Highly efficient oxalate-degrading enzyme for degradation of insoluble and soluble oxalic acid

Abstract

The present invention discloses enzymes for inducing oxalate by pH and thermal stability and methods for using conditions associated with oxalate in food processing.

Description

Field of the Invention

[0001] The present invention relates to a catalytic and highly efficient oxalate degrading enzyme (oxalate decarboxylase, OxDC). The present invention solves the problem of effectively degrading insoluble and soluble calcium oxalate, wherein the OxDC enzyme has a higher affinity (K) for oxalate than previously discovered and reported. m The invention also provides evidence why some OxDC enzymes are more stable and more active under acidic conditions, such as pH 1.5-5.0, and gives the reason for the instability as being caused by the loss of the quaternary structure of the protein. The invention also provides evidence for the first oxalate decarboxylase encapsulated in a trimer. The invention also relates to reducing the concentration of oxalate (also known as oxalic acid) from foods (such as flour, bread, canned vegetables, pies, etc.) and beverages (such as tea, beer, juice, etc.) and / or completely removing oxalic acid, thereby reducing the intake of oxalic acid from daily foods. This essentially establishes a series of low-oxalate and oxalate-free foods and beverages that can help people better manage oxalate-related disease conditions and / or allow healthy people to have a more nutritious diet (oxalic acid is considered an anti-nutrient). The invention further relates to a method for immobilizing the enzyme, which is both to stabilize the enzyme when it tends to be high temperature, and also to reuse the same enzyme to treat multiple batches of food and beverages (i.e., to achieve recycling of the enzyme). The present invention further relates to a method for formulating the enzyme, both to stabilize the enzyme and to prepare the pH environment by a new formulation. The present invention further relates to the use of the enzyme and the formulation in the preparation of enzyme granules for therapeutic, industrial, biotechnological, chemical, physical or other related applications, in particular therapeutic preparations (such as pharmaceutical preparations and nutraceutical preparations). The present invention also includes pharmaceutical and food compositions containing unformulated or formulated high-efficiency enzymes. The present invention further relates to the use of the enzyme as a food processing aid, food additive, industrial or other related application fields. The present invention includes the use of the enzyme in food manufacturing processes. The present invention also includes the use of the enzyme in industrial production processes (pulping and papermaking, chemistry, etc.). The present invention also relates to the use of these compositions in methods for treating a subject in need thereof, wherein the method comprises taking a pharmaceutical or nutraceutical composition containing one or more of the high-efficiency enzymes or one or more of the formulated enzymes. Background Art

[0002] Oxalate is a salt of a small organic dicarboxylic acid. It has two acid dissociation constant points: pH 1.25 and 4.14. Therefore, at lower pH values, more oxalate will exist as monoprotonated or oxalic acid, and the affinity with divalent counterions such as calcium will decrease, resulting in increased solubility. Insoluble oxalate refers to the oxalate ion (C2 O 4 2+ ) is strongly bound to, for example, calcium (Ca) by ionic interactions. 2+ ) on such counter ions.

[0003] Since oxalate is a weak organic acid, its solubility is highly dependent on pH. The acid dissociation constant of an acid is equal to the pH at which the acid and its corresponding base are present in equal amounts. Oxalic acid is a dicarboxylic acid (two acid groups), and therefore, has two acid dissociation constant points: pH 1.25 and pH 4.14. Therefore, as pH decreases, oxalate will dissolve in the monoprotonated (HC 2 O 4 1- ) or oxalate, and its affinity with calcium is reduced, resulting in increased solubility.

[0004] Oxalate is a metabolic end product in mammals. Oxalate can also be ingested through the diet, as it is commonly found in plants, primarily in leaves, nuts, fruits, and bark. Therefore, mammals have two sources of oxalate: endogenous (derived from the body's metabolism) or exogenous (derived from the diet). Oxalate absorption begins in the stomach and reaches maximum absorption in the small intestine. Studies have shown that oxalate excretion in urine increases immediately 20 minutes after oxalate ingestion, and excreta have two distinct peaks at 40 and 180 minutes after ingestion. Supporting the claim of absorption in the stomach is the fact that the first peak of oxalate absorption was not detected in patients who had undergone gastrectomy. 12 Due to the tight junctions of the gastric epithelium 3 , therefore, maximal absorption occurs in the small intestine, which may be affected by the fact that oxalate transport in the stomach may be limited to transcellular transport (across epithelial cells) rather than intercellular transport (between cells).

[0005] The body cannot degrade or metabolize oxalate; therefore, it is primarily excreted through the kidneys. When oxalate is not adequately removed, oxalate levels accumulate in the blood and concentrate in the urine, leading to hyperoxaluria (elevated oxalate levels in the urine) and, in severe cases, hyperoxalatosis (deposition of oxalate in tissues), which can cause tissue damage. SUMMARY OF THE INVENTION

[0007] The invention described herein relates to catalytically efficient oxalate degrading enzymes (oxalate decarboxylases, OxDCs) and their use in the degradation of soluble and insoluble oxalates, wherein the enzymes are found to have a much higher affinity (K) for oxalate than previously disclosed. mThe present invention also provides evidence for methods to keep free radicals from inhibiting the OxDC enzyme, such as replacing residue 340 with glutamic acid and adding vitamins (Cb6301, Fig.10 ).

[0008] The present invention also provides evidence for why some OxDC enzymes are more stable and more active under acidic conditions such as pH 1.5-5.0, and the instability caused by the loss of the quaternary structure of the protein. The present invention also provides evidence for the first OxDC that is naturally packaged into a trimer and the reasons for its inclusion into a trimer. Cb6301 has the smallest amount of ionic charged residues and the largest amount of hydrogen bond residues at the trimer interface. Due to the reduced number of ionic interactions and the increased number of hydrogen bonds, Cb6301 will be more stable in nature. The enzymes that are naturally packaged into trimers (Cb6301, Cb6312 and Cb6803) have enhanced stability and activity under extremely acidic conditions of pH 1.5. It was found that the remaining enzymes packaged into hexamers are mainly bound together as hexamers through ionic interactions at the hexamer interface. In addition, these enzymes also have higher ionic interactions at the trimer interface. The higher ionic interactions make the enzyme have poor stability at acidic pH (especially below pH 3.0). The reason for the decreased stability is due to the protonation of aspartic acid (acid dissociation constant = 3.65) and glutamic acid (acid dissociation constant = 4.25) under acidic pH conditions. When these amino acids are protonated, the protein quaternary structure of OxDC dissociates, leading to the unfolding of the enzyme and subsequent loss of activity (an irreversible event). The high ionic interactions at the hexamer and trimer interfaces make these interfaces prone to dissociation under acidic pH conditions.

[0009] The present invention also describes methods for recombinantly expressing these enzymes, formulations of these enzymes, and pharmaceuticals, specialty foods, or medical food compositions prepared from the formulated or unformulated enzymes. Another embodiment of the present invention is the use of these compositions in therapeutic purposes such as pharmaceuticals, specialty foods, or medical foods. The present invention also describes the use of these enzymes in food processing to degrade oxalate from foods (i.e., bread, flour, canned vegetables), beverages (i.e., beer, tea, juice), and industrial processes (i.e., pulp and paper, chemicals). One embodiment of the present invention is recombinantly expressed enzymes and immobilization of these enzymes for easy recovery and / or reuse. Another embodiment of the present invention is the use of these immobilized compositions in food processing. Another embodiment of the present invention is the use of these immobilized compositions in industrial applications. Other embodiments relate to the immobilization of oxalate degrading enzymes, thereby recycling and reusing the degrading enzymes for desired applications. The immobilization describes how the enzymes can increase stability when thermophilic.

[0010] The present invention describes formulations of oxalate degrading enzymes and other pH sensitive enzymes to reduce the loss of activity when the pH sensitive enzymes are placed in a non-optimal pH environment. Even under non-optimal pH conditions, this new formulation can still maintain activity by maintaining a microenvironment around the enzyme that is conducive to activity. A further embodiment of the present invention is to use these prepared compositions for the treatment and prevention of diseases, particularly diseases related to oxalate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 .OxDC subunit unit arrangement. OxDC packs into dimers of trimers to form hexamers. Trimeric subunit A interacts with both subunits B and C. Hexameric subunit A interacts with subunit D at the hexameric interface and with B and C at the trimer interface.

[0012] Figure 2 Thermostability of three OxDC enzymes: Agrocybe spp. ("A0" and "A8"), Bacillus cereus ("Bce"), and Synechococcus elongatus ("Cb6301_D29"). The relative activity of OxDC was calculated as described in Example 4 by normalizing all activity results to the activity at 25°C.

[0013] Figure 3 pH stability of two OxDC enzymes: Bacillus cereus ("Bce") and Synechococcus elongatus ("Cb6301_D29"). The relative activity of OxDC was calculated as described in Example 5 by normalizing all activity results to the activity at 25°C.

[0014] Figure 4 Oxalate degradation of OxDC in "A0" from pH 2 to pH 7.5 in 9 different foods containing different amounts of calcium as described in Example 6. The percentage of oxalate degradation refers to the percentage of total (insoluble + soluble) starting oxalate at time T=0.

[0015] Figure 5 OxDC activity of the "Cb6301_D29" recombinant enzyme at various pH and incubation times as described in Example 7. One unit (U) is defined as 1 micromole of oxalate degraded per minute.

[0016] Figure 6 OxDC activity of the "Bce" recombinant enzyme at various pH and incubation times as described in Example 7. One unit (U) is defined as 1 micromole of oxalate degraded per minute.

[0017] Figure 7. OxDC activity of Bce, Bpu, Bam, Bcl, Cb6301, Cb6803, A8 and YvrKOxDC recombinases at various pH values ​​as described in Example 7.

[0018] Figure 8 . As described in Example 8, the net ionic charge at the hexamer interface under acidic conditions of pH 1.5 relative to the most acidic pH value, where Bce, Bpu, Bam, Bcl, Cb6301, Cb6803, A8 and YvrKOxDC recombinant enzymes showed oxalate degradation activity. The number of amino acids or charges at the interface between two subunits. For example, according to Figure 1 It can be seen that it will be the interface between subunits A and D. The OxDC enzyme forms dimers of trimers, so an OxDC hexamer has three interfaces. Therefore, the y-axis should be multiplied by 3 to get the total charge between the entire hexameric structure. According to Figure 1 , with three hexameric interfaces between subunits A and D, B and E, and C and F.

[0019] Fig. 9 As described in Example 8, the net ionic charge at the trimer interface under neutral conditions relative to the most acidic pH conditions, where Bce, Bpu, Bam, Bcl, Cb6301, Cb6803, A8 and YvrK OxDC recombinant enzymes showed oxalate degradation activity. Two subunits (according to Figure 1 is the net ionic charge at the interface between A and B). The OxDC enzyme forms a dimer of trimers. Figure 1 As shown, each trimer has three interfaces between A and B, B and C, and C and A.

[0020] Fig.10 A partial multiple amino acid sequence alignment of Cb6301, A8, Bcl, Bce, Bpu, Bam, and YvrK OxDC enzymes was performed using the Clustal multiple sequence alignment of MUSCLE 3.8. The underlined regions are amino acids at the hexamer interface as described in Example 8. The bold and underlined residues are located at the trimer interface as described in Example 8. The residues highlighted in bold are important for maintaining the oxalate degradation activity of Cb6301, Cb6803, and Cb6312.

[0021] Fig.11 Native-PAGE gels of Cb6301, Bce and YvrK enzymes at different pH as described in Example 8.

[0022] Fig.12OxDC activity of "Bce" after incubation with different chemicals at 40°C for 6 days as described in Example 9. One unit (U) is defined as the degradation of 1 micromole of oxalate per minute.

[0023] Fig.13 OxDC activity of "Cb6301_D29" after incubation with different chemicals at 40°C for 6 days as described in Example 9. One unit (U) is defined as 1 micromole of oxalate degraded per minute.

[0024] Fig.14 Enzyme kinetics of Cb6301_D29OxDC: The reaction rate (v) per oxalate concentration (mM) was specified as described in Example 10. 0 ).

[0025] Fig.15 .A8 enzyme kinetics as a function of pH: K m Perform the calculations as described in Example 10.

[0026] Fig.16 . Relative activity percentage of unformulated Bce enzyme (solid bars) and formulated Bce (open bars) at different pH values ​​according to Example 11.

[0027] Fig.17 . Percent oxalate degraded in 1 hour: unformulated Bce enzyme (black circles) and formulated Bce (white circles) according to Example 11.

[0028] Fig.18 Mean oxalate per creatinine during low oxalate diet (LOD), high oxalate diet (HOD), and high oxalate diet with small dose of A0 as described in Example 12. Error bars represent SEM (p value = 0.00001, t critical value = 5% = 1.81246).

[0029] Fig.19 Mean oxalate per creatinine during low oxalate diet (LOD), high oxalate diet (HOD), and high oxalate diet with low dose of Ox1-CY (Cb6301) (CY L) as described in Example 12. Error bars represent SEM (p value = 0.00064, t critical value = 5% = 1.81246).

[0030] Fig. 20Mean oxalate per creatinine during low oxalate diet (LOD), high oxalate diet (HOD), and high oxalate diet with low dose Ox1-BC (Bce) (BC L) as described in Example 12. Error bars represent SEM (p value = 0.05188, t critical value = 5% = 1.81246).

[0031] Fig.21 Mean oxalate per creatinine during low oxalate diet (LOD), high oxalate diet (HOD), and high oxalate diet with low dose of Yvrk (Yvrk) as described in Example 12. Error bars represent SEM (no significant reduction in urine oxalate).

[0032] Fig. 22 . Percentage of formate produced per oxalate-calcium reaction: normalized calcium reaction under equimolar conditions (oxalate:calcium = 1:1) Bce was tested neat at 1 / 2x dilution and 1 / 4x dilution as described in Example 13.

[0033] Fig.23 . Percentage of formate produced per oxalate-calcium reaction: normalized calcium reaction under equimolar conditions (oxalate:calcium = 1:1) Cb6301 was tested neat at 1 / 5x, 1 / 10x and 1 / 20x dilutions as described in Example 13.

[0034] Fig.24 . Percentage of formate produced per oxalate-calcium: calcium reaction normalized under equimolar conditions (oxalate:calcium = 1:1) Yvrk was tested neat at 1 / 2x and 1 / 4x dilutions as described in Example 13.

[0035] definition

[0036] All words used in the present invention are intended to have the meanings commonly given in the art. For the sake of clarity, some words are defined below.

[0037] Oxalate degrading enzymes:

[0038] The term "oxalate degrading enzyme" is understood to mean any enzyme capable of reducing oxalate. The enzyme should catalyze a reaction that converts oxalate itself into products, rather than simply acting in an oxalate reduction pathway. According to the definition, oxalate degrading enzymes include oxalate decarboxylase, oxalate oxidase and oxalyl-CoA decarboxylase. The term "oxalate" includes oxalic acid and any salt thereof.

[0039] Cofactors:

[0040] The term "cofactor" should be understood as a non-enzymatic compound necessary for enzyme activity, including, for example, NAD + , NADP + , FAD, CoA, ATP and ADP.

[0041] Substrate:

[0042] The term "substrate" is to be understood as the incoming compound of an enzyme-catalyzed reaction. For a reaction catalyzed by an oxalate-degrading enzyme, this shall mean oxalate.

[0043] Subunit:

[0044] An enzyme subunit is a single enzyme molecule that combines (or "co-combines") with other enzyme molecules to form an enzyme complex. OxDC is typically composed of six subunits, thus forming a hexamer (a dimer of a trimer). For an animated description, see Figure 1 However, Cb6301, Cb6803 and Cb6312 described herein are naturally synthesized as three subunit trimers.

[0045] Enzymatic or catalytic efficiency:

[0046] The efficiency of an enzyme in catalyzing a reaction. Defined as k cat / K m The unit is conversion / M / s. Conversion refers to the conversion of substrate into reaction product.

[0047] Oxalate-related diseases and / or oxalate-related imbalances:

[0048] The term "oxalate-related diseases and / or oxalate-related imbalances" should be understood as diseases caused by an imbalance in systemic oxalate levels, including primary hyperoxaluria, hyperoxaluria, absorptive hyperoxaluria, enterogenic hyperoxaluria, idiopathic calcium oxalate nephrolithiasis (urolithiasis), vulvodynia, oxalopathy associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and disorders / conditions caused by / associated with gastrointestinal surgery, bariatric surgery (obesity surgery), and / or antibiotic treatment.

[0049] pH Insensitive Enzymes:

[0050] pH-insensitive enzymes are defined as enzymes formulated to exhibit higher activity at a specific pH than unformulated enzymes; thereby, making them less sensitive to the surrounding pH than unformulated enzymes.

