methods under each such heading also apply to the compositions and methods under other headings.

[0018] As used herein, the singular articles "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The entire disclosures of all references cited herein are incorporated herein by reference. The following abbreviations / acronyms have the following meanings, unless otherwise specified: °C degrees Celsius g or gm gram g / L grams per liter g / mol grams per mole mol / mol mole to mole ratio μmol micromole hr or h hour kg kilogram mg milligram mL or ml milliliter min minute M molarity mM millimolarity μm micrometer (micron) μL and μl microliter UFC ultrafiltration concentrate Dissolved solids active oxidase protein + other non-oxidase fermentation solids RH relative humidity wt weight % wt / wt weight percent

[0019] III. Oxidases That Can Be Stabilized by Glycine The discovery that glycine is a preferred amino acid stabilizer for the exemplified oxidase enzymes is predicted to apply to a wide range of oxidases. Furthermore, testing glycine for its ability to stabilize a particular oxidase is conventional and does not require more experimentation than necessary.

[0020] Certain oxidases that are expected to be stabilized using glycine are those that use molecular oxygen (O2) as their receptor and are classified as EC 1.1.3. Exemplary oxidases include those listed in Table 1.

[0021] [Table 1]

[0022] The oxidase enzyme is preferably stabilized with glycine in an aqueous mixture, and can then be dried, for example, by spray drying, spray agglomeration, or spray granulation. The oxidase enzyme can be subjected to strong shearing and extrusion, followed by drying, and also by flow It can be used in dynamic layer coatings. Glycine-stabilized oxidase can be incorporated into granules, pellets, films, pads, gels, or any other solid or liquid composition, preferably one that does not separate glycine from the oxidase.

[0023] IV. Ratio of glycine to oxidase enzyme Different oxidases are likely to require different amounts of glycine for stabilization, and this amount is easily determined. These amounts are best expressed as the molar ratio or weight percentage of glycine to active oxidase enzyme protein, or as glycine (in defined units) relative to oxidase enzyme activity. Based on the attached examples, the recommended ratio is approximately 1 to 10 g of glycine per gram of active oxidase protein, for example, approximately 1.6 to 8.4 g of glycine per gram of active oxidase protein. In some cases, this ratio is at least approximately 6.4 g of glycine per gram of active oxidase protein.

[0024] The origin of glycine, i.e., whether it is naturally occurring or synthetic, is not important. It should be understood that the glycine used as a stabilizer is free glycine, not a glycine residue incorporated into a protein (including the oxidase protein that will be stabilized). Glycine is the primary determinant of oxidase stabilization, meaning it is the only additive necessary and sufficient for improved stability; however, glycine can also be mixed with other amino acids, other stabilizers, or other beneficial agents. [Examples]

[0025] The following examples are illustrative and do not limit the scope of the present invention to the exemplified oxidases. Glucose oxidase (GOx), also known as notatin (EC number 1.1.3.4), is an oxidase that catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. This enzyme is produced by certain species of fungi, such as black mold (Aspergillus niger). The oxidation reaction is carried out by flavin adenine dinucleotide (FAD), a redox cofactor that deeply penetrates and tightly binds non-covalently between two identical GOx monomers. The GOx dimer contains two FAD cofactors responsible for the enzyme's redox properties. GOx is a heat-unstable enzyme and is sensitive to drying processes, including heating. Under denaturing conditions, such as those present during heat drying, the dimer dissociates into its subunits, leading to irreversible loss of cofactors, a transition from a "bound" state to a "free" state, followed by enzyme inactivation and aggregation.

[0026] Example 1. Thermal deactivation of GOx during heat drying. Two samples of unadulterated (unprocessed) GOx UFC (25 mg of active protein per gram of UFC) were obtained from the DuPont / Danisco fermentation plant. These samples were incubated in a 50°C oven for 4, 8, and 24 hours. The experimental procedure involved adding 80 μL of each sample to the inner wells of a 96-well plate (to avoid edge effects), sealing the plate with a permeable seal, and incubating the plate at 50°C. To ensure all water was removed, the weight of the plate was recorded before and after drying.

[0027] To measure activity, the dried contents of each well were resuspended in 80 μL of purified water. After resuspending, activity was measured using standard glucose oxidase analysis. In this analysis, glucose oxidase catalyzes the conversion of glucose and oxygen to hydrogen peroxide and gluconic acid. The reaction of hydrogen peroxide with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), i.e., ABTS, changes the appearance of the reaction medium from colorless to green. This reaction is catalyzed by the enzyme peroxidase. The green color is measured at 405 nm with a spectrophotometer. This method is prepared from standard materials of known concentration. Calibration was performed using linear regression of standard dilutions. Oxidase enzyme activity decreased over time, as shown in Table 2.

