Stabilization of oxidases by glycine

Abstract

Compositions and methods involving the use of the amino acid glycine to stabilize oxidase enzymes are described. These compositions and methods allow for drying of compositions containing oxidase enzymes with increased yields of enzyme activity and for long-term storage of dried compositions containing oxidase enzymes, in some cases without the need for refrigeration.

Description

Technical Field

[0001] The compositions and methods of the present invention involve the use of the amino acid glycine to stabilize oxidase enzymes. These compositions and methods allow heat drying of compositions containing oxidase enzymes with increased yield of enzyme activity, as well as long-term storage of dried compositions containing oxidase enzymes, in some cases without the need for refrigeration. Background Art

[0002] Oxidase (EC 1.1.3) is an enzyme that catalyzes redox reactions, typically involving diatomic oxygen (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. Many 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 a particularly wide range of commercial uses. GOx (sometimes referred to as GOD) is widely used to control microbial contamination (e.g. in winemaking), as well as for biochemical analysis and biosensors to measure free glucose in blood and urine. GOx is also used to produce stronger doughs in baking, to remove oxygen from food packaging, and to prevent browning of certain foods such as egg whites.

[0004] GOx is a heat-sensitive enzyme, and commercial production is expensive and inefficient due to the large loss of activity during production and storage. Processes used to make animal feed, such as spray drying and pelleting, are particularly destructive to GOx. Given the large number of uses for GOx and other oxidases, methods to improve stabilization in a cost-effective manner are needed.

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

[0006] GOx and HOx are thermosensitive enzymes, and their commercial production becomes expensive and inefficient due to substantial activity losses during production and storage. There is a need to improve stabilization in a cost-effective manner. Summary of the invention

[0007] The compositions and methods of the present invention involve the use of the amino acid glycine to stabilize oxidase enzymes. These compositions and methods allow for heat drying of compositions containing oxidase enzymes with increased enzyme activity yield, as well as long-term storage of dried compositions containing oxidase enzymes with reduced need for refrigeration. Various aspects and embodiments of these compositions and methods are described in the following independently numbered paragraphs.

[0008] 1. In one aspect, a method for improving the stability of an oxidase in a composition is provided, the method comprising: mixing the free amino acid glycine with the oxidase at a ratio of at least 1 gram of glycine per gram of oxidase, wherein the mixed oxidase has improved stability in the composition compared to the oxidase in a composition lacking glycine but being the same in other aspects.

[0009] 2. In some embodiments of the method of paragraph 1, the ratio is at least 6.4 grams of glycine per gram of oxidase.

[0010] 3. In some embodiments of the method of paragraph 1 or 2, glycine is the primary stabilizer of the oxidase in the formulation.

[0011] 4. In an embodiment of the method of any preceding paragraph, glycine is present in the substantial absence of free acidic amino acids.

[0012] 5. In an embodiment of the method of any preceding paragraph, glycine is the only free amino acid present.

[0013] 6. In an embodiment of the method of any preceding paragraph, the oxidase is glucose oxidase or hexose oxidase.

[0014] 7. In an embodiment of the method of any of the preceding paragraphs, the oxidase and glycine are mixed in an aqueous solution or suspension.

[0015] 8. In some embodiments of the method of paragraph 7, the mixed oxidase and glycine is subsequently dried.

[0016] 9. In another aspect, a composition is provided comprising an oxidase and free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase, wherein the mixed oxidase has improved stability in the composition compared to the oxidase in a composition lacking glycine but being otherwise identical.

[0017] 10. In some embodiments of the composition of paragraph 9, the ratio is at least 6.4 grams of glycine per gram of oxidase.

[0018] 11. In some embodiments of the composition of paragraph 9 or 10, glycine is present in the substantial absence of acidic amino acid residues.

[0019] 12. In an embodiment of the composition of any of the preceding paragraphs 9-11, glycine is the only free amino acid present.

[0020] 13. In some embodiments of the composition of any of paragraphs 9-12, the oxidase is glucose oxidase or hexose oxidase.