[0051] Microenvironment:

[0052] Microenvironment is defined herein as the environment in contact with and / or in the closest proximity to the active enzyme. In certain embodiments, the microenvironment is within the boundaries of the particle; thus, it starts from the outer surface of the particle and reaches the core of the same particle. Since the present invention also contemplates nanoscale particles, it is understood that the microenvironment also refers to the environment within the microparticle or nanoparticle. The microenvironment described herein is considered to be the environment that is closest to the active enzyme formulated in comparison to the surrounding environment surrounding the microenvironment. The term "surrounding environment" is considered to be the environment surrounding the microenvironment. For embodiments in which the enzyme is associated with a particle, it is outside the boundaries of the particle associated with the active enzyme.

[0053] pH activity distribution:

[0054] The pH activity profile of the enzyme is the distribution obtained when the activity of an unformulated enzyme is measured under different pH conditions and the enzymes are plotted against each other (i.e., the x-axis represents pH and the y-axis represents activity). The effective pH activity profile is defined as the pH activity profile obtained when the activity of an unformulated enzyme is measured under different pH conditions and the enzymes are plotted against each other (i.e., the x-axis represents pH and the y-axis represents activity). Therefore, the effective pH activity profile does not describe the characteristics of the unformulated enzyme, but shows the activity detected when the formulated enzyme is placed in different surrounding environments. The pH of the effective pH activity profile describes the pH of the surrounding environment, not the microenvironment.

[0055] pH Active Compounds:

[0056] pH-active compounds are compounds that have a direct or indirect effect on the pH of their environment.

[0057] Protein quaternary structure:

[0058] Protein quaternary structure is the number and arrangement of multiple folded protein subunits in a multisubunit complex. It includes organizations ranging from simple dimers to large homo-oligomers as well as complexes with a defined or variable number of subunits.

[0059] Hexamer interface:

[0060] At the interface of the hexamer, amino acids from one subunit interact with residues from the second subunit. These interactions can consist of hydrogen bonds, ionic and / or hydrophobic interactions. In the presence of OxDC, ionic interactions contribute most to maintaining the hexameric structure. The residues at the hexameric interface are Figure 8 As described in the present invention, the hexameric interface will consist of interactions that occur between two subunits. For example, between subunits A and D. There are two additional hexameric interfaces between subunits B and E and subunits C and F. For an animated description, see Figure 1 .

[0061] Entire hexamer interface:

[0062] The total hexamer interface is the total number and type of amino acids that interact at all three hexamer interfaces. Since OxDC is packaged into a hexamer with six identical subunits, it has three separate hexamer interfaces; therefore, the total hexamer interface is calculated by multiplying the hexamer interface by three. These interactions can consist of hydrogen bonds, ionic and / or hydrophobic. In the presence of OxDC, the interactions involved are primarily ionic. The residues on the hexamer interface are Figure 8 The multiple sequence alignment shown in is marked. For a description of the animation see Figure 1 The interface of the entire hexamer is the sum of the interactions between A and D, B and E, and C and F.

[0063] The trimer interface:

[0064] At the trimer interface, amino acids from one subunit interact with residues from the second subunit. These interactions can consist of hydrogen bonds, ionic and / or hydrophobic interactions. In the presence of OxDC, ionic and hydrogen bonding interactions contribute most to maintaining the trimer structure. Residues at the trimer interface are underlined and boldfaced. Figure 8 In the multiple sequence alignment shown in . As described in the present invention, the trimer interface will consist of interactions that occur between two subunits. For example, between subunits A and B. There are two additional trimer interfaces between subunits B and C and subunits C and A. For an animated description, see Figure 1 .

[0065] The entire trimer interface:

[0066] The entire trimer interface is the total number and type of interacting amino acids at all three trimer interfaces. Since Cb6301, Cb6803, and Cb6312 are packed into trimers with three identical subunits, they have three separate trimer interfaces; therefore, the entire trimer interface is calculated by multiplying the trimer interface by three. These interactions can consist of hydrogen bonding, ionic, and / or hydrophobic interactions. In the presence of OxDC, the interactions involved are primarily ionic and hydrogen bonding. Residues at the trimer interfaces are underlined and boldfaced in Figure 8 For animation description see Figure 1 The interface of the entire trimer is the sum of the interactions between A and B, B and C, and C and A.

[0067] Net ionic charge:

[0068] The net ionic charge is the overall charge at the hexameric or trimer interface between two interacting subunits under defined pH conditions. It can be calculated either when all aspartic and glutamic acids are protonated (acidic pH) or at neutral pH, where these residues are ionic. Regarding OxDC, most homologues are incorporated into dimers of trimers. Thus, three subunits interact with three others, see Figure 1 In the presence of Cb6301, as shown in Table 4 and Fig. 9 As shown, the net ionic charge of -3 corresponds to only one of the three subunit interactions. If the entire trimer molecule is considered, the total net ionic charge would be -3x3=-9.

[0069] stability:

[0070] An enzyme is defined as stable under specific conditions (pH, temperature, etc.) when the oxalate degradation activity is 80-125% of that under control conditions.

[0071] Enzyme Nomenclature:

[0072] Yvrk = oxalate decarboxylase from Bacillus subtilis

[0073] Cb6301 = Oxalate decarboxylase from Synechococcus elongatus 6301

[0074] Cb6301_D29 = Oxalate decarboxylase from Synechococcus elongatus 6301, in which the first 29 amino acids at the N-terminus have been removed

[0075] Cb6803 = Oxalate decarboxylase from Synechococcus elongatus 6803

[0076] Bce = oxalate decarboxylase from Bacillus cereus

[0077] Bc1 = oxalate decarboxylase from Bacillus clausii

[0078] Bam = oxalate decarboxylase from Bacillus amyloliquefaciens

[0079] A8 / A0=oxalate decarboxylase from Agrocybe aegerita

[0080] Bpu = oxalate decarboxylase from Bacillus pumilus DETAILED DESCRIPTION OF THE INVENTION

[0082] Overview

[0083] There are two types of hyperoxaluria, primary hyperoxaluria (PH) and secondary hyperoxaluria (SH). Primary hyperoxaluria (PH) is an inborn error of glyoxylate metabolism, with an incidence of 0.1-0.2 per million. Primary hyperoxaluria is divided into three types: I, II and III, wherein type I is caused by insufficient or lack of activity of liver-specific peroxisomal alanine / glyoxylate aminotransferase (AGT), and the oxalate content in urine is about 88-352 mg per 24 hours (equivalent to 1-4 mmol per 24 hours). Type II primary hyperoxaluria (PH) is caused by insufficient or lack of activity of glyoxylate reductase / hydroxypyruvate reductase (GRHPR), and the oxalate content in urine is about 88-176 mg per 24 hours (equivalent to 1-2 mmol per 24 hours). Primary hyperoxaluria (PH) type III is a newly identified congenital defect that also presents with severe hyperoxaluria, with urinary oxalate excretion >0.8 mmol / 24 h.

[0084] If chronic or end-stage renal failure (ESRF) develops, patients in either state of PH type I and II can develop plasma oxalate concentrations greater than 100 μmol / L. Transient saturation of the blood with CaOx in PH patients leads to systemic hyperoxalatosis: CaOx crystals are deposited in multiple organs, including the kidneys, thyroid, myocardium, bones, skin, blood vessels, and eyes. If untreated, systemic hyperoxalatosis eventually leads to end-stage renal failure (ESRF) and death.

[0085] There are no approved therapies to treat or prevent PH types I-III. Current recommended treatment options focus solely on increasing the solubility of calcium oxalate deposits through supplementation with magnesium, citrate, and orthophosphate and encouraging urine output of at least 2 L per 24 hours. Pyridoxine is a cofactor for AGT deficiency and has a positive effect in PH type I in reducing urinary oxalate levels. Unfortunately, the only current treatment is combined kidney and liver transplantation; however, many transplanted organs are rejected or damaged by consistent plasma oxalate levels even after transplantation.

[0086] Secondary hyperoxaluria includes oxalate-related disorders such as, but not limited to, hyperoxaluria, absorptive hyperoxaluria, enteric hyperoxaluria, idiopathic calcium oxalate kidney stone disease (urolithiasis), vulvodynia, oxalopathy associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and disorders and conditions caused by / associated with gastrointestinal surgery, bariatric surgery (including obesity surgery), and / or antibiotic therapy.

[0087] Urolithiasis (kidney / urinary stone disease) is a common consequence of hyperoxaluria and a major health problem around the world. The risk of kidney stone formation revolves around a number of factors that are not fully understood. In Western countries, kidney or urinary stone disease accounts for up to 12%, of which approximately 70% are composed of calcium oxalate or calcium oxalate (CaOx) plus calcium phosphate. This disease occurs due to increased levels of oxalate in the kidneys and urine, the most common form of hyperoxalemia in humans, called enteric hyperoxaluria.

[0088] CaOx nephrolithiasis is highly prevalent, and there is evidence that slightly elevated urinary oxalate concentrations may be an important factor in a subgroup of patients with idiopathic CaOx urolithiasis. 4 Some have suggested that this is partly due to the widespread belief that stone-forming individuals have higher average urinary calcium levels than the general population: hypercalciuria is 5-10 times more common in stone-forming individuals than in the general population, and the relative supersaturation of calcium oxalate is higher in hypercalciuric individuals than in the general population. 5 The ratio of calcium to oxalate in normal urine is 5:1; therefore, a small increase in oxalate will have a large effect on the possible crystals produced due to the higher availability of calcium. Within the normal range of urine volume, small changes in oxalate will have a greater effect on CaOx supersaturation than changes in calcium.

[0089] The urine chemistry of many stone-recurrent populations differs from that of healthy people, and when urine chemistry is assessed in stone-forming populations, it has been demonstrated that oxaluria and calcium oxalate supersaturation can be controlled by metabolic diet manipulation. This strongly supports the key role of diet as a determinant of oxaluria and calcium oxalate supersaturation. 8

[0090] The importance of the calcium to oxalate ratio was also particularly evident. As calcium levels in the controlled metabolism diet decreased, oxalate in the urine tended to increase, indicating that more was being absorbed. 9

[0091] Zellweger range disorders (ZSD) are characterized by a generalized loss of peroxisome function due to insufficient peroxisome synthesis, and these patients have a high prevalence (83%) of hyperoxaluria. Although the mechanism of oxalate synthesis in ZSD patients is unclear, some ZSD patients have urine oxalate levels comparable to those of patients with primary hyperoxaluria (PH).

[0092] ESRF patients with chronic renal failure and chronic hemodialysis may develop hyperoxaluria because they cannot fully eliminate oxalate due to complications of their renal failure. In addition, vitamin C is often injected intravenously as a hemodialysis antioxidant and is subsequently metabolized to oxalate in the human body. The plasma oxalate concentration of these patients is between 30 and 90 μmol / L. In the United States alone, there were 345,000 hemodialysis patients in 2006.

[0093] The oxalate balance in the human body is complex and not yet fully understood. Oxalate is primarily excreted through the kidneys, but another way for the body to excrete oxalate is through the intestines. It has been shown that oxalate can be secreted into the intestines as an alternative excretion route to reduce the burden on the kidneys. Therefore, oxalate flow in the intestines can play an important role in the development of urolithiasis. 6 It has been demonstrated that oxalate transport is accomplished by solute-related carrier (SLC) transporters, particularly the SLC26 family of transporters. 78 This gene family encodes transporters that have been shown to have oxalate affinity and are found in the intestine (SLC26A1 (SAT1), SLC26A2 (DTDST), SLC26A3 (DRA), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9).

[0094] Oxalate Foods:

[0095] Various foods contain oxalates. Foods such as spinach, rhubarb, and nuts are known to contain high levels of oxalate. However, many other foods and beverages such as beets, chocolate, strawberries, bran, and tea are also high in oxalate. Other foods that contain oxalate include, but are not limited to, beans, grapefruit, oranges, onions, beets, potatoes, lettuce, plums, raspberries, pineapples, kiwis, kale, and tomatoes (see Table 1). The most common recommendation by doctors for patients with calcium oxalate kidney stones is to follow a low-oxalate diet. However, because the amount of oxalate in foods is affected by the environment in which the plant was grown, climate, season, and origin, maintaining a low-oxalate diet is often beyond the control of the dieter. A low-oxalate diet often conflicts with other more serious medical conditions, such as diabetes, making it difficult for patients to achieve a low-oxalate diet.

[0096] Table 1: Oxalate content in food (example)

[0097] Food name Total oxalate (mg) / 100g Sesame seeds 3800 Edible rhubarb 1235 Baby spinach 1063 Almond meal flour 519 sweet potato 354 sweet potato 278 Wheat bran flour 269 Special K Cereal 189 Hershey Milk Chocolate 107 black tea 78

[0098] Oxalate Bioavailability:

[0099] The bioavailability of oxalate depends on the solubility of oxalate. In soluble form, oxalate exists as oxalic acid (low pH) or oxalate ions (lacking strong affinity counterions). Therefore, the major factors that directly affect the solubility and bioavailability of oxalate are high affinity counterions (calcium, iron, and to some extent magnesium), and those that have indirect effects include phosphate (bound calcium), fat (high pH), and phytate (bound calcium). 9 Therefore, as the pH decreases, oxalate will be in the monoprotonated (HC 2 O 4 1- ) or oxalic acid (H 2 C 2 O 4 ) and has a reduced affinity for calcium, resulting in increased solubility.

[0100] Insoluble oxalates are all C 2 O 4 2- Oxalates, and they dissolve by lowering the pH. Calcium is the main factor in the insolubility of oxalate in the gastrointestinal (GI) tract. The recommended daily calcium intake for adults is about 1000 mg per day. For adults, most (72%) of this calcium is supplied by dairy products. As mentioned above, as the pH becomes more acidic, the amount of oxalate increases. Jaeger and Robertson proposed the concentration of oxalate that can be obtained under different concentrations of calcium and pH conditions, and demonstrated that at a pH of 2 and an average calcium concentration of 5mM (200mg / L), the maximum soluble oxalate concentration was 0.49mM (43mg / L), which is close to the expected amount of oxalate in a normal diet. 10 Thus, at the expected concentrations of calcium and oxalate in a normal adult diet, most oxalate is soluble at pH 2 and thus available for absorption or degradation by acid-stable enzymes. This fact is also supported by studies of oxalate excretion 20 minutes after ingestion, demonstrating that oxalate is bioavailable in the stomach. Oxalate solubility varies with changes in the calcium to oxalate ratio; therefore, the two most important characteristics of oxalate-degrading enzymes for therapeutic purposes are low pH tolerance and enzyme catalysis or catalytic efficiency.

[0101] Oxalate degrading enzymes:

[0102] Currently, three classes of enzymes have been identified as oxalate degraders: (1) oxalate decarboxylases (OxDC, oxalate carboxylate degraders, EC 4.1.1.2), (2) oxalate oxidases (OXO, oxalate:oxidoreductase, EC 1.2.3.4), and (3) oxalyl-CoA decarboxylases (oxalyl-CoA carboxylate degraders, EC 4.1.1.8). OxDC degrades oxalate (as oxalate) in a one-step electron withdrawal reaction to produce formate and carbon dioxide and requires Mn. 2+ and O 2 In order to facilitate catalysis. 2 Before the OXO enzymes are 2 Oxidation and generation of H 2 O 2 The third enzyme is present in bacteria and uses thiamine pyrophosphate as a cofactor to convert oxalyl-CoA to formyl-CoA and carbon dioxide.

[0103] Enzyme catalytic efficiency:

[0104] Enzymes can be efficient degraders of oxalate and are generally easy to produce in large quantities. For an enzyme to be an efficient degrader of oxalate in the gastrointestinal tract, it needs to be resistant to proteases, pH-tolerant, and have high enzymatic efficiency.

[0105] It is well known to those skilled in the art that the catalytic efficiency of an enzyme is usually described as k aat / K m . K m It can be described as the substrate concentration at which the enzyme exhibits 50% of the reaction rate. Therefore, K m The lower the value, the faster the enzyme works at a lower concentration of substrate. cat can be described as the rate of the catalytic reaction in units of per second (conversions per second, i.e., conversion of substrate to product). Thus, k cat should be as high as possible, and the rate k aat / K m It should also be high, thereby describing an enzyme that can rapidly degrade substrate at low substrate concentrations.

[0106] Enzyme efficiency is particularly important in the degradation of oxalate in vivo. The reason is that oxalate can be either soluble or insoluble (both exist as salts). Soluble oxalate is freely available to the enzyme, but insoluble oxalate requires the enzyme to compete with an ionic interaction with a counter ion such as calcium. The soluble and insoluble species are in a constant state of equilibrium, see the equation below. As the ratio of calcium to oxalate increases, more oxalate will be bound and not freely available to the enzyme ("soluble"). When oxalate is removed in the equilibrium below, the ratio of calcium to oxalate will increase, further reducing the amount of soluble oxalate available.