[0028] [Table 2]

[0029] Example 2. Thermal stabilization of GOx with glycine Unadulterated (unprocessed) GOx UFC (25 mg of active protein per gram of UFC) samples were obtained from the DuPont / Danisco fermentation plant. A series of heat stress experiments were performed on GOx concentrates prepared with glycine at varying concentrations ranging from 0% to 16%. A correlation was observed between GOx denaturation (based on the ratio of bound to free FAD cofactors) and glycine concentration after heating the samples at 50°C for 8 hours (Table 3). FAD profiling of GOx samples was performed by hydrophobic interaction chromatography using UV detection at 450 nm. The area under the absorption curve against retention time was determined for "bound" and "free" FAD cofactors, and their respective proportions were then calculated. The addition of glycine to GOx UFC reduced the degree of enzyme protein denaturation under heat stress. Based on graph analysis of denaturation profiles, the preferred glycine concentration is more than approximately 1g of glycine per gram of oxidase protein, for example, more than 1.6g, and even more than 6.4g, per gram of oxidase protein. Amounts of glycine exceeding approximately 8.4g per gram of oxidase protein do not appear to offer any further additional benefits.

[0030] [Table 3]

[0031] Samples of unadulterated GOx UFC and uncompounded GOx UFC containing cationic or neutral amino acids were prepared for drying in a 96-well plate. Approximately 20 μL of each GOx preparation was added to six wells, and the plate was sealed with a breathable cover. The plate was incubated at 50°C for 4 hours to dry the samples. The enzyme activity of the dried samples was measured according to the protocol described in Example 1 above.

[0032] Cationic amino acids included arginine and lysine, while neutral amino acids included alanine, glycine, proline, and threonine. Of the amino acids tested, alanine, glycine, and proline showed the highest solubility in GOx UFC. Histidine, a neutral amino acid, was not included despite its high solubility due to its tendency to increase the pH of the concentrate.

[0033] Table 4 shows the restored activity of GOx when formulated with different cationic and neutral amino acids. Anionic amino acids, including aspartic acid, D-glutamic acid, and L-glutamic acid, had considerably lower solubility in GOx UFC and exhibited inferior enzyme activity compared to neutral and cationic amino acids. Therefore, these are not preferred additives for thermal stabilization of the GOx composition of the present invention.

[0034] [Table 4]

[0035] The results of the activity analysis showed that the dry formulation containing glycine exhibited the highest recovered activity compared to the unadulterated sample without glycine, as well as samples formulated with other neutral and cationic amino acids. Among the other amino acids tested, glycine has the lowest molecular weight, allowing for its incorporation as a heat stabilizer at the highest molar concentration compared to active enzyme proteins.

[0036] Example 3. Storage stability of GOx spray-dried composition The addition of glycine at 14% (glycine (wt) / UFC (wt)), i.e., 5.6 g of glycine per gram of active protein, to GOx concentrate, UFC dissolved solids 8.2%, improved the storage stability of the dried enzyme composition produced by spray drying. Spray-dried GOx powder compositions with and without glycine (control) were subjected to a 6-month stability study. Samples were contained in plastic bottles with sealed caps and incubated at 37°C and 65% relative humidity for 180 days. Enzyme activity was monitored by analyzing the samples at 0, 3, 7, 14, 30, 60, 90, and 180 days during this period. The recovered enzyme activity of the tested samples is summarized in Table 5. The GOx / glycine composition maintained up to 55% of its enzyme activity after 180 days of storage.

[0037] [Table 5]

Claims

1. A method for increasing the stability of an oxidase enzyme in a composition, comprising mixing the oxidase enzyme with the free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase enzyme, wherein the mixed oxidase enzyme has increased stability in the composition compared to an oxidase enzyme in a composition that is otherwise identical but lacks glycine.

2. The method according to claim 1, wherein the ratio is at least 6.4 grams of glycine per gram of oxidase enzyme.

3. The method according to claim 1 or 2, wherein glycine is the first stabilizer of the oxidase enzyme in the preparation.

4. The method according to any one of claims 1 to 3, wherein glycine is present in the substantially absent presence of free acidic amino acids.

5. The method according to any one of claims 1 to 4, wherein glycine is the only free amino acid present.

6. The method according to any one of claims 1 to 5, wherein the oxidase enzyme is glucose oxidase or hexose oxidase.

7. The method according to any one of claims 1 to 6, wherein the oxidase enzyme and glycine are mixed in an aqueous solution or suspension.

8. The method according to claim 7, wherein the mixed oxidase enzyme and glycine are subsequently dried.

9. A composition comprising an oxidase enzyme and the free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase enzyme, wherein the oxidase enzyme has increased stability in the composition compared to an oxidase enzyme in a composition that lacks glycine but is otherwise identical.

10. The composition according to claim 9, wherein the aforementioned ratio is at least 6.4 grams of glycine per gram of oxidase enzyme.

11. The composition according to claim 9 or 10, wherein glycine is present in the substantially absent presence of acidic amino acid residues.

12. The composition according to any one of claims 9 to 11, wherein glycine is the only free amino acid present.

13. The composition according to any one of claims 9 to 12, wherein the oxidase enzyme is glucose oxidase or hexose oxidase.

14. A method or composition according to any one of claims 9 to 13, wherein the oxidase enzyme and glycine are incorporated into granules, films, pads, gels, or other solid and / or liquid compositions.

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