[0021] 14. In some embodiments of the method or composition of any of paragraphs 9-13, the oxidase and glycine are incorporated into a particle, film, pad, gel, or other solid and / or liquid composition.

[0022] These and other aspects and embodiments of compositions and methods will be apparent from this description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a graph showing the ratio of bound FAD to free FAD in the presence of increasing amounts of glycine per gram of active GOx. DETAILED DESCRIPTION

[0024] I. Introduction

[0025] The inventors of the present invention have discovered that incorporation of the amino acid glycine improves the heat drying yield of the enzyme composition of the formulated oxidase concentrate more than any other amino acid. Although the use of amino acids to stabilize oxidases, such as glucose oxidase (GOx) has been described (see, e.g., U.S. Pat. No. 4,543,326), such studies have consistently identified different amino acid residues, such as acidic amino acid residues (i.e., aspartic acid and glutamic acid and salts thereof), as the most preferred amino acids for stabilization. The present invention relates to the use of glycine (a neutral amino acid), particularly as a preferred stabilizer for oxidases.

[0026] II. Definitions and Abbreviations

[0027] Before describing the compositions and methods of the present invention in detail, the following terms are defined for the sake of clarity. Undefined terms shall be given the ordinary meanings used in the relevant art.

[0028] As used herein, the term "granule" refers to a small particle of a substance. The particle comprises a core optionally having one or more optional coating layers.

[0029] As used herein, "weight percent," "weight fraction," "mass fraction," or simply "fraction" refers to the relative amount of mass on a % wt / wt or fractional wt / wt basis, e.g., the relative amount of a component compared to the mass of the entire particle.

[0030] As used herein, the terms "pellets" and "granulation" refer to solid, round, spherical and cylindrical tablets or pellets and methods of forming such solid shapes, particularly feed pellets and solid, extruded animal feeds.

[0031] As used herein, the term "recovered activity" or "activity recovery rate" refers to the ratio of (i) the activity of an enzyme after treatment involving one or more of the following stressors: heating, pressurization, 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 can be expressed as a percentage. The percentage of recovered activity is calculated as follows:

[0032]

[0033] As used herein, the term "about" refers to ±15% of a reference value.

[0034] For ease of reference, the elements of the compositions and methods of the present invention may be arranged under one or more headings. It should be noted that the compositions and methods under each heading are also applicable to the compositions and methods under other headings.

[0035] As used herein, the singular articles "a / an" and "the" include plural referents unless the context clearly indicates otherwise. All references cited herein are hereby incorporated by reference in their entirety. Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0036] ℃ Celsius

[0037] g or gm gram

[0038] g / L grams / liter

[0039] g / mol gram / mole

[0040] mol / mol molar ratio

[0041] μmol micromole

[0042] hr or h hours

[0043] kg kilogram

[0044] mg milligram

[0045] mL or ml milliliters

[0046] min

[0047] M Moore

[0048] mM millimole

[0049] μm micrometer (micron)

[0050] μL and μl microliter

[0051] UFC Ultrafiltration Concentrate

[0052] Dissolved solid active oxidase protein plus other non-oxidase fermentation solids

[0053] RH Relative humidity

[0054] wt weight

[0055] %wt / wt weight percentage

[0056] III. Oxidase that can be stabilized by glycine

[0057] Glycine is a preferred amino acid stabilizer for the exemplary oxidase, a finding that is expected to be applicable to a wide range of oxidases. Furthermore, testing the ability of glycine to stabilize a particular oxidase is routine and does not require undue experimentation.

[0058] Specific oxidases expected to be stabilized using glycine are those that use molecular oxygen (O2) as an acceptor and are classified as EC 1.1.3. Exemplary oxidases include those listed in Table 1.

[0059] Table 1. EC 1.1.3 enzymes

[0060]

[0061]

[0062] The oxidase is preferably stabilized with glycine in an aqueous mixture, but can then be dried, such as by spray drying, spray agglomeration, spray granulation, etc. The oxidase can be subjected to high shear and extrusion, followed by drying, and can be used for fluid bed coating. The glycine-stabilized oxidase can be incorporated into granules, pellets, films, mats, gels, or any other solid or liquid composition, preferably a composition in which the glycine is not separated from the oxidase.