[0107] Calcium Oxalate <-> Calcium + Oxalate (soluble)

[0108] More calcium oxalate can be dissolved by removing soluble oxalate from the right side of the equation. The equilibrium is also affected by calcium concentration, so at high calcium concentrations the amount of soluble oxalate will also decrease. Therefore, the K of the enzyme m This becomes important because the enzyme needs to be able to have high activity at low concentrations of the substrate (small amounts of soluble oxalate). 11 Its importance in a therapeutic setting will be apparent to those skilled in the art, since dietary calcium is part of the normal diet and effectively reduces the amount of available soluble oxalate. Enzymes used for therapeutic purposes or for removing oxalate in an environment of high calcium concentration must be highly efficient, being able to catalyze the oxalate degradation reaction even when the amount of soluble oxalate is very low. This requires that the enzyme have a high affinity for its substrate and, among other things, a high k cat / K m .

[0109] Acid resistance of enzyme:

[0110] Recent advances in biotechnology allow the selection and preparation of new macromolecular compounds such as peptides and proteins as drugs for therapeutic purposes. Such compounds show strong and selective therapeutic activity, however, the therapeutic activity of proteins is highly dependent on optimal environmental factors such as pH, temperature and surface interactions.

[0111] As is well known to those skilled in the art, certain macromolecules have a high resistance to acidic pH values, which is inherent to the natural enzyme structure itself. However, the pH range for obtaining high activity in many enzymes is extremely limited. For example, the OxDC enzymes from Bacillus subtilis (YvrK) or Bacillus cereus (Bce) have optimal activity around pH 4 and 2.5, respectively; however, the enzyme completely loses activity at pH>7. This reduces the possibility of these enzymes being used in body fluids or other fluids at neutral or alkaline pH values.

[0112] The use of enzymes in industrial and biotechnological processes often requires that the enzymes must function under very specific and sometimes completely non-physiological conditions and that the treatments in many cases be adjusted to suit the properties of the enzymes used. Several industrial processes will continue to benefit in the future from the use of enzymes with re-engineered pH-dependencies (e.g. starch liquefaction for the production of ethanol and high fructose syrups (Shaw, Bott, & Day, 1999), detergent applications (Ito et al., 1998), and dye bleaching (Cherry et al., 1999). Therefore, there is a strong interest in developing experimental and theoretical methods to modify the pH-dependency of enzymes.

[0113] Advances have been made in the field of protein engineering and directed evolution, and now the performance of enzymes under various conditions can be routinely optimized using rational engineering or screening / selection-based approaches. Enzyme properties and the pH activity profile of enzymes have been altered under mutagenic effects. However, this has proven to be a very difficult task. Despite decades of research on enzyme structure-function relationships, the likelihood of success in rationally redesigning enzymatic pH activity profiles remains low. There are some experimental examples where the value of the active site acid dissociation constant has been changed and the pH-activity profile has been redesigned, but these shifts are modest and, typically, the critical mutations have been found using comparative protein engineering strategies (i.e., mutations are introduced based on comparisons with homologous enzymes that possess the desired pH activity profile). The conclusion of two decades worth of work is that very specific point mutations in the active site can change the pH dependence of enzyme activity, but unless this specific active site point mutation is known (e.g., from comparative studies), there is little hope of achieving a significant shift in the pH activity profile using rational engineering methods without inactivating the mutant enzyme or significantly reducing its activity. On the other hand, distant point mutations confer mostly wild-type activity to the mutant enzyme, but also produce very small shifts in the pH activity spectrum (Tynan-Connolly & Nielsen, 2006).

[0114] Protein quaternary structure:

[0115] Most enzymes are assemblies of multiple polypeptide chains. Therefore, protein quaternary structure refers to the number and arrangement of enzyme subunits relative to each other. Regarding OxDC, it is known from the literature that this particular enzyme is naturally enclosed in a hexamer, which is essentially a dimer enclosed in a trimer.

[0116] Enzyme immobilization:

[0117] Once the most suitable enzyme is identified, it can be formulated for better integrated processes. One of the most widely considered approaches is enzyme immobilization. Immobilization allows: (1) high enzyme loadings with high activity, thereby achieving large volumetric productivity; (2) controlled scale-up of the reaction; (3) simplification of downstream processes due to easy recovery and reuse of the biocatalyst; (4) product stream removal from the biocatalyst; (5) continuous operation (or batch operation on a drain and replenishment basis) and process automation are possible; and (6) substrate inhibition can be minimized. In addition, if the immobilization is designed properly, it can also prevent autolysis or denaturation caused by organic solvents and can provide thermal stability, handling and storage stability. If the enzyme used is expensive, immobilization can prove to be a key factor for economic feasibility. Enhanced stability that allows continuous reuse can achieve high specific productivity, thereby affecting the production costs associated with the biocatalyst. A typical example is the yield of immobilized glucose isomerase, which allows the use of 12,000-15,000 kg of high fructose corn syrup (containing 42% fructose) of dried fruit products per kg of biocatalyst over the lifetime of the biocatalyst. The improved thermal stability allows the above-mentioned conventional reactor operation with minimal risk of microbial growth, thus reducing the risk of microbial growth and reducing the requirements for hygiene, as the reactor does not require high cleaning requirements.

[0118] Oral administration and dosage forms of enzymes:

[0119] In certain embodiments, the composition can be administered alone or in combination with other treatments simultaneously or sequentially in a variety of ways depending on the condition to be treated. A typical mode of administration is oral administration, so that the administered composition is delivered to the gastrointestinal tract. The route of administration can be selected based on the disease or condition, the desired therapeutic effect, and the properties of the cells used. The actual method of preparing the dosage form is known or will be obvious to those skilled in the art. (See Remington's Journal of Pharmaceutical Sciences, 20th edition, published in 2000. Lippincott, Williams and Wilkins.) When the composition described herein is administered to an individual, the dosage is preferably a "preventive effective amount" or "therapeutically effective amount" that is fully demonstrated to be beneficial to the subject. In the case of treating oxalate-related diseases, the therapeutically effective amount is the amount that reduces oxalate in the subject and / or alleviates the symptoms of the disease.

[0120] Oral administration is the preferred and most widely used route of administration. However, due to its low bioavailability, this route is generally not suitable for transporting macromolecules such as proteins. The reduction in bioavailability is due to its inherent instability and low absorbability in the harsh environment of the gastrointestinal tract. Therefore, the technology that has been used to improve the bioavailability of oral delivery proteins is based on specific methods of preventing degradation by acid and the gastrointestinal tract or increasing protein permeability through the epithelial layer of the gastrointestinal tract (K. Park, Kwon, and Park, 2011). Due to the greater difficulty of the oral route of administration, many therapeutic proteins are administered parenterally. In order to minimize discomfort and improve patient compliance, protein drugs with long-term continuous delivery of sustained-release dosage forms are expected. The most widely used method for long-term delivery of protein drugs is the injection of protein drugs in microspheres made of biodegradable polymers.

[0121] The U.S. Food and Drug Administration has approved biodegradable and / or biocompatible polymers in many products. Among the family of synthetic polymers, polyesters have been widely attracted and studied. Their attractive features include ease of degradation by hydrolysis of the ester chain, absorption of degradation products through metabolic pathways in some cases, and possible changes in structure that affect degradation rate. Examples of biodegradable and biocompatible polyesters are poly(glycolic acid) and poly(lactic acid) and a series of copolymers thereof such as poly(lactic-co-glycolic) acid (PLGA). PLGA has been extensively studied as a carrier for controlled protein and peptide delivery (Ding and Schwendeman, 2008), (Cohen, Yoshioka, Melissa, Hwang, and Langer, 1991), (Gupta, Singh, and O'Hagan, 1998), (van de Weert, Hennink, and Jiskoot, 2000), (Schwendeman, 2002), resulting in several commercially available injectables (Okada, Doken, Ogawa, and Toguchi, 1994), (Ogawa, Okada, Heya, and Shimamoto, 1989), (Johnson et al., 1996) with a good safety record (Chasin and Langer, 1990). PLGA degrades into lactic acid and glycolic acid monomers, which are then converted to CO 2 It is eliminated from the body via the Krebs cycle in the form of water and water. Other examples of biodegradable and biocompatible polyesters or copolyesters are: poly(orthoesters), polycaprolactone and poly(propylene fumarate).

[0122] Polypropylene fumarate is a biodegradable unsaturated linear polyester. The degradation products are propylene glycol, poly (acrylic acid-co-fumaric acid) and fumaric acid. When in composite materials, the degradation time depends on the structure of the polymer and other components (Temenoff and Mikos, 2000).

[0123] Another example of biodegradable polymers and controlled drug delivery applications is polyanhydrides (Brem et al., 1995). Polyanhydrides are degraded by hydrolysis of anhydride chains, and the degradation products are nontoxic and produce minimal inflammatory reactions (Gunatillake and Adhikari, 2003). By selecting suitable diacid monomers, the degradation rate can be changed simply by changing the structure in the polymer backbone. For example, poly (sebacic acid) degrades rapidly (about 54 days in saline), while the degradation time of poly (1,6-bis (p-carboxyphenoxy)) hexane is about one year. Therefore, the combination of these different number monomers will produce polymers with degradation characteristics designed for specific applications (Temenoff and Mikos, 2000). Other examples of biodegradable polymers are poly (ethylene sulfonic acid) and poly (acrylic acid).

[0124] It is well known that there are different levels of water-soluble acidic impurities in PLGA, which can affect its solid state stability, drug encapsulation efficiency and drug release response (Yamamoto, Okada, Yasuaki, and Miyagawa, 1993). Moreover, it is generally believed that the degradation mechanism of aliphatic polyester microspheres is a hydrolysis mechanism, and the ester main bond undergoes hydrolysis in an aqueous environment such as body fluids, and in the presence of PLGA, the polymer eventually degrades into lactic acid and glycolic acid monomers, thereby reducing the pH value of the current environment (Freitas, Merkle, and Gander, 2005), (Fu, Pack, Klibanov, and Langer, 2000), (Zhu, Mallery, and Schwendeman, 2000). This has been regarded as a problem of encapsulated protein stability in the art.

[0125] Vert and coworkers have extensively studied the size dependence of hydrolytic degradation of devices based on lactic and glycolic acid polymers. Factors that can modulate the hydrolytic degradation behavior of lactide / glycolide homopolymer and copolymer microspheres include, but are not limited to: water permeability and solubility (hydrophilicity / hydrophobicity), chemical composition, hydrolysis mechanism (non-catalytic, autocatalytic, enzymatic), additives (acidic, basic, monomer, solvent, drug), morphology (crystalline, amorphous), device or particle size (size, shape, surface area-to-volume ratio), matrix porosity, glass transition temperature (glassy, ​​rubbery), molecular weight and molecular weight distribution, physicochemical factors (ion exchange, ionic strength, pH) and properties of the preparation process (ion exchange, ionic strength, pH), sterilization, and implantation site (Anderson and Shive, 1997), (TGPark, 1995), (SMLi, Garreau, and Vert, 1990), (Grizzi, Garreau, Li, and Vert, 1995), (TGPark, 1995). Some of these factors are also relevant to the hydrolytic degradation behavior of the other types of polymers discussed above, and the factors involved are further described below.

[0126] Additives can affect the degradation rate through their acidity or alkalinity and loading. Maulding et al. reported on accelerating the degradation of thioridazine tertiary amine compounds. Catalysis is attributed to the nucleophilic nature of the amino group (Maulding et al., 1986). Therefore, basic compounds can catalyze the cleavage of ester chains, thereby accelerating the degradation of polymers. On the other hand, an appropriate amount of basic compounds can neutralize the carboxyl end groups, thereby reducing the acid-induced degradation rate.

[0127] The degree of crystallinity of the homopolymer or copolymer used can play an important role in the degradation rate. Long-term studies in animals have shown that implantation of amorphous structures results in a decrease in the molecular weight of the implant compared to semi-crystalline samples; therefore, it is believed that the amorphous component degrades, in part due to autocatalytic degradation behavior (Pistner et al., 1994).

[0128] The porosity of the microspheres plays a major role as it can enhance the diffusion of low molecular weight degradation products at the oligomers and carboxyl chain ends that can promote autocatalytic degradation (Shive and Anderson, 1997). Microspheres made from solutions with lower polymer concentrations usually have an internally porous structure (Yang, 2001), which may lead to a more efficient diffusion of acidic degradation products through the polymer matrix and thus promote their release (Liu and Schwendeman, 2012).

[0129] The molecular weight distribution of the monomers can also affect the autocatalytic process, as large molecular weights or a wide range of molecular weight distributions have more carboxylic acid end groups available for autocatalysis.

[0130] It has been shown that degradation products are not only monomers; in PLGA films, the main components of water-soluble acids after 3 weeks of culture are glycolic acid, lactic acid and lactoyl lactic acid, as well as an unknown polymer assumed to be a lactic acid tetramer (Ding and Schwendeman, 2004). The acid content increases sharply after three weeks, which is due to the continuous accumulation of acids caused by polymer degradation and the accelerated degradation rate caused by these acids that self-catalyze polyester hydrolysis (Pearce and Schaefgen, 1992). The linear dimer of glycolic acid is unstable and rapidly hydrolyzes to glycolic acid, while lactoyl lactic acid can remain intact for a longer period of time. In addition, it has been observed that the release of glycolic acid is 3-4 times faster than that of lactic acid (Marcato, Paganetto, Ferrara, and Cecchin, 1996), (Giunchedi, Conti, Scalia, and Conte, 1998). As the lactide content of the polymer increases from 50% to 100% (PLGA to PLA 50:50), the corresponding decrease in monomer acidity can be observed and explained by the decreased degradation rate of the lactide-rich copolymers and homopolymers (Tamada and Langer, 1993), (Shih, Waldron, and Zentner, 1996).

[0131] In addition, PLGA was co-doped with Mg(OH) 2 MgCO 3 and ZnCO 3 Antacids such as (Zhu et al., 2000) strongly inhibit the loss of acid-sensitive protein structure and aggregation for more than one month; (Zhu and Schwendeman, 2000), (Jiang and Schwendeman, 2008), (Kang and Schwendeman, 2002).

[0132] In recent years, the monitoring of the microclimate pH distribution within PLGA microspheres over a wider range has been improved by pH mapping using confocal scanning microscopy and pH-sensitive probes (Sansdrap and Moes, 1997), (Ding and Schwendeman, 2008). In addition, a basic model for predicting the microenvironment has been established for PLGA films (Liu and Schwendeman, 2012), and it will prove to be useful for predicting other structures as well.

[0133] Further description of the embodiments

[0134] Protein quaternary structure:

[0135] The present invention is based on the inventors' pursuit of developing novel compositions for degrading oxalate in subjects, industrial processes, and / or food processes. The activity of OxDC has been evaluated for enzymes found in seven bacterial species and a number of variants of one bacterial species, Cb6301. In addition, the activity has been evaluated from a fungal species, Agrocybe spp. (A8 / A0). The activity has been tested according to the procedure outlined in Example 1. Figure 5-7 As shown, the OxDC activity in many of these homologues is stable and active at least at pH 1.5 (Cb6301, Cb6312 and Cb6803), pH 2.0 (A8 and Bcl), pH 2.5 (Bce), pH 3.0 (Bam) and pH 3.5 (Yvrk and Bpu). Cb6301, Cb6312 and Cb6803 show comprehensive protection from pepsin to pH 1.5. Since the protein quaternary structure of these enzymes is a trimer, Cb6301, Cb6312 and Cb6803 are active and stable at pH 1.5. The lower number of ionic interactions at the trimer interface and the increased number of hydrogen bonding interactions result in an increased resistance of the trimer protein quaternary structure to pH changes. These three enzymes are the first enzymes in the oxalate decarboxylase family of enzymes to be found to be naturally encapsulated in trimers. Due to the amino acid composition at the hexamer interface, all remaining enzymes are included in the hexamer, as shown in Table 4. The interactions that hold the hexamer together are primarily ionic interactions, i.e., the negative charges from glutamate and aspartate interact with the positive charges from lysine and arginine. Upon protonation of aspartate (pKa 3.65) and glutamate (pKa 4.25), under acidic conditions, the protein quaternary structure of OxDC dissociates, leading to enzyme unfolding and subsequent loss of activity (an irreversible event), see Fig.11 . YvrK and Bpu contain more glutamate than Bam and Bce, respectively, see Table 4, making the pKa of glutamate a key driver of protein quaternary structure dissociation; therefore, being the reason why Yvrk and Bpu are active and stable only at pH 3.5. Enzymes naturally enclosed in hexamer have a higher number of ionic interactions not only at the hexamer interface, but also at the trimer interface. The combined number of ionic interactions per subunit is as follows:

[0136] Cb6301:25

[0137] Bcl:29

[0138] A8:32

[0139] Bce:32

[0140] Bam: 44

[0141] YvrK:45

[0142] Bpu:47

[0143] There was a direct correlation between the number of total ionic interactions and acidic pH stability, with Cb6301 having the smallest amount but the highest stability, and Bpu having the largest amount but the lowest stability.