[0063] IV. Glycine to Oxidase Ratio

[0064] Different oxidases may require different amounts of glycine for stabilization, and these amounts are readily determined. Such amounts are best expressed as a molar ratio or weight percent of glycine to active oxidase protein, or a molar ratio or weight percent of glycine to oxidase activity (expressed in defined units). Based on the additional examples, a recommended ratio is about 1 to about 10 g glycine / g active oxidase protein, such as about 1.6 to about 8.4 g glycine / g active oxidase protein. In some cases, the ratio is at least about 6.4 g glycine / g active oxidase protein.

[0065] The source of glycine, i.e., 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 to be stabilized. Glycine is the primary determinant of oxidase stability, meaning that it is the only excipient necessary and sufficient to improve stability; however, glycine may also be mixed with other amino acids, other stabilizers, or other beneficial agents.

[0066] Examples

[0067] The following examples are intended to illustrate, but not to limit the scope of the invention to the oxidases exemplified. 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 fungal species, such as Aspergillus niger. The oxidation reaction is carried out by flavin adenine dinucleotide (FAD), which is a redox cofactor that is tightly bound (not covalently) between two identical GOx monomers. The GOx dimer contains two FAD cofactors that are responsible for the redox properties of the enzyme. GOx is a heat-labile enzyme and is sensitive to drying processes involving heating. Under denaturing conditions, such as those occurring during heat drying, the dimer dissociates into its subunits, resulting in irreversible cofactor loss, from a "bound" state to a "free" state, which in turn leads to enzyme inactivation and aggregation.

[0068] Example 1. Thermal inactivation of GOx during heating and drying

[0069] Two samples of pure (unformulated) GOx UFC (25 mg active protein / g UFC) were obtained from the DuPont / Danisco fermentation plant. These samples were cultured in an oven at 50°C for four, eight, and twenty-four hours. The experimental method involved adding 80 μL of each sample to the inner well of a 96-well plate (to avoid edge effects), sealing the plate using a breathable seal, and incubating the plate at 50°C. The weight of the plate was recorded before and after drying to ensure that all moisture was removed.

[0070] To measure activity, the dried contents of each well were resuspended in 80 μL of purified water. After resuspension, the activity was determined using a standard glucose oxidase assay. Glucose oxidase catalyzes the conversion of glucose and oxygen in the assay into hydrogen peroxide and gluconic acid. Hydrogen peroxide is reacted with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to change the appearance of the reaction medium from colorless to green. The reaction is catalyzed by peroxidase. The green color is measured at 405 nm on a spectrophotometer. The method is calibrated using linear regression of standard dilutions prepared from known concentrations of standard substances. As shown in Table 2, the enzymatic activity of the oxidase decreases over time.

[0071] Table 2. Oxidase activity yield of pure GOx UFC samples incubated at 50 °C for 4, 8 and 24 h; n = 8, mean ± SD

[0072]

[0073] Example 2. Thermal stabilization of GOx by glycine

[0074] Pure (unformulated) GOxUFC (25 mg active protein / g UFC) samples were obtained from the DuPont / Danisco fermentation plant. A series of heat stress experiments were performed on GOx concentrates formulated with different concentrations of glycine ranging from 0% to 16%. After heating the samples at 50°C for 8 hours, a correlation was observed between GOx denaturation (based on the ratio of bound FAD cofactor to free FAD cofactor) and glycine concentration (Table 3). The FAD distribution of the GOx samples was performed by hydrophobic interaction chromatography with UV detection at 450nm. The area under the absorption curve and the retention time of the "bound" and "free" FAD cofactors were determined, and then their respective ratios were calculated. The addition of glycine to GOx UFC reduced the degree of denaturation of the enzyme protein under heat stress. Based on the graphical analysis of the denaturation curves, the preferred glycine concentration is greater than about 1 g glycine / g oxidase protein, for example greater than 1.6, or even greater than 6.4 g glycine / g oxidase protein. Amounts greater than about 8.4 g glycine / g oxidase protein do not appear to provide additional benefit.