[0144] Since the active site is very close to the subunit interface, therefore, the enzyme that is itself a hexamer needs to have a hexameric active protein quaternary structure. The enzyme with more than 10 ionic amino acid residues (D, E, R and K) on the hexamer interface (interaction between 2 of 6 subunits) is only active under the conditions above pH 3.0, referring to Table 4. The enzyme with 5-9 ionic residues (D, E, R and K) on the hexamer interface (interaction between 2 of 6 subunits) only shows activity above pH 2.0, and the enzyme with less than 5 ionic residues (interaction between 2 of 6 residues) shows activity below pH 2.0. It is also related to the total cationic net charge at the hexamer interface, which is only due to the number of arginine and lysine residues in the interface, because all aspartic acid and glutamic acid are protonated. These results show the following inevitable trend:

[0145] 1) Enzymes with an overall net ionic charge of +8 and above have oxalate degradation activity only above pH 3.0 (charge between 2 of the 6 subunits).

[0146] 2) Enzymes with an overall net ionic charge of +4 to +7 have oxalate degrading activity above pH 2.0 (charge between 2 of the 6 subunits).

[0147] 3) Enzymes with an overall net ionic charge of less than +4 have oxalate degrading activity below pH 2.0 (charge between 2 of the 6 subunits).

[0148] At pH conditions where most, if not all, of the aspartic and glutamic acids are protonated, the hexamer interface has an overall net positive charge. The amount of the overall positive charge increases with the number of lysine and arginine amino acid residues. Since a larger overall net positive charge is produced with protonation of the acids and with decreasing pH, enzymes with a greater proportion of ionic residues at the hexamer interface are more sensitive to changes in pH than enzymes with fewer ionic residues. In fact, Figure 7It was shown that at pH values ​​where all aspartates and glutamates were protonated, the total net ionic charge correlated directly with the total net ionic charge at the most acidic pH at which the YvrK, Bam, Bpu, Bcl, Cb6301, A8 / A0, and Bce enzymes were shown to have oxalate degradation activity. 2 The values ​​show strong correlation with a large amount of data, greater than 0.95.

[0149] It should be noted that the number of amino acids or charges at an interface between two subunits, e.g. subunits A and D, cf. Figure 1 The OxDC enzyme forms dimers of trimers; therefore, one OxDC hexamer has three interfaces. Therefore, to calculate the total charge across the "entire hexamer interface", the ionic charges mentioned above should be multiplied by 3.

[0150] There is a direct correlation not only within the hexamer interface, but also within all interfaces (hexamer and trimer). For example, the enzymes with the lowest acid stability (Bam, Bce, and Bpu) have more than 44 ionic amino acids at both the hexamer and trimer interfaces. Bel, Bce, and A8 have 29-32 ionic amino acids, and Cb6301 has 25. Although the number of ionic interactions of Bcl, Bce, A8, and Cb6301 is reduced, they still have a large number of hydrogen bonding interactions. These hydrogen bonding interactions increase the stability at the interface and make the interface less susceptible to acid denaturation.

[0151] Therefore, based on the amino acid sequence of any OxDC to be found or the amino acid sequence that has been found using the following critical values, we can predict whether the enzyme is a trimer or a hexamer, as well as the acid stability of the enzyme. This information also provides sequence modification strategies that change the characteristics of the enzyme, i.e., the pH activity distribution. For example, equipped with this knowledge, a person skilled in the art can determine the position of the enzyme that changes the stability and pH distribution. The definition of the critical value is as follows:

[0152] Total charged amino acids of one subunit at the hexamer and trimer interface:

[0153] 1) > 39: The enzyme will be a hexamer and will only be active above pH 3.0

[0154] 2) Between 29-39: The enzyme will be a hexamer and will only be active at pH 2.0 and above

[0155] 3) <29: The enzyme will be a trimer and will only be active below pH 2.0 and above

[0156] Total charged amino acids of one subunit at the hexamer interface:

[0157] 1) > 10: The enzyme will be a hexamer and will only be active above pH 3.0

[0158] 2) Between 5 and 9: the enzyme will be a hexamer and will only be active at pH 2.0 and above

[0159] 3) <5: the enzyme will be a trimer and will only be active below pH 2.0 and above

[0160] Total arginine and lysine across the hexamer interface:

[0161] 1) > 22: The enzyme will be a hexamer and will only be active at pH 3.0 and above

[0162] 2) 10-21: The enzyme will be a hexamer and will only be active at pH 2.0 and above

[0163] 3) 9 or less: The enzyme will be a trimer and will only show activity below and above pH 2.0

[0164] Catalytic efficiency:

[0165] Certain embodiments of the present invention relate to oxalate degrading enzymes with high catalytic efficiency. Their high catalytic efficiency makes it possible for them to compete with the strong ionic interaction between calcium and oxalate, thereby degrading oxalate even in the presence of high concentrations of calcium ions in the surrounding environment. The catalytic efficiency is inherent to the amino acid sequence and structure of the enzyme and is generally measured in terms of k cat and K m Measurement.

[0166] The novel enzyme is disclosed to have high catalytic efficiency and stability even in the absence of a dosage form.

[0167] The catalytic efficiency ranged from 871 to 77,000 turnovers / M / s, see Table 5. The enzymes were highly stable even under acidic pH conditions, such as pH 1.5-5.0 (Cb6301, Cb6312, and Cb6803). The affinity of enzymes A8, Cb6301, Cb6312, Cb6803, and Bce for oxalate was much stronger than previously discovered / reported. m The K of these enzymes is 8.4 mM for the Yvrk enzyme. m In fact, when oxalate degradation was monitored using insoluble oxalate, Cb6301 and Bce were more efficient in degrading both soluble and insoluble oxalate compared to the Yvrk enzyme, see Figure 22-24These enzymes have such a high affinity for oxalate that they outcompete calcium and can therefore degrade the entire oxalate, not just the soluble fraction. These in vitro results were confirmed in a beagle dog study where A8, Cb6301, Bce and Yvrk were evaluated using the same number of oxalate-degrading units. The results showed that the A8 and Cb6301 enzymes were able to reduce the oxalate content in urine by 60% and 40%, respectively, see Figure 18-21 Bce reduced urine oxalate by 24%, while Yvrk did not show a significant reduction in urine oxalate ( Figure 18-21 ).

[0168] Monoprotonated oxalate (pKa = 3.81 and pKa = 1.25) binds to the unprotonated glutamate in the active site. The unprotonated glutamate in the intact active site is more likely to remain unprotonated than the equivalent residue in a disrupted active site (e.g., the active site of a disrupted hexamer). Therefore, as the pH drops from approximately 6 to 3, the proportion of monoprotonated oxalate compared to unprotonated oxalate will be maximized. This will therefore increase the binding of oxalate to the intact active site, resulting in a lower K m and higher catalytic efficiency, see Fig.15 Glutamate also needs to remain unprotonated, thus shifting the pKa to a lower value, and since it occurs in a hydrophobic environment (e.g., an undisrupted active site), the residue will remain unprotonated, thus enhancing binding. Since the structure of the YvrK enzyme is unstable below pH 3.5, the K must be determined at pH 4.0. m , where less monoprotonated oxalate is obtained, which further leads to K m However, enzymes such as Cb6301, A8 / A0, and Bce can obtain K values ​​measured under more acidic conditions (below pH 3.0). m Under these more acidic pH conditions, a larger proportion of monoprotonated oxalate is obtained, further resulting in K values ​​of less than 1 mM. m In addition, these acid-stable enzymes provide a stable structure around the active site, allowing glutamate to remain unprotonated under more acidic conditions.

[0169] stability:

[0170] Most of the enzymes tested were stable and showed OxDC activity at temperatures above 60°C, see Figure 1. This property is very helpful for predicting stability. Therefore, the present invention includes these highly catalytically efficient, pH and heat stable oxalate degrading enzymes. Therefore, the highly catalytic enzyme has a structure that can show high stability in proteases, acids and temperatures. For example, when administered orally, this stable distribution reduces the risk of loss of activity. This stable and catalytically efficient enzyme does not require a stable dosage form and can maintain high activity in harsh environments such as the human stomach. In another embodiment of the present invention, a simple formulation containing sugars such as glucose, fructose, trehalose, glucose or lactose is used to prepare the enzyme. The enzyme can then be dried using generally known methods (including spray or freeze drying methods).

[0171] Cb6301, Cb6803, and Cb6312 all have a small amount of oxalate oxidase activity, which can generate free radicals that are harmful to these particular enzymes. The generation of these free radicals leads to a loss of activity as a function of time. We found that if the isoleucine residue at position 340 ( Fig.10 In addition, we found that the introduction of vitamins such as o-phenylenediamine, hydroquinone and ascorbic acid into the enzyme solution can keep the enzyme active for a longer time.

[0172] Enzyme modification

[0173] 1.) pH activity distribution under acidic conditions : To design enzymes with acidic activity profiles, ionic amino acids in the hexamer interface can be replaced with polar or hydrophobic residues and / or the enzyme can be truncated to remove the first 10-30 amino acids at the n-terminus. In addition, the trimer interface will be designed to have approximately 10-14 (D / E) and 8-11 (R / K) amino acids with a variability of 3+ / -. Based on the crystal structure, these ionic amino acids will be positioned and designed to interact with each other and with polar amino acids that form hydrogen bonds. This will make aspartic acid and glutamic acid less susceptible to acidic conditions. Embodiments include enzymes modified to cover this standard.

[0174] 2.) Sustained pH activity as a function of time : An important structural feature of Cb6301, Cb6312 and Cb6803 is that amino acid 340 is hydrophobic. Due to the formation of free radicals, an enzyme that loses activity as a function of time appears. If amino acid 340 is mutated to glutamic acid, the enzyme remains fully active as a function of time; therefore, the enzyme is stable and not susceptible to inhibition by free radicals. Therefore, this residue will mutate to glutamic acid to maintain continuous activity. Therefore, embodiments relate to Cb6301, Cb6312 and Cb63803, wherein residue 340 is replaced by glutamic acid.

[0175] 3.) Broad pH activity profile :In order to design an enzyme with acidic activity characteristics, the ionic amino acids in the hexamer interface can be composed of about 4-5 (D / E) and 4-5 (R / K) amino acids + / - 2 amino acids. According to the crystal structure, these ionic amino acids will be positioned and designed to interact with each other and with the polar amino acids that form hydrogen bonds. This will make aspartic acid and glutamate less susceptible to acidic conditions. According to certain embodiments, the enzyme is designed to have the amino acids at the hexamer interface. In addition, the trimer interface will be designed to have about 16 (D / E) and 7 (R / K) amino acids (+ / - 5 amino acids). Similarly, according to the crystal structure, these ionic amino acids will be positioned and designed to interact with each other and with the polar amino acids that form hydrogen bonds. This will make aspartic acid and glutamate less susceptible to acidic conditions. Certain embodiments relate to enzymes modified to include the amino acid residue content at the trimer interface.

[0176] 4.) Low K m / High catalytic efficiency : In order to obtain a low K m / Enzymes with high catalytic efficiency would employ the same strategy as described in point 3 above (“broad pH activity profile”).

[0177] Recombinant expression:

[0178] The enzymes described herein can be recombinantly expressed using any sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the sequences of SEQ ID Nos: 1-47, a variety of expression systems and host cells, many of which are commercially available and well known to those skilled in the art, or can be custom formulated. The original sequence can be altered to improve expression, such as codon optimization, or to include sequences that facilitate downstream processing, such as the inclusion of secretion sequences. In addition, changes in gene sequences can be envisioned by those skilled in the art. The host strain will be transformed with a suitable vector, wherein the coding will encode a promoter for gene expression of the enzyme. The expressed gene sequence may also include coding for sequences useful downstream, such as affinity tags used in affinity purification, etc.

[0179] The recombinant enzyme can be expressed in a variety of hosts known to those skilled in the art of protein expression, including but not limited to: Escherichia coli, Lactobacillus, Bacillus, Aspergillus, etc.

[0180] For the recombinant production of enzymes, the host should include a construct in the form of a plasmid, vector, phagemid or transcription or expression component containing an enzyme or protein or its functional fragment. A variety of constructs are available, including structures maintained with single or multiple replications. Many recombinant expression systems, components and reagents for recombinant expression can be purchased on the market, such as from Invitrogen (Carlsbad, California, USA); USBiological (Swampscott, Massachusetts, USA); BD Biosciences sPharmingen (San Diego, California, USA); Novagen (Madison, Wisconsin, USA); Stratagene (La Jolla, California, USA); and Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) (Braunschweigh, Germany).

[0181] Heterologous promoters, including constitutive and / or inducible promoters, can control the recombinant expression of the protein at will. T7 or other promoters are suitable for the host and are well known to those skilled in the art.

[0182] The recombinant nucleic acid sequence of the enzyme or protein may include nucleic acids for purposes other than expressing the protein, including but not limited to purification purposes, folding purposes, etc. These examples include: secretion sequences, signal sequences, linkers, expression control elements, affinity tags, etc. The amino acids produced by these nucleic acid sequences may or may not be removed after protein expression. All of the constructs mentioned above can be used to express the enzymes and proteins to be used in the methods described herein.

[0183] As described above, the host cells are transformed / transfected with the selected expression system. The cells will be cultured using methods known to those skilled in the art, including liquid cultures in shake flasks, bioreactors and fermenters, solid cultures in plates, and the like.

[0184] Prior to use of the methods described herein, proteins can be purified from natural or recombinant sources. Purification can include extraction from host cells by sonication, French press, glass beads or other physical dissolution or chemical cell lysis, and separation by precipitation, centrifugation or chromatography steps or other means known to those skilled in the art. Optionally, a concentration step can be used, such as by dialysis, diafiltration, tangential flow filtration (TFF), chromatofocusing chromatography and / or steps associated with buffer exchange.

[0185] Immobilization:

[0186] Thermostable OxDC enzymes with broad pH activity profiles, K m Less than 1 mM and stable over a wide pH range, it is an ideal candidate for immobilization. Therefore, the A8 enzyme is an ideal candidate because it has a thermal dissolution temperature of approximately 77 degrees Celsius, is active at pH 2.0-6.0 and is stable at pH 2.0-11.0. Immobilization can achieve: (1) high enzyme loading with high activity; (2) controlled scale-up of the reaction; (3) easy recovery and reuse; (4) a biocatalyst-free product; (5) continuous operation (or batch operation on a drain and replenishment basis) and process automation are possible; and (6) inhibition of the substrate can be minimized. In addition, if the immobilization is designed properly, it can also prevent autolysis or denaturation caused by organic solvents, and can bring thermal stability, handling and storage stability. Immobilization can prove to be crucial in terms of economic feasibility. Enhanced stability that allows continuous reuse can achieve high unit productivity, thereby affecting the production costs associated with biocatalysts. The improved thermal stability allows conventional reactor operation as described above, thereby reducing the risk of microbial growth and reducing the need for sanitary conditions, as the reactor does not require high cleaning requirements.

[0187] Oxalate Degradation in Food:

[0188] To evaluate the effectiveness of the OxDC enzyme from Agrocybe oleraceus (A0) in degrading oxalate in human food, several conventional Western meals (pre-made "Lean Cuisine" meals) were cooked in a microwave oven, homogenized, and used as substrates in a screening of oxalate degradation activity of the A0 enzyme according to the instructions on the package. Table 3 lists the meals evaluated and the approximate calcium concentrations in the final reaction mixture. These experiments were performed to demonstrate the effectiveness of oral administration of the OxDC enzyme to remove oxalate from a diet being digested in the human stomach.

[0189] like Figure 4 As shown, OxDC from A0 can degrade more oxalate at acidic pH than at alkaline pH and in diets with lower calcium content. 2+ ) diet, approximately 90% of oxalate was reduced from pH 2 to 5 within 60 minutes. 2+ ) diet, greater than 70% of oxalate was degraded within 60 min at pH between 2 and 4. 2+ ) diet, A0 OxDC enzymes can degrade 60-80% of total oxalate within 60 minutes at pH 2 to 3, and 50% at pH 4.2+ ), at pH 2 and 3, the enzyme degraded 40-60% of the total oxalate within 60 minutes. The reduction in degradation percentage can be attributed to the reduced solubility of oxalate in moderate to high calcium content diets. m =8.4 mM), A0 for oxalate (K m =0.08 mM) has a high affinity, making A0 more capable of degrading the low oxalate content in the human stomach. In order for the OxDC enzyme to effectively degrade oxalate in the human stomach, the enzyme needs to be compatible with the fed human stomach (pH 1.0-4.5) and less than 1.0 mM K m Therefore, Cb6301, Cb6312, and Cb6803 are ideal candidates as well as the A0 / A8 and Bce enzymes for oral oxalate lowering.