[0075] Table 3. Changes of free FAD and bound FAD with glycine concentration

[0076]

[0077] Pure GOx UFC and formulated GOx UFC samples containing cationic or neutral amino acids were prepared and dried in 96-well plates. About 20 μL of each GOx formulation was added to 6 wells and the plate was sealed with a breathable cover. The plate was incubated at 50 ° C for 4 hours to dry these samples. The enzyme activity of the dried samples was measured by the protocol described in Example 1 above.

[0078] Cationic amino acids include arginine and lysine, and neutral amino acids include 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 higher solubility because it would increase the pH of the concentrate.

[0079] Table 4 shows the recovery activity of GOx formulated with different cationic and neutral amino acids. Anionic amino acids including aspartic acid, D-glutamic acid, and L-glutamic acid have lower solubility in GOx UFC and show lower enzyme activity yields compared to neutral and cationic amino acids. Therefore, they are not preferred excipients for the thermal stability of the GOx composition of the present invention.

[0080] Table 4. GOx recovery activity after drying at 50 °C for 4 h; n = 6, mean ± SD

[0081]

[0082] Activity assay results showed that the dried formulation containing glycine had the highest recovered activity compared to pure samples without glycine and samples formulated with other neutral and cationic amino acids. Glycine has the lowest molecular weight of the other amino acids tested and provides the highest molar concentration as a thermal stabilizer relative to active enzyme protein.

[0083] Example 3. Storage stability of GOx spray-dried compositions

[0084] Glycine was added to the GOx concentrate, and UFC dissolved 8.2% of the solids at 14% (wt glycine / wt UFC), i.e., 5.6g glycine / g active protein, improving the storage stability of the dry enzyme composition produced by spray drying. A 6-month stability study was conducted on the spray-dried GOx powder composition containing glycine and without glycine (control). The samples were placed in plastic bottles with closed lids and incubated at 37°C and 65% relative humidity for 180 days. During this period, the enzyme activity was monitored by analyzing the samples after 0, 3, 7, 14, 30, 60, 90 and 180 days. The recovered enzyme activity of the test samples is listed in Table 5. After 180 days of storage, the GOx / glycine composition retained up to 55% of the enzyme activity.

[0085] Table 5. Recovered enzyme activity (%) of glucose oxidase / glycine spray-dried powder compositions compared to unformulated glucose oxidase after 6 months at 37°C / 65%RH

[0086]

Claims

1. A method for improving the stability of an oxidase in a composition, the method comprising: The free amino acid glycine is mixed with the oxidase in a ratio of at least 1 gram of glycine per gram of oxidase, wherein the mixed oxidase has increased stability in the composition compared to the oxidase in an otherwise identical composition lacking glycine, wherein the oxidase is glucose oxidase.

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

3. The method of claim 1 or 2, wherein glycine is the major stabilizer of the oxidase in the formulation.

4. The method of claim 1 or 2, wherein glycine is present in the absence of free acidic amino acids.

5. The method of claim 1 or 2, wherein glycine is the only free amino acid present.

6. The method of claim 1 or 2, wherein the oxidase and glycine are mixed in an aqueous solution or suspension.

7. The method of claim 6, wherein the mixed oxidase and glycine is subsequently dried.

8. A composition comprising an oxidase and the free amino acid glycine in a ratio of at least 1 gram of glycine per gram of oxidase, wherein the oxidase has increased stability in the composition compared to the oxidase in an otherwise identical composition lacking glycine, wherein the oxidase is glucose oxidase.

9. The composition of claim 8, wherein the ratio is at least 6.4 grams of glycine per gram of oxidase.

10. A composition as claimed in claim 8 or 9, wherein glycine is present in the absence of acidic amino acid residues.

11. A composition as claimed in claim 8 or 9, wherein glycine is the only free amino acid present.

12. The composition of claim 8 or 9, wherein the oxidase and glycine are incorporated into particles, films, pads, gels or other solid and / or liquid compositions.

Images