[0190] It is well known that oxalates can cause problems in human health, such as acidifying foods, making dietary calcium unavailable, causing chemical burns, dental damage, and causing urinary tract stones and kidney stones. Therefore, it is beneficial to provide foods that are low in oxalate or free of oxalate, as this would allow people who are prone to oxalate-related conditions and symptoms to avoid consuming oxalate. This would lead to individuals enhancing their health values ​​for foods and beverages. In the food processing industry, enzymes are widely used in many different production stages; therefore, it is feasible to include OxDC enzymes. In fact, several foods were evaluated to demonstrate the effectiveness of OxDC enzymes in degrading oxalate in individual foods. These foods were ready-to-drink tea, beer, and juice. However, it is conceivable to use OxDC enzymes in food processing for a wide range of food types, such as: canned foods (vegetables, fruits, and soups), chocolate, flour, and spice processing.

[0191] Addition of the Cb6301 OxDC enzyme to these beverages resulted in complete removal of oxalate from most of the beverages. The range of oxalate reduction was between 75-100%. These experiments were performed to demonstrate the usefulness of using the OxDC enzyme in food processing. Furthermore, the results showing the above removal of oxalate from dietary contents demonstrate that the enzyme is effective not only in beverage manufacturing, but also in more complex food manufacturing processes and matrices, such as canned foods, soups, flour, chocolate, spice processing, etc.

[0192] As in Example 9 and Figure 10-11 As shown, no molecules were tested that completely inhibited the enzyme activity. These molecules were selected for their potential to inhibit OxDC activity. This suggests that the OxDC enzyme can be used in a wide range of foods and effectively removes oxalate.

[0193] formula:

[0194] Due to the incorporation of pH active compounds, therefore, the novel formulation of the present invention produces a microenvironment pH value that directly surrounds the enzyme and changes relative to the surrounding pH value. This change makes the pH value of the microenvironment different from the suboptimal environment, and is optimal relative to each enzyme. Therefore, the pH value under the microenvironment falls within the optimal pH range of the corresponding enzyme. For example, under the conditions of pH being about 1.5-4.5, a highly active free acid stable enzyme will have low activity when pH 6 (if any). However, by creating a pH of about 1.5-4.5 microenvironment around the enzyme, even if the pH of the turnover is 6, high activity can still be maintained. Therefore, the formulated enzyme is made more active under different pH values ​​of a wider range, is less restricted by pH activity distribution, and is less sensitive to surrounding pH values.

[0195] The high activity maintained by the formulated enzyme at suboptimal pH can be measured and monitored by the formation of enzyme catalytic reaction products. The effective pH activity profile of the formulated enzyme is defined herein as the range of ambient (surrounding) pH where the enzyme formulated according to the invention maintains ≥20% activity relative to the optimal pH conditions.

[0196] The inventors surprisingly discovered that the dosage forms described herein maintain the activity of the exemplary enzyme, Bacillus oxalate decarboxylase (OxDC), at ambient pH conditions, whereas the unformulated exemplary enzyme was never observed to be active at around and above pH 7.0.

[0197] As an example of a specific application of the present invention, the present invention describes the formulation of YvrK, Bce or A8; however, these examples should not limit the scope of the present invention. It is clear to those skilled in the art that the present invention can be applied to any enzyme, which is actually applied to the site of action representing the suboptimal pH value of the unformulated enzyme, which is more acidic or alkaline relative to the optimal pH value of the respective unformulated enzyme.

[0198] According to some specific embodiments, a microenvironment pH value different from the surrounding pH value is provided. In one embodiment, compared with the surrounding pH value, the pH value is reduced. Any compound that has an acidifying effect on its microenvironment and does not reduce enzyme activity can be used for this purpose. In addition, according to the present invention, it should be recognized that under the conditions of using a compound with this effect, by raising pH from a lower surrounding pH value to adjust the microenvironment pH value, enzyme activity will be maintained under pH conditions higher than the surrounding pH value. In order to further describe the present invention, the example of a neutral or alkaline surrounding environment will be used, and an acidic microenvironment pH value will be used.

[0199] Acidic substances that exhibit acidification in the preparation can be introduced into the preparation by many means (including but not limited to: adding, generating, degrading, reacting and / or as impurities). Therefore, acidic substances can be added to the preparation in the form of separate compounds. Acidic substances can also be the result of chemical reactions or degradation of any compound that is a part of the preparation. In addition, acidic substances can be regarded as the result of the method for preparing a separate compound that is a part of the preparation, thereby, being regarded as an impurity of the original material. All situations that lead to acidification in the final preparation are considered to be part of the present invention. The resulting degradation products or conversion or reaction products can have the structure of monomers or polymers, and this structure has the common feature of acidifying the surrounding environment of the prepared enzyme. The acidic substances of the preparation are released, contained, concentrated, developed, generated, dissolved and / or suspended in the microenvironment, and local pH conditions are produced thereby, under which, when the preparation is placed in an environment where the suboptimal pH value of the enzyme substrate is available, it is beneficial to the activity of the enzyme.

[0200] The acidification of the formulation compound may be instantaneous or develop over time. For example, if the acidifying compound is an impurity from the formulation raw material, the acidification may be instantaneous at the time of formulation; however, if the acidification is caused by a degradation product, the effect may develop over time. The length of time depends on many factors, including but not limited to the type of raw materials, the environment and formulation additives, and the time span may range from instantaneous acidification to significant acidification after a few weeks to months or years.

[0201] Acidifying compounds and their effects are well known to those skilled in the art, but examples of these compounds are given herein without limiting the scope of the invention. Acidifying compounds having an acidifying effect on the microenvironment include, but are not limited to, organic acids, inorganic acids, acidic side chains, and acidic functional groups. Examples of small organic acids that can be used in formulations include, but are not limited to, L-tartaric acid, citric acid, fumaric acid, toluenesulfonic acid, maleic acid, adipic acid, DL-malic acid, succinic acid, L-aspartic acid, and glutamic acid. Examples of acidic side chains and functional groups include, but are not limited to, carboxyl, phenolic, ammonium ions, and the like.

[0202] There are many types of polymers that can generate acidic degradation products. Such polymers and degradation products can be included in the embodiments of enzyme preparations to adjust the pH of the microenvironment. These polymers are well known to those skilled in the art, and the present invention provides examples without limiting the scope of the invention. Examples of polymers that generate acidic degradation products include, but are not limited to, polyesters and polyanhydrides such as poly(glycolic acid) (PGA), poly(dextrorotatory lactic acid) (PLA), poly(lactic acid), poly(lactic acid), poly(lactic acid-co-hydroxyethyl) (PLGA), poly(orthoesters), polycaprolactone. Another example is poly(ethylene sulfonic acid), poly(acrylic acid) and poly(propylene fumarate).

[0203] Several different types of poly(orthoesters) have been developed. Each design is inherently different and has specific properties, for example, type I poly(orthoesters) form suitable alkanediols and gamma-butyrolactone. The lactone is easily hydrolyzed to form gamma-hydroxybutyric acid. The acid affects the pH of the microenvironment and accelerates further degradation of the polymer.

[0204] Biodegradable polymer polyanhydrides degrade by hydrolysis of their anhydride chains. Examples of polyanhydrides include, but are not limited to, those composed of adipic acid, fumaric acid, pimelic acid, suberic acid, azelaic acid, dodecanedioic acid, dodecanedicarboxylic acid, isophthalic acid, terephthalic acid, p-carboxyphenoxyacetic acid, 5-(p-carboxyphenoxy)pentanoic acid, 8-(p-carboxyphenoxy)octanoic acid, suberic acid, ricinoleic maleic acid, ricinoleic acid succinate, 12-hydroxystearic acid succinate, caprylic acid, lauric acid, myristic acid, stearic acid, oleic acid, fatty acid esterified ricinoleic acid and / or methacrylated sebacic acid.

[0205] Many copolymers of the polymers and / or poly(ethylene glycol) or imides can also be used in the present invention. In many cases, copolymers can provide properties that are conducive to microenvironment formation. The following paragraphs further illustrate these features. The copolymers referred to are made from lactic acid and glycolic acid. The two main series are the (I) LA / GA and (dL) LA / GA series. As further described below, the compositions may be different.

[0206] The properties and degradation rate of the polymer can be adjusted by selecting the polymer, the block copolymer or copolymer, the ratio of monomers and monomer classes to each other, the hydrophilicity and hydrophobicity of the monomers, the molecular weight of the monomers, the end groups of the polymers, the ratio of monomer classes and in some cases the crystallization rate of the polymer (Brunner, Mader, and Gopferich, 1999), (S. Li, 1999), (Gopferich and Tessmar, 2002). Thus, a large number of different characteristics can be obtained from the same molecular building block or monomer, and the scope of the present invention should not be limited to any particular combination or ratio of different monomers.

[0207] In addition, the change of microenvironment pH value can also be achieved by impurities in the above-mentioned polymers. These impurities can be degradation products of the above-mentioned polymers, or originate from the production of the polymers. The characteristics of the impurities depend on the original materials and their manufacture or preparation methods, but for example, these impurities can include but are not limited to acidic oligomers such as lactic acid, gluconic acid, lactyl lactic acid and oligomers of lactic acid.

[0208] In addition to the one or more enzymes, the one or more polymeric materials and the one or more acidifying compounds, the particles may also contain one or more additives, such as buffers, solubilizers, stabilizers, preservatives, vitamins or cofactors for the enzymes or one or more pharmaceutically acceptable excipients, such as fillers, bulking agents, diluents, carriers, etc. The additives may be any molecule that protects the enzyme from high temperatures, dehydration and storage, such as carbohydrates, amino acids, surfactants, salts, etc. The additives may also be any molecule that indirectly affects acidification by affecting the degradation rate of the polymer and the rate at which an acidic environment is generated. For example, the rate of hydration and the rate of external water exchange both affect the degradation rate of certain polymers; therefore, the incorporation of hydration additives may change the degradation rate and thereby the acidic environment over time. Polymer degradation and subsequent changes in microenvironmental pH are also affected by the initial microenvironmental pH, because autocatalytic chain scission is accelerated under acidic pH conditions (Witschi and Doelker, 1998); therefore, the degradation rate may be adjusted by the incorporation of free organic acids such as fumaric acid and succinic acid. For example, the retention of these acids can be adjusted by taking into account their solubility. These additives that are able to accelerate the degradation process are called degradation accelerators and can control the degradation of the formulation.

[0209] In addition, the enzyme incorporated into the polymer itself can also have an effect on the degradation rate of the polymer. This has been confirmed by the results of incorporating 2% bovine serum albumin and accelerating the degradation rate of the formulation components.

[0210] Acidifying compounds, polymers and additives can be encapsulated with the enzyme and coated by a polymer network. The polymer network can be made of the same polymer that degrades into the acidifying compound, or made of a separate polymer. Such polymers include, but are not limited to, artificial or natural polymers, including, but not limited to: i) polysaccharides: alginates, including alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propane-1,2-diol alginate, gum arabic, carrageenan, chitosan and its derivatives, chondroitin sulfate, dextran derivatives, heparin, hyaluronic acid, pectin, inulin, cellulose or cellulose derivatives, including methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl methylcellulose, etc. or a combination thereof; ii) mucopolysaccharides; iii) including locust bean gum, guar gum, tragacanth gum, agar, gum arabic, xanthan gum, karaya gum, tara gum, iv) gelling agents or swelling agents, including hydrocolloids and hydrogels, such as agar, carrageenan, gelatin, polyvinyl pyrrolidone, etc. or their combinations; v) others such as proteins and polyamides: collagen, albumin, protamine, spermine and synthetic polymers, including: polyacrylic acid, polyphosphoric acid, tripolyphosphoric acid, poly-L-lactic acid, poly-lactic acid, poly-d-lactic acid, poly(hydroxyacetic acid), poly(vinyl alcohol), poly(lactic acid-co-hydroxyacetic acid), poly(orthoester), polycaprolactone, propylene fumarate, polyanhydride, etc. or their combinations; and acrylic resin compounds, including but not limited to L-100, L-100-55, RS, RL or copolymers or mixtures and combinations of the above.

[0211] Other polymer materials that can be added to the preparation or used to encapsulate the formulated enzyme and concomitants can be biopolymers or synthetic polymers. Examples of biopolymers include, but are not limited to, proteins, polysaccharides, mucopolysaccharides, heparin, heparan sulfate, heparanoid derivatives, dermatan sulfate, pentosan polysulfate, chondroitin sulfate, cellulose, agarose, chitin, carrageenan, linoleic acid and allantoin, cross-linked collagen, fibronectin, laminin, elastin, cross-linked elastin, collagen, gelatin, hyaluronic acid, alginate chitosan, dextran, methylcellulose, polylysine and natural rubber.

[0212] In the formulations forming polymer matrices of the present invention, these matrices are designed so that small water-soluble molecules can enter and leave the polymer matrix, including but not limited to oxalate, oxalic acid, formate, formic acid, carbon dioxide, oxygen and enzyme cofactors, etc. These matrices can have many shapes, including but not limited to granules, sheets, blocks or films.

[0213] In addition, the polymer matrix of the present invention does not release the enzyme into the environment substantially. In other words, the enzyme is kept in the optimal microenvironment for a period of time to degrade a sufficient amount of substrate in the environment, and the level of the substrate is also reduced accordingly.

[0214] Within the polymer matrix, the polymer material may serve as both a generator of acidifying species and a protective and retaining carrier for the enzyme, while allowing the matrix to diffuse or otherwise be transported into the composition for in situ degradation. All functions need not be attributed to the above polymers, but may be a common feature of particles containing different polymers.

[0215] In one embodiment, the present invention uses biodegradable components whose degradation products do not cause irritation or damage to biological tissues or fluids; thus, ensuring safe application in biological systems such as humans or animals. In addition, the present invention contemplates a design of a formulation that ensures high compatibility with the target application or delivery site, thereby enhancing the beneficial effects of the formulated pH-insensitive enzyme. For example, particles intended for delivery in the gastrointestinal tract may also have mucoadhesive properties. Preferably, they have mucoadhesive properties but are not absorbed. Such final particles will be micron-sized and therefore unlikely to be absorbed. Mucoadhesive properties will be obtained by coating with cationic charged polymers or copolymers with additional amino groups (Bivas-Benita, Romeijn, Junginger, and Borchard, 2004), the latter of which also show abnormally short degradation times, once again indicating the opportunity to modify degradation properties through creative polymer synthesis, thereby reducing the acidification rate of the microenvironment. Microparticles or nanoparticles for intravenous delivery can be designed in the same way to reduce any immune response. Such designs are well known to those skilled in the art and may involve PEGylation of the particles.

[0216] Particle formation:

[0217] Other methods of the present invention relate to methods for preparing respective enzymes by producing micron particles or nanoparticles. For the continuous and pH-insensitive activity of the prepared enzyme, particle formation (combining the use of a specific method for preparing particles and specific polymers or copolymers used) is considered to protect the enzyme and produce a local microenvironment suitable for pH. The present invention contemplates the particle formation of the above-mentioned enzyme (one or more), polymeric materials, acidifying substances and other additives. As used herein, particle formation refers to the combination of enzymes with polymer or copolymer solutions and other materials to ensure local suitable pH and stabilize protein as required to form small particles comprising active enzymes, polymers or copolymers, acidulants, stabilizers, vitamins and other above-mentioned additives. This method for forming active enzyme particles increases the amount of active enzymes in the particles, and can improve the efficacy of the granular dosage form containing the particles when used for disease treatment or prevention programs. Particle formation can also help protect the enzyme from digestion by proteases.

[0218] There are many methods for particle formation, such as coacervation, phase separation, polymerization, spray drying, electrostatic methods, and air suspension methods. Spray drying is a mechanical microencapsulation method developed in the 1930s and is one of the suitable methods for preparing active enzyme particles in the embodiments of the present invention. In this method, the enzyme, polymer, acidifying substance and additives are dispersed or dissolved in an aqueous medium, a solvent medium or an emulsion and dispersed or dissolved through a nozzle installed in a suitable spray drying device. It may also be related to other methods, but the premise is that the activity of the enzyme is not severely reduced (at least 20% activity is maintained relative to the maximum activity).

[0219] According to certain embodiments, the preparation comprises the composition that enzyme, acidifying compound and additive are combined in polymeric material. Those skilled in the art can find other methods suitable for preparing and being used to prepare the composition of the present invention. By incorporating enzyme into polymeric material, enzyme is thereby protected to a certain extent and isolated from the local pH environment mainly affected by the above-mentioned acidifying compound. The composition of the prepared enzyme generated appears in discrete units of micrometer or nanometer size. Without limiting the scope of the present invention, discrete units of micrometer or nanometer size will be simply referred to as "particles". However, many different shapes, forms, designs and structures may be apparent to provide a suitable microenvironment for pH-sensitive enzymes, and are therefore considered herein.

[0220] Granules can be formed by known methods, preferably by spray drying. After forming granules comprising one or more enzymes, one or more polymeric materials and one or more acidulants or acidifying polymers and one or more additives, the granules can be further processed, for example by drying, freeze drying or lyophilization. Although freeze drying does not form granules, it can dry the granules comprising enzymes and polymeric materials that have been formed. Such granules can be in a suspended, dispersed or emulsified state, and then under freeze drying conditions. Freeze drying avoids heating enzymes and makes the drying process suitable for heat-sensitive proteins. Freeze drying or other methods (such as coating) can be omitted, and only spray drying can be used to form the granules. Then, such granules can be formulated into oral drugs or food preparations in the following manner, for example, by mixing with fillers and filling granules into pouches, adding to capsules, pressing into tablets, adding to chewable tablets, adding to fast-dissolving or oral dissolving tablets, or adding to liquids, syrups, elixirs or foods.

[0221] The morphology and size of the particles will have a significant impact on hydration, acid retention, substrate migration into the particles, and mucoadhesive properties; therefore, the morphology resulting from spray drying parameters such as spray airflow, feed rate, solvent, and concentration will have a significant impact on the final pH activity profile of the formulated enzyme. It is well known that other parameters such as spray drying feed viscosity, density, surface tension, and atomization conditions can all affect droplet size and therefore final particle size. Therefore, a complex combination of spray drying process parameters and feed characteristics will have an impact on the final pH activity profile of the formulated enzyme, and it should be considered obvious to change the manufacturing process to change the pH activity profile of the formulated enzyme.

[0222] Some of the methods described above may pose a risk of loss of enzyme activity due to exposure to hazardous reagents, solvents, temperature, equipment, etc. The effects of the hazardous conditions may be reduced by the introduction of protein stabilizing compounds known to those skilled in the art.

[0223] In some cases, a polymeric material can be applied to the particles (e.g., as a coating) to increase the storage stability of the particles or inhibit the degradation of the enzyme. Suitable coating materials are materials that allow aqueous compositions containing substrates and / or reaction products to diffuse into the particles of the present invention or otherwise enter and flow out of the particles of the present invention. As described above, the substrate enters the granular composition of the present invention, thereby causing enzyme degradation. Therefore, coating materials that cause diffusion coatings or other permeable coatings (e.g., coatings containing substantially water-soluble pore-forming substances) can be applied. Examples of suitable coating materials include, but are not limited to, materials considered as polymeric materials. Coating materials different from those used as polymeric materials can be selected, but the polymeric materials and coating materials can also be the same. The specific example of coating is a film former, for example, polyvinyl pyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, hydroxypropyl cellulose, polydextrose, maltodextrin or other polysaccharides including chitosan, alginate and hyaluronic acid.

[0224] In addition to the oxalate reductase, polymer, acidifying compound and additive, the above particles may also contain other particles. These internalized particles may contain other enzymes, polymers, acidifying substances and / or additives. Therefore, the present invention contemplates several layers of entities described herein.

[0225] Embodiments may involve using the final uncoated or coated particles in a drug or other composition that transmits the enzyme in an active form to a specific environment. These environments can be biological, environmental, industrial and / or chemical environments. In particular, as an example, the method can be used to spray dry or otherwise prepare OxDC particles from Bacillus subtilis, Bacillus cereus or Camellia sinensis, and these particles can be used to degrade oxalate in the stomach, intestines or vascular system of humans or animals. Therefore, the present invention also provides methods for treating and preventing oxalate-related diseases by administering formulated proteins or pharmaceutical compositions comprising them.

[0226] Composition:

[0227] According to certain embodiments, compositions comprising particles as described above are disclosed. These particles comprise one or more catalytically efficient oxalate reductases, one or more polymeric materials, acidifying substances and / or additives. The composition may also contain other particles including other enzymes, polymers, acidifying substances and / or additives. The composition of the present invention may also comprise one or more additional factors that can improve enzyme activity. These additional factors may be oxalyl CoA, MgCl 2 , and / or thiamine diphosphate (vitamin B 1 activated forms of), other vitamins, or pH buffering compounds.

[0228] The composite embodiments may contain one type of particles as described above or different types and amounts of particles. The particles of the composite may be provided individually or together in an oral or intravenous dosage form.

[0229] According to a specific embodiment, a pH-insensitive active enzyme with high catalytic efficiency is provided in the composition and administered at an effective dose. An effective dose is an amount that significantly reduces the oxalate content to present a beneficial clinical result. Compared with the amount of current oxalate before the composition is applied, an effective dose includes an amount of an enzyme activity unit of oxalate reductase activity that reduces a portion of the current oxalate or begins to reduce the amount of oxalate or maintains the level of an enzyme activity unit of oxalate reductase activity of the amount of oxalate that decreases the individual. The number of activity units of oxalate reductase activity that can be used in a single dose composition is generally in the range of about 0.001 units to about 20,000 units, and all ranges are included therein. The unit of enzyme is defined as the amount of enzyme that degrades 1 micromole of oxalate per minute at 37°C.

[0230] In order to deliver the particles as described above to humans or animals, the particles can be formulated into a suitable dosage form for administration. The dosage form depends on the route of administration. For the OxDC enzyme, the suitable route of administration is oral or intravenous administration, depending on the targeted disease condition, and both types of compositions will be described herein.

[0231] The composition is provided as an oral drug, nutraceutical, special dietary food or medical food preparation, which can be delivered to the oral cavity, mouth, oral patch, stomach or attached to the gastric mucosa in a sachet, capsule, tablet, chewable tablet, fast dissolving tablet, orally dissolving tablet, powder, granule, pill, liquid, syrup, elixir, sustained release solution, fast release tablet or other oral dosage forms known to those skilled in the art of medicine and food. The composition is delivered with food before or after ingestion of food.

[0232] Oral formulations may optionally include buffering capabilities. For example, the composition may include a buffering compound that adjusts the pH of the composition once it is ingested, and thereby adjusts the surrounding environment such as the stomach. Such buffering compounds may be acetates, citrates, phosphates, or other buffering compounds.

[0233] The composition to be administered is usually in a solid form such as a powder or a solid dosage form such as a sachet, capsule or tablet (for example, the particles are further processed into a suitable dosage form by methods well known to those skilled in the art). For this purpose, suitable pharmaceutically acceptable excipients such as fillers, binders, disintegrants, colorants, flavoring agents, pH regulators, stabilizers, etc. may be added. In addition, one or more therapeutic and / or preventive substances, and / or other enzymes, cofactors, vitamins, substrates, coenzymes, minerals and other agents that help reduce oxalate may also be added.

[0234] Examples of suitable pharmaceutically acceptable excipients include: dextrin, maltodextrin, dextrose, fructose, glucose, lactose, including carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), microcrystalline cellulose (e.g., various grades of ), cellulose derivatives including starch or modified starch (such as potato starch, corn starch, rice starch, pregelatinized starch), polyvinyl acetate, polyvinyl pyrrolidone, agar, sodium alginate, cross-linked sodium carboxymethyl cellulose, calcium hydrogen phosphate, calcium phosphate (such as basic calcium phosphate, calcium hydrogen phosphate), calcium sulfate, carboxyalkyl cellulose, dextrates, calcium hydrogen phosphate, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, polyethylene glycol, polyethylene oxide, and lubricants such as talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oil.

[0235] The composition comprising particles of other enzymes, polymers, cofactors, vitamins, coenzymes, acidifying substances or additives can be administered simultaneously, sequentially, before or after the composition comprising particles of oxalate reductase. The composition comprising particles containing other enzymes, cofactors, coenzymes, acidifying substances or additives can be combined with the composition comprising particles containing oxalate reductase to form a single dosage for administration to provide an effective amount of oxalate at the site of action.

[0236] Oral compositions as described above, under conditions found after consumption of food, or in the presence of proteases, reduce the amount of soluble oxalate throughout the gastrointestinal tract. Specific compositions of the invention are designed to reduce oxalate in the gastrointestinal tract of humans and other animals. The compositions reduce oxalate in the gastrointestinal tract, particularly in the intestine, and prevent exogenous oxalate (e.g., from food) from entering the systemic circulation, and create a suitable transepithelial gradient to promote oxalate secretion from the blood into the intestine, thereby reducing oxalate in the gastrointestinal tract as well as throughout the body.

[0237] According to a specific embodiment, a synthetic compound suitable for reducing oxalate levels in humans or animals is provided. They can also be applicable to treating or preventing conditions associated with oxalate, including but not limited to hyperoxaluria, absorptive hyperoxaluria, enteric hyperoxalate hyperoxalate, primary hyperoxaluria, idiopathic calcium oxalate nephrolithiasis (urolithiasis), vulvodynia, oxalic acid disease associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and patients undergoing gastrointestinal surgery and bariatric surgery (obesity surgery), and / or patients who have received antibiotic treatment. Embodiments of the present invention relate to treating and preventing oxalate-related conditions in humans and animals by administering a therapeutically effective amount or a preventive effective amount of the synthetic compound taught herein, respectively. A therapeutically effective amount is the amount of oxalate reduced in a subject diagnosed with an oxalate-related condition. A preventive effective amount is the amount provided to a subject who is in danger, has preliminary symptoms, or has previously suffered from an oxalate-related condition.

[0238] Embodiments of the oxalate degrading particles or compositions of the invention can be administered in a desired amount, e.g., an amount sufficient to effectively reduce oxalate levels in human tissues or fluids to an amount that has been shown to have a beneficial clinical effect. A reduction in oxalate absorption can be indicated by a reduction in oxalate levels found in blood, serum, plasma, or urine, or other body fluids, tissues, and organs.

[0239] Use of granules and synthetics - Treatment methods:

[0240] According to further embodiments, methods are disclosed involving providing a particulate composition to the intestinal tract of a human or animal, for example, providing a composition capable of reducing oxalate in the stomach and intestines, thereby reducing oxalate absorption from the gastrointestinal tract, and creating a suitable transepithelial gradient that favors oxalate secretion from the blood to the intestines. The particulate formulation and composition can further protect oxalate reductase from the environment in the stomach that is damaging to the enzyme.

[0241] In other embodiments, methods are provided involving adding one or more OxDC enzymes to food and beverages during food processing, thereby reducing oxalate in urine by reducing or removing food-derived oxalate. Thus, in a specific embodiment, a method is provided involving contacting a food or beverage with an oxalate reductase as taught herein under conditions and in an amount sufficient to reduce the oxalate present in the food or beverage.

[0242] The particles and compositions of the present invention are suitable for use in methods of reducing the absorption of oxalate in the body and reducing the level of endogenous oxalate in the body, and are used to treat or prevent oxalate-related conditions, including but not limited to hyperoxaluria, absorptive hyperoxaluria, enteric hyperoxalate, primary hyperoxaluria, idiopathic calcium oxalate kidney stone disease (urolithiasis), vulvodynia, oxalic acid diseases associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and patients undergoing gastrointestinal surgery and bariatric surgery (obesity surgery), and / or patients who have received antibiotic treatment.

[0243] According to certain embodiments, a method is provided involving administration of a composition capable of reducing oxalate in food, stomach and / or intestines to avoid absorption of oxalate by the human or animal body by reducing oxalate in food sources. The method of providing active oxalate reductase to the intestine is to provide oxalate reductase in a polymer material in the form of an oral drug, wherein the polymer material is capable of maintaining the enzyme with a suitable microenvironmental pH value.

[0244] Certain methods of the invention include administering a composition of an embodiment capable of degrading oxalate by one or more oxalate degrading enzymes at a pH commonly found in biological tissues, organs, and fluids. Certain method embodiments involve administering a composition capable of reducing oxalate in blood to reduce oxalate levels in the fluid and oxalate levels in plasma, serum, and urine derived from the fluid.

[0245] Reduction in oxalate absorption can be achieved by providing oxalate degrading enzymes to the gastrointestinal tract or bloodstream, thereby reducing the concentration of absorbed dietary oxalate as well as endogenously produced oxalate. In addition to absorption pathways, oxalate secretion pathways have been identified in the human gastrointestinal tract. The synthetic embodiments are also useful in degrading oxalate secreted into the intestine from the circulatory system, thereby achieving an overall reduction in the oxalate load in an individual.

[0246] Reduction in oxalate absorption can be achieved by providing oxalate-degrading enzymes during food processing and thereby reducing the concentration of available dietary oxalate for absorption.

[0247] The method of reducing oxalate in humans or animals may include administering an effective amount of a composition to a subject, a human or an animal, thereby reducing the oxalate present locally or systemically, and the composition comprises one or more oxalate reductases or fragments having oxalate reducing activity in the particle composition of the present invention. The reduction can be measured in any tissue or body fluid environment of the subject. Body fluids include body secretions, such as nasal or gastric secretions, saliva, blood, serum, urine, chyme or digestive matter, tissue fluid, and other fluids or semisolid materials produced by humans or animals. For example, the oxalate reductase particle composition can be administered to humans or animals in oral form, and the activity of the oxalate reductase reduces the oxalate present in the intestinal tract of humans or animals. When maintained under the local microenvironment pH conditions of the present invention, the particle composition of the present invention can be mixed in a liquid, food or other dietary material, and provided to humans or animals, so that the oxalate reductase activity of the particles is effective in the intestinal environment. The particle composition of the present invention can also be mixed with food or other materials in which oxalate is found, and the oxalate reductase activity of the particles reduces the oxalate present in the food or other materials.

[0248] Other methods for reducing oxalate absorption in humans or animals and treating and preventing oxalate-related conditions involve administering a composition comprising particles containing an effective amount of active oxalate reductase. The effective amount includes an amount of oxalate reductase activity units that reduce a portion of the current oxalate level or a level of oxalate reductase activity units that begin to reduce the amount of oxalate present in the diet or in the subject's tissues or fluids or maintain a reduced amount of oxalate in the subject, compared to the current amount of oxalate prior to administration of the composition.

[0249] In the treatment method, an effective amount of the granule composition taught herein is administered orally or intravenously to the subject at least once a day, and if necessary, the administration can be one or several days, or a week, or a month, or several years or continued throughout the patient's life. This treatment can continue to maintain the desired oxalate level in the subject.

[0250] All patents, patent applications and references included in the present invention are expressly incorporated by reference in their entirety. Of course, it should be understood that the foregoing only relates to exemplary embodiments of the present invention, and that many modifications or substitutions may be made therein without departing from the spirit and scope of the present disclosure.

[0251] Although exemplary embodiments of the present invention are provided herein, the present invention is not limited to these embodiments. Many modifications or changes may be suggested to those skilled in the art. As examples of preferred applications of the present invention, formulations of YvrK, Bce, A8 or Cb6301 are described herein; however, these examples should not limit the scope of the present invention.

[0252] The present invention is further illustrated by the examples included herein which are provided for ease of understanding. The exemplary embodiments should not be construed as imposing limitations on the scope thereof. On the contrary, it should be fully understood that after reading the description herein, it is suggested that those skilled in the art make changes to other embodiments, modifications and equivalent forms without departing from the spirit of the present invention and / or the scope of the appended claims.

[0253] Example

[0254] Example 1:

[0255] Activity test:

[0256] The removal of substrate (oxalate) and the formation of product (formate) were monitored to determine the oxalate degradation activity of the enzyme. The activity was tested in 50 mM citrate or phosphate buffer at pH 3 or in 10 mM oxalate ion (C 4) buffer at pH 4. 2 O 4 2 -) buffer solution. To determine the pH activity profile of the enzyme, a combination of citrate and phosphate buffers (50 mM) was used to determine the enzyme activity at pH from 1.5 to 8.0. The test sample was added to the preheated reaction buffer and incubated at 37°C with shaking at 1100 rpm for a specified time point (t). The reaction was stirred at t ± 5 seconds with 2.5 NH4OH at a rate of 10% acid to the reaction mixture. 2 SO 4 The quenched reaction mixture was filtered and analyzed for formate concentration using an isocratic ion exclusion HPLC method. Specific activity was defined as μmol oxalate and mg protein degraded per minute.

[0257] HPLC Method:

[0258] The quenched reaction mixture was filtered and concentrated in a 4% flask equipped with Rezex purchased from Phenomenex. TM ROA-organic acid H+ (8%), LC column (300×7.8 mm) Agilent 1100 series HPLC system was used for analysis. The injection volume was 40 μl and the mobile phase was 5 mM HCl. 2 SO 4 (isocratic), flow rate 0.6 mL per minute, column temperature 40°C. Oxalic acid and formic acid standards were analyzed in each batch to prepare a standard curve. The run time between each injection was 20 minutes, oxalic acid and formic acid were eluted at 8 minutes and 16 minutes respectively, and the detection was at a wavelength of 210 nm.

[0259] Example 2:

[0260] Amino acid sequence of OxDC enzyme

[0261] Serial ID Number: 1

[0262] Oxalate decarboxylase [Bacillus cereus, Bce]

[0263] MKKRTVNEAGRNVPQPIRSDGAGAIDSGPRNVMRDIQNPNMLVPPITDAGLVPNLKFSFSDTSMILKQGGWSREITARELPVSTTIAGVNMSLTAGGVRELHWHKEAEWAYMLLGRARITAVDQNGRNFIADVGPGDLWYFPPGIPPHSIQGLEHCEFLLVFDDGHFSDLSTLAISDWFAHTPKEVLSANFGVPESVFRS LPSDQVYIYQGEVPGSLESQEVQSPKGEVPLTFKHELLKQKPVKTPGGSVRIVDSTNFPISKTIAAALVEVEPGGMRELHWHPNNDEWQYYLTGEARMT VFLGNGTARTFDYRAGDVGYVPFATGHYIQNTGTETLWFLEMFRSSNRFEDVSLNQWMALTPKEIVESNIHVGPQVMDSLRKEKWPVVKYPGFSYSPKSDE

[0264] Serial ID Number: 2

[0265] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] full length native sequence 12

[0266]

[0267] Serial ID Number: 3

[0268] Oxalate decarboxylase [Synechococcus elongatus, Cb6301]-D29 sequence 13

[0269] MQTQTWRSLSNVVWGKDLPAFSYPFSKTPLVDYDGGVTKQVGTYNFPVSKGMAGVYMTLKPGAIRELHWHANAAEWAYVIEGRTRVTLTNPDGQVQIADVDQGGLWYFPRGWGHSIEGIGPGTAKFLLVFNDGTFSEGATFSITDWLSHTPISWVQQNFGWSQDEVEKLPKKQVYISRYN PEVKPLDKTQSRNPKVSRIVLPYTHNLLAEKPRTSQAGNTLKLASAKEFPASFNMAGALLRLEPGAMRQLHWHPNADEWQYVLNGSMDLAVFASEGKASMSRLQKGDVGYVPKGYGHALRNSSDQPLDVLIVFNDGDYQSIDLNDWIMSNPNTVLDDVFQLSPQLLDKLPKESEILIPRS

[0270] Serial ID Number: 4

[0271] Oxalate decarboxylase [Synechococcus elongatus, Cb6301]-D10 sequence 14

[0272] MLGVITCFVLIGSFCLPSLAQTQTWRSLSNVVWGKDLPAFSYPFSKTPLVDYDGGVTKQVGTYNFPVSKGMAGVYMTLKPGAIRELHWHANAAEWAYVIEGRTRVTLTNPDGQVQIADVDQGGLWYFPRGWGHSIEGIGPGTAKFLLVFNDGTFSEGATFSITDWLSHTPISWVQQNFGWSQDEVEKLP KKQVYISRYNPEVKPLDKTQSRNPKVSRIVLPYTHNLLAEKPRTSQAGNTLKLASAKEFPASFNMAGALLRLEPGAMRQLHWHPNADEWQYVLNG SMDLAVFASEGKASMSRLQKGDVGYVPKGYGHALRNSSDQPLDVLIVFNDGDYQSIDLNDWIMSNPNTVLDDVFQLSPQLLDKLPKESEILIPRS

[0273] Serial ID Number: 5

[0274] Oxalate decarboxylase [Bacillus cereus, Bce][Synechococcus elongatus, Cb6301]-fusion sequence 15

[0275]

[0276] Serial ID Number: 6

[0277] Oxalate decarboxylase [Synechococcus elongatus, Cb6301]-D20 sequence 16

[0278] MGSFCLPSLAQTQTWRSLSNVVWGKDLPAFSYPFSKTPLVDYDGGVTKQVGTYNFPVSKGMAGVYMTLKPGAIRELHWHANAAEWAYVIEGRTRVTLTNPDGQVQIADVDQGGLWYFPRGWGHSIEGIGPGTAKFLLVFNDGTFSEGATFSITDWLSHTPISWVQQNFGWSQDEVEKLPKKQVY ISRYNPEVKPLDKTQSRNPKVSRIVLPYTHNLLAEKPRTSQAGNTLKLASAKEFPASFNMAGALLRLEPGAMRQLHWHPNADEWQYVLNGSMDLAVFASEGKASMSRLQKGDVGYVPKGYGHALRNSSDQPLDVLIVFNDGDYQSIDLNDWIMSNPNTVLDDVFQLSPQLLDKLPKESEILIPRS

[0279] Serial ID Number: 7

[0280] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 6 C5N 17

[0281]

[0282] Serial ID Number: 8

[0283] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] SEQ ID NO: 6 C5S 17

[0284]

[0285] Serial ID Number: 9

[0286] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] SEQ ID NO: 6 C5A 17

[0287]

[0288] Serial ID Number: 10

[0289] Oxalate decarboxylase [Synechococcus elongatus, CB6301] "Ring Mutant" Sequence ID Number: 2 G 167N, A 168S, S171Q, I172L 17

[0290]

[0291] Serial ID Number: 11

[0292] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340E 17

[0293]

[0294] Serial ID Number: 12

[0295] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID No: 2 I340E, G167N, A 168S, S171Q, I172L 17

[0296]

[0297] Serial ID Number: 13

[0298] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] SEQ ID NO: 2 I340A

[0299]

[0300] Serial ID Number: 14

[0301] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340C 17

[0302]

[0303] Serial ID Number: 15

[0304] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340D 17

[0305]

[0306] Serial ID Number: 16

[0307] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340E 17

[0308]

[0309] Serial ID Number: 17

[0310] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340F 17

[0311]

[0312] Serial ID Number: 18

[0313] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340G 17

[0314]

[0315] Serial ID Number: 19

[0316] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340H 17

[0317]

[0318] Serial ID Number: 20

[0319] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340K 17

[0320]

[0321] Serial ID: 21

[0322] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340L 17

[0323]

[0324] Serial ID Number: 22

[0325] Oxalate decarboxylase [Synechococcus elongatus Cb6301] Sequence ID number: 2 I340M 17

[0326]

[0327] Serial ID Number: 23

[0328] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340N 17

[0329]

[0330] Serial ID Number: 24

[0331] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340P 17

[0332]

[0333] Serial ID Number: 25

[0334] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340Q 17

[0335]

[0336] Serial ID Number: 26

[0337] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340R 17

[0338]

[0339] Serial ID Number: 27

[0340] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340S 17

[0341]

[0342] Serial ID Number: 28

[0343] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340T 17

[0344]

[0345] Serial ID Number: 29

[0346] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340V 17

[0347]

[0348] Serial ID Number: 30

[0349] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340W 17

[0350]

[0351] Serial ID: 31

[0352] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 I340Y 17

[0353]

[0354] Serial ID: 32

[0355] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID number: 2 V291Y 17

[0356]

[0357] Serial ID Number: 33

[0358] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] SEQ ID NO: 2 L312Y 17

[0359]

[0360] Serial ID Number: 34

[0361] Oxalate decarboxylase [Synechococcus elongatus Cb6301] Sequence ID number: 2 V338P 17

[0362]

[0363] Serial ID: 35

[0364] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID No: 2 V291Y, L312Y 17

[0365]

[0366] Serial ID: 36

[0367] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID No: 2 V291Y, V338F 17

[0368]

[0369] Serial ID: 37

[0370] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID No: 2 L312Y, V338F 17

[0371]

[0372] Serial ID: 38

[0373] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] Sequence ID: 2 V291Y, L312Y, V338F 17

[0374]

[0375] Serial ID: 39

[0376] Oxalate decarboxylase [Synechococcus elongatus, Cb6301] SEQ ID NO: 2 E194K, V291Y, L312Y, V338F 17

[0377]

[0378] Serial ID Number: 40

[0379] Oxalate decarboxylase [Synechococcus elongatus] "6803"

[0380] MVNSVIGWLRRRFLLVGLSVLLITFLGIFTPTIAQSEQWRSLSNVVWGKDLPAFTYAFSKTPLVLYDGGTTKQVGTYNFPVSKGMAGVYMSLEPGAIRELHWHANAAEWAYVMEGRTRITLTSPEGKVEIADVDKGGLWYFPRGWGHSIEGIGPDTAKFLLVFNDGTFSEGATFSVTDWLSHTPIAWVEENLGWTAA QVAQLPKKQVYISSYGPASGPLASATPQGQTAKIEVPHTHNLLGQQPLVSLGGNELRLASAKEFPGSFNMTGALIHLEPGAMRQLHWHPNADEWQYVL DGEMDLTVFASEGKASVSRLQQGDVGYVPKGYGHAIRNSSQKPLDIVVVFNDGDYQSIDLSTWLASNPSSVLGNTFQISPELTKKLPVQDTIFSLPTQP

[0381] Serial ID: 48

[0382] Oxalate decarboxylase [Synechococcus elongatus] "6312"

[0383] MASLSRLFKPYSQLFSKFRLFLICLVLLLIGSSCWLLPALSQSSQWHSLSGVVWGKDLPAFSYPFHQTPLTLYDGGTTKQVGTYNFPVSKGMAGVYMTLEPGAIRELHWHANAAEWAYVISGRTRITLTSPDGNVQIADVDQGGLWYFPRGWGHSIEGLGPGTAKFILVFNDGTFSEGATFSITDWVSHMPISWVQDALGLTATQVQGLPNKQVYISRRPPAPGPLATTQPRNPNIPRLEVTHVHDIAAQPFFAVEDQNTILLASNKEFPASFNMAGGIIHLEPGAIRQPHWHPNADEWQYILDGEMELTVFASEGKASISTLKTGDVGYIPKGYGHALRNPSHKPMDVLLVFDAGEYESIELTGWIASNPDSVVGNTFQVPANLLSRLPRQKKLFARPGK

[0384] Sequence ID No.: 41

[0385] Oxalate decarboxylase [Clostridium kluyveri] "Bcl"

[0386] MKRGDNVKPLKGNPNIPQPIRADGAGGVDRGPRNLMRDLQNPNILVPPETDRGLIPNLRFSFSDAHMQLNHGGWSREITQRDLPIATTLAGVNMSLTPGGVRELHWHKQAEWSYMLLGHARITAVDQNGRNFIADVGPGDLWYFPPGIPHSIQGLDDGCEFLLVFDDGMFSDLSTLSLSDWMAHTPKDVLSANFGVPESVFATIPTEQVYIYQDEVPGPLQSQQINSPYGAVPQTFKHELLKQPPLVTPGGSVRIVDSRNFPVSKTIAAALVEVEPGAMREMHWHPNNDEWQYYLTGQARMTVFTGNGVARTFDYRAGDVGYVPFATGHYIQNTGNESVWFLEMFKSDRFEDVSLNQWLALTPTELVQHNIHVDSKFTNKLRKEKWPVVKYPTI

[0387] Sequence ID No.: 42

[0388] Oxalate decarboxylase [Agrocybe aegerita] "A0" / "A8"

[0389] MISVASCTIALLLSSVAFAAPAPSSAASSIVVSATSSSTVSSAPVSVSSFLPTTSIAAATPSSIAVALSSTATVPFIDLNPNGPLWDPSVSGVPQAERGSLGATIMGPTDVDTTKANPDLLAPPTTDHGSVDNAKWAFSLSHNRLQTGGWAREQNIGAMPIATEMASVNMRLEPGAIRELHWHKTAEWAYVLKGNTQVTAVDQNGKNFIGTVGPGDLWYFPPGIPHSLQATGDDPEGSEFILVFDSGAFSEDSTFLLTDWMSHVPVEVLAKNFQTDISAFARIPAEELYIFPAAVPPDSQQDPTSPEGTVPNPFTFALSKVPPMQLSGGTAKIVDSTTFTVSKAIAAAEVTIEPGAIRELHWHPTQDEWSFFIEGRARMTIFAAQSNARTFDYQAGDIGYVPATMGHYVENIGNTTVRYLEIFNTAVFEDISLSNWLALTPPELVKAHLGFDDATMAHLAKVKPIVVGPA

[0390] Sequence ID number: 43

[0391] Oxalate decarboxylase [Agrocybe aegerita] "A0" / "A8 D-18"

[0392] MAPAPSSAASSIVVSATSSSTVSSAPVSVSSFLPTTSIAAATPSSIAVALSSTATVPFIDLNPNGPLWDPSVSGVPQAERGSLGATIMGPTDVDTTKANPDLLAPPTTDHGSVDNAKWAFSLSHNRLQTGGWAREQNIGAMPIATEMASVNMRLEPGAIRELHWHKTAFWAYVLKGNTQVTAVDQNGKNFIGTVGPGDLWYFPPGIPHSLQATGDDPEGSEFILVFDSGAFSEDSTFLLTDWMSHVPVEVLAKNFQTDISAFARIPAEELYIFPAAVPPDSQQDPTSPEGTVPNPFTFALSKVPPMQLSGGTAKIVDSTTFTVSKAIAAAEVTIEPGAIRELHWHPTQDEWSFFIEGRARMTIFAAQSNARTFDYQAGDIGYVPATMGHYVENIGNTTVRYLEIFNTAVFEDISLSNWLALTPPELVKAHLGFDDATMAHLAKVKPIVVGPA

[0393] Sequence ID No.: 44

[0394] Oxalate decarboxylase [Bacillus amyloliquefaciens] "Bam"

[0395] MSKENNCNIPQPIRGDKGATVTIPRNLERDRQNPDMLTPPETDHGTVDNMKFSFSDVHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKEAEWAYMLTGKARVTIVDEQGRSFIDDVKEGDLWYFPSGLPHSIQALKEGCEFLLVFDDGSFSENSTFQVTDWLAHTPLDVIASNFGVSEKDLAGLPGKEKYIFEEPVPGKLKDDIVEGPNGEVPYPFTYRLLDEGPTAETDGGKVYIADSTNFKVSKTIASALVVVEPGAMRELHWHPNTHEWQYYISGKGRMTVFASDGHARTFNYQAGDVGYVPFAMGHYVENLGDEPLVFLEIFKDDHYADVSLNQWLAMLPEKFVQQHLDLGKDFTDILSKEKHPVVKKKC

[0396] Sequence ID No.: 45

[0397] Oxalate decarboxylase [Bacillus pumilus] "Bpu"

[0398] MSEKQNGVPQPIRGEKGATVKIPRNLERDRQNPDMLTPPETDHGTVPNMKYSFSDTHNRLEKGGYAREVTVRELPISKSLASVNMRLKPGAIRELHWHKEAEWAYMIYGEARITSVDAEGRNFTEDVTEGDLWYFPSGLPHSIQALEPGAEFLLVFDDGSFSENSTFQVTDWLAHTPEEVVLQNFGMTKEQFEKLPEKEKYIFQKGIPGSLECDKVKTGQGEVPNSFKYELLKQEPITSSGGQVWIADSTNFKASKTIASALVKVDPGAIRELHWHPNTDEWQYFISGKARMTVFASDGHARTFNYQAGDVGYVPFAMGHYVENTGDEPLYFLEIFKSDHYADISLNQWLAVTPKQLILDHLDQGEEFLKLLDTEKHPVIAAPKKED

[0399] Sequence ID No.: 46

[0400] Oxalate decarboxylase [Clostridium botulinum]

[0401] MYIQNQYQNLCNLLMSGCIPQPIRDGAGATDIGPRDILRDLENPDMLVPPSTDTGLIPNLKFSFSDTNMTIRPGGWSREITVRELPIATTMAGVNMRLTPGGVREVHWHQQSEWSYMLKGSARITAVDDRGRNFIADIGPGDLWFFPPLFPHSIQGLEEGCEFLLLFDDGNFSDLRTFSLSEFFAHYPKDVLAANFGVTKNCFNCLPEGQVYIYQDTIPGPLESEAIESPYGTIPQSYKHSLLAQKPMTTPGGSVRIADTSNFPVAKTTAAALVEIKPGGMREIHWHPNDEFQYFLTGQSRMTVFADTGASRTFDYRAGDVGYVPTGYGHYVQNIGNETVWFLEAFRSDRFKSISLSQMMAITPQQLIASNLNVGPGFLNALSRSKFQCSVGPCFHQTECSD

[0402] Sequence ID No.: 47

[0403] Oxalate decarboxylase [Bacillus subtilis, YvrK]

[0404] MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMKFSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKEAEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGAEFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEIS NLPGKEKYIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADSTNFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGHARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWLAMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK

[0405] Each OxDC subunit has two active sites. In the full-length sequence of Cb6301, the residues that are critical for activity are as follows:

[0406] 97-HWHXXXXE-104 H-143

[0407] 280-HWHXXXXE-287 H-326

[0408] In order to maintain the activity of the variant, the residues highlighted in red should be 100% conserved. As long as the modified enzyme has at least 85%, 90%, 95% or 99% of the native amino acid sequence, other regions of the enzyme may be modified to replace amino acids with similar types of amino acids, or to modify regions that can affect enzyme properties as described above.

[0409] Example 3:

[0410] Expression, fermentation and enzyme extraction:

[0411] OxDC-A0 was produced by fermenting Agrocybe oleracea (“A0”) by lowering the pH to 3.0 and adding MnCl 2OxDC-A0 was induced to a final concentration of 5 mM. Most of the OxDC protein was present in the fungal cells and was obtained by centrifugation. The pellet was resuspended in 50 mM phosphate buffer at pH 3 and homogenized, and the resulting mixture was used for testing. OxDC-A8 was produced by fermenting Agrocybe spp. ("A8") by lowering the pH to 3.0 and adding MnCl 2 Induce OxDC-A0 to a final concentration of 5mM. Most of the OxDC protein is present in the culture supernatant and is separated from the cells by centrifugation. The protein in the supernatant is purified and concentrated by ammonium sulfate precipitation and tangential flow filtration (TFF). The final protein solution is in 50mM citrate buffer at pH 3. All enzymes and variants (including A8) are recombinantly expressed in the constructed Escherichia coli strain. The full-length gene is inserted between the NdeI and BamHI sites of pColdIV or pOTIpr or pET vectors, and the sequence-verified plasmid is transformed into competent cells of Escherichia coli Origami or BW25113 or BL21 to construct an expression cell line. Protein expression is carried out in batch fed fermentation and induced according to the induction conditions listed in Table 2. Cells are collected and lysed by homogenization or ultrasonic treatment. After washing in 50mM citrate buffer at pH 5, the protein is dissolved in 50mM arginine buffer at pH 9.5.

[0412] Table 2: Induction conditions for different expression constructs.

[0413]

[0414] All enzymes were expressed in soluble form. Enzymes were not crystallized in this process and all evaluations of enzymes in the following examples were from soluble forms of the enzymes.

[0415] Example 4:

[0416] Thermal stability:

[0417] OxDC enzymes from four different source organisms (A0, A8, Bce and Cb6301_D29) in solution obtained as described in Example 3 were incubated at different temperatures from 25 to 95°C for 20 minutes. At the end of the 20-minute incubation, the remaining oxalate degradation activity of each sample was tested as described for the enzyme activity assay. The activity of the samples incubated at 25°C (ambient temperature) was considered to be 100%. Figure 1 As shown, OxDC enzymes extracted from fungi A0 and A8 were determined to be more stable than enzymes Bce and Cb6301_D29 from bacterial sources.

[0418] Example 5: pH stability:

[0419] As described in Example 3, OxDC enzymes from two different source organisms (Bce and Cb6301_D29) were incubated in solutions at different pH values ​​of 1-13 for 120 minutes. At the end of the 120-minute incubation, the remaining oxalate degradation activity of each sample was tested as described for the enzyme activity assay. The activity of the samples incubated at 25°C (ambient temperature) was considered to be 100%. Figure 3 As shown, the Cb6301_D29 enzyme is more stable than the Bce enzyme, with a stability range of 2.5 to 11, compared to the stability range of 3.0 to 10 for the Bce enzyme. As described in Example 8, the number and composition of ionic residues at the hexamer and trimer interfaces determine the stability of the protein quaternary structure. Cb6301 lacks the necessary residues to be included in the hexamer; therefore, unlike the Bce enzyme (hexamer), Cb6301 is a trimer. In addition, Cb6301 has a minimum amount of ionic charged residues at the trimer interface and a maximum amount of hydrogen bond residues. Due to the reduced number of ionic interactions and the increased number of hydrogen bonds, Cb6301 will be more stable in nature. Therefore, Cb6301, as a trimer, has enhanced pH stability under acidic conditions compared to other enzymes included in hexamers.

[0420] Enzymes that are hexamer themselves meet the condition that the hexameric protein quaternary structure is active.

[0421] Example 6:

[0422] Degradation of oxalate in different meals under simulated gastric environment (simulating meal content in human stomach):

[0423] To evaluate the efficacy of the OxDC enzyme from Agrocybe oleraceus (A0) in degrading oxalate in human food, several conventional Western meals (pre-prepared "Lean Cuisine" meals) were cooked in a microwave oven, homogenized and used as substrates in the screening of oxalate degradation activity of the A0 enzyme according to the instructions on the package. Table 3 lists the meals evaluated and the approximate calcium concentrations in the final reaction mixture. Calcium concentrations were estimated from the instructions (labels) of the dietary ingredients. Fresh uncooked spinach produced by Fresh Express was added to each meal at 30 g / L to provide oxalate, resulting in a final concentration of approximately 3 mM oxalate.

[0424] Table 3: Evaluated human foods and their calcium concentrations

[0425]

[0426] *Total Daily Value (DV) of Calcium for Spinach (based on Fresh Express content) and Meal (based on Lean Cuisine content).

[0427] **Calculated using the equation [Ca] (mM) = (percentage of daily nutritional intake of Ca) * 1000 / 40. The daily nutritional intake of calcium is 1000 mg and the molecular weight of calcium is 40 grams per mole.

[0428] All foods were prepared according to the instructions of Lean Cuisine and cut into small pieces by a food processor. Foods were combined with 400 mL of 50 mM citric acid (final concentration 20 mM) and the final volume was adjusted to 800 mL with deionized (DI) water. The pH of the food mixture was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.5 by adding 6N HCl and / or 10 M NaOH.

[0429] For each reaction, 0.8 mL of the above food mixture, 0.1 mL of 30 g / L pepsin (final concentration 3 g / L), and 0.1 mL of 800 U / L OxDC (final concentration 80 U / L) were mixed together and reacted at 37°C with shaking at 1000 rpm for 60 min. Then, 0.1 mL of 2.5 NH 2 SO 4 Quench (stop). The remaining oxalate and the generated formate concentrations were analyzed by ion exclusion HPLC method, see Example 1. The percentage of oxalate degradation was calculated using the following formula:

[0430] Oxalate degradation percentage = formate concentration / (oxalate concentration + formate concentration) × 100%

[0431] Spinach alone (no meal) was used as a low calcium control. As a negative control (no enzyme), 0.1 mL of 50 mM citric acid was added to each reaction instead of OxDC solution.

[0432] like Figure 4 As shown, OxDC from A0 can degrade more oxalate at acidic pH than at more basic pH and in diets with lower levels of calcium. 2+ In the diet with a pH between 2 and 5, more than 90% of the total oxalate was degraded within 60 minutes. 2+ In diets containing moderate levels of calcium (3-5 mM Ca 2+ ) diet, A0 OxDC enzymes can degrade 60-80% of total oxalate within 60 minutes at pH 2 to 3, and 50% at pH 4. 2+), the enzyme degraded 40-60% of the total oxalate within 60 minutes at pH 2 and 3. The reduction in degradation percentage can be attributed to the reduced solubility of oxalate in diets containing moderate to high amounts of calcium. m =8.4 mM), A0 for oxalate (K m =0.08 mM) has a high affinity, which makes A0 more capable of degrading low levels of oxalate in the human stomach. In order for the OxDC enzyme to effectively degrade oxalate in the human stomach, the enzyme needs to match the fed human stomach (pH 1.0-4.5) and have a K of less than 1.0 mM. m Therefore, Cb6301, Cb6803, Cb6312 and Bcl, as well as A0 / A8 and Bce are ideal candidates.

[0433] Example 7:

[0434] pH and time profile of OxDC enzyme:

[0435] The activity of the OxDC enzymes of Bce and Cb6301 obtained as described in Example 3 was tested as described in Example 1, but the pH range in the tested reactions was 1.5 to 7.0. After reacting at 37° C. for 5 minutes, 10 minutes, 20 minutes and 40 minutes, the reaction was terminated as described in Example 1. The concentration of formate generated was determined by HPLC and the activity of the OxDC enzyme was calculated as described in Example 1.

[0436] like Figure 5 and Figure 6 As shown, Cb6301_D29 is active in the pH range of 1.5 to 4.5, a wider pH range than bce (pH 2.4 to 4.5). However, Bce remains active for a long time under these conditions. Figure 7 The pH activity profiles of many OxDC enzymes are shown in . Each of these enzymes has a unique pH activity profile, and only four enzymes, namely, A8 / A0, Cb6301, Cb6803, Cb6312, and Bcl, are active at pH 2.0 or lower.

[0437] Cb6301, Cb6803 and Cb6312 all have a small amount of oxalate oxidase activity, which produces free radicals that are harmful to these particular enzymes. These free radicals produced lead to a loss of activity as a function of time. We found that if the isoleucine residue at position 340 ( Fig.10 In addition, we found that the introduction of vitamins such as o-phenylenediamine, hydroquinone and ascorbic acid into the enzyme solution can maintain the activity of the enzyme for a longer time.

[0438] As described in Example 8, the number and composition of ionic residues at the hexamer interface determine the stability of the protein quaternary structure. Cb6301, Cb6312 and Cb6803 lack most of these residues; therefore, unlike other enzymes, these enzymes are naturally encapsulated in trimers. Compared with hexamers, trimers have improved pH stability, especially when the pH is lower than 2.0. Due to the lower number of ionic interactions at the trimer interface and the increased number of hydrogen bond interactions, the quaternary structure of the trimer protein is more resistant to pH changes. The enzyme that is a hexamer itself requires the protein quaternary structure of the hexamer to be active. Enzymes with more than 10 ionic amino acid residues (D, E, R and K) on the hexamer interface specified in Example 8 are only active under conditions above pH 3.0. Enzymes with 5-9 ionic residues (D, E, R and K) at the hexamer interface are only active above pH 2.0 and enzymes with less than 5 ionic residues show activity below pH 2.0. This corresponds to the total ionic net charge at the hexamer interface (the pH at which all aspartates and glutamates are protonated):

[0439] 1) Enzymes with a total net ionic charge of +8 or higher have oxalate degradation activity only above pH 3.0.

[0440] 2) Enzymes with a total ionic charge of +4 to +7 have oxalate degradation activity above pH 2.0.

[0441] 3) Enzymes with a total ionic charge less than +4 exhibit oxalate degradation activity below pH 2.0.

[0442] Enzymes that are naturally packaged into hexamers have a higher number of ionic interactions not only at the hexamer interfaces, but also at the trimer interfaces. The combined number of ionic interactions for each subunit is as follows:

[0443] Cb6301:25

[0444] Bcl:29

[0445] A8:32

[0446] Bce:32

[0447] Bam: 44

[0448] YvrK:45

[0449] Bpu:47

[0450] There is a direct correlation between the number of total ionic interactions and acidic pH stability, with the highest stability when the amount of Cb6301 is the smallest, and the lowest stability when the amount of Bpu is the largest. The enzymes with the lowest acid stability (Bam, Bce, and Bpu) all have more than 44 ionic amino acids at the hexamer and trimer interfaces. Bcl, Bce, and A8 have 29-32 ionic amino acids, and Cb6301 has 25. Although the number of ionic interactions of Bcl, Bce, A8, and Cb6301 is reduced, they still have a large number of hydrogen bonding interactions. These hydrogen bonding interactions increase the stability at the interface and make the interface less susceptible to acid denaturation.

[0451] Based on the above information, a screening method can be provided for selecting an enzyme from a plurality of enzymes, wherein the selected enzyme meets one or more of the above criteria.

[0452] Example 8:

[0453] Protein quaternary structure:

[0454] 1. Amino Acid Sequence Analysis

[0455] By analyzing the crystal structure and amino acid sequence, it was determined that the reason why some OxDC enzymes have enhanced stability in acidic environments depends on the number and composition of ionic interactions at the hexamer and trimer interfaces. The amino acids at the hexamer interface are underlined. Fig.10 In the multiple sequence alignment shown. Fig.10 In the multiple sequence alignment found in , the amino acids at the trimer interface are underlined and shown in bold. Except for Cb6301, Cb6312 and Cb6803, all known OxDC enzymes are hexamers. When the environment becomes more acidic, enzymes with a larger proportion of ionic interactions (D, E, K and R amino acids) at the hexamer interface lose their protein quaternary structure. Similarly, enzymes with more ionic interactions at the hexamer and trimer interfaces are more likely to lose protein quaternary structure under acidic conditions. For example, Bam, Yvrk and Bpu are inactive at pH 3.0 and below due to the dissociation of the protein quaternary structure. This loss of protein quaternary structure is irreversible, and after the hexamer / trimer dissociation, the enzyme no longer has oxalate degradation activity. This is attributed to the protonation of aspartic acid and glutamic acid at the hexamer and trimer interfaces; therefore, the ionic interactions that hold these interfaces together are destroyed. Aspartic acid and glutamic acid have acid dissociation constants of 3.65 and 4.25, respectively, which can drop by about 0.5-1.0 pH units if the surrounding environment is predominantly hydrophobic.

[0456] On the contrary, due to the lack of ionic interactions required to form hexamers, Cb6301 does not form a hexameric structure, and this is reasonable. Therefore, Cb6301 is included in trimers and is active under more acidic conditions down to pH 1.5. Although much weaker than Yvrk, Bam and Bpu enzymes, Bcl and Bce enzymes still form hexameric structures. This is due to fewer ionic interactions that keep the interface structure together. Due to fewer ionic interactions, acidic pH stability is enhanced and activity is retained. Therefore, under this trend, Bcl and Bce are active when pH values ​​drop to 2.0 and 2.5, respectively. For example, the enzymes with the lowest acid stability (Bam, Bce and Bpu) all have more than 44 ionic amino acids on the hexameric and trimer interfaces. Bcl, Bce and A8 have 29-32 ionic amino acids, and Cb6301 has 25. Although the number of ionic interactions of Bcl, Bce, A8 and Cb6301 is reduced, they still have a large number of hydrogen bond interactions. These hydrogen bonding interactions increase the stability at the interface and make the interface less susceptible to acid denaturation.

[0457] Enzymes that are hexamers themselves require the protein quaternary structure of the hexamer to be active. Enzymes with more than 10 ionic residues (D, E, R and K) on the hexamer interface (interactions between 2 of the 6 subunits) are only active above pH 3.0. Enzymes with 5-9 ionic residues (D, E, R and K) on the hexamer interface (interactions between 2 of the 6 subunits) are only active above pH 2.0 and enzymes with less than 5 ionic residues (interactions between 2 of the 6 residues) are active below pH 2.0. This corresponds to the total net ionic charge at the hexamer interface (pH where all aspartic acid and glutamic acid have been protonated). These results show the following inevitable trends:

[0458] 1) Enzymes carrying an overall net ionic charge of +8 and above have oxalate degradation activity only above pH 3.0 (charge between 2 of the 6 subunits).

[0459] 2) Enzymes carrying an overall ionic charge of +4 to +7 have oxalate degradation activity above pH 2.0 (charge between 2 of the 6 subunits).

[0460] 3) Enzymes carrying an overall net ionic charge of less than +4 have oxalate degrading activity below pH 2.0 (charge between 2 of the 6 subunits).

[0461] At pH conditions where most, if not all, aspartates and glutamates are protonated, the hexamer interface has an overall positive net charge. Enzymes with a greater proportion of ionic residues at the hexamer interface are more sensitive to changes in pH than enzymes with fewer ionic residues. In fact, Figure 7 The total net ionic charge at a pH where all aspartates and glutamates are protonated is shown to be directly correlated with the total net ionic charge at the most acidic pH where the YvrK, Bam, Bpu, Bcl, Cb6301, A8 / A0, and Bce enzymes were shown to be active. In fact, the R2 values ​​show a strong correlation of greater than 0.95 with a large number of data.

[0462] Based on the above information, a screening method can be provided for selecting an enzyme from a plurality of enzymes, wherein the selected enzyme meets one or more of the above criteria.

[0463] Table 4: Analysis and activity of hexamer and domain boundary amino acids

[0464]

[0465]

[0466] AA = amino acid

[0467] * Number of amino acids or charges at one interface between two subunits. OxDC enzyme forms dimers of trimers; therefore, one OxDC hexamer has three interfaces. Therefore, all values ​​in Table 4 should be multiplied by 3.

[0468] ^Total net ionic charge equals the total number of lysine and arginine residues at the pH at which all glutamate and aspartate residues are protonated. 1 The ionic charge is the total charge on the trimer interface at neutral pH.

[0469] 2. Size Exclusion Chromatography

[0470] SEC-HPLC was used to monitor the formation of dimers / polymers.

[0471] Draw a molecular weight standard curve for SEC-HPLC. First, 2 The gel filtration molecular weight standard was reconstituted in 50 mM arginine buffer to a concentration of 20 mg / ml and then diluted in 50 mM arginine buffer according to the supplier's recommendations. To determine the molecular weight of OxDC, the enzyme was diluted in 50 mM arginine buffer to concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml using the constructed standard curve.

[0472] Molecular weight standards are as follows:

[0473] Dextran blue: 1 mg / ml

[0474] Thyroglobulin: 5mg / ml

[0475] Ferritin: 0.3mg / ml

[0476] Aldolase: 4mg / ml

[0477] Conalbumin: 3mg / ml

[0478] Ovalbumin: 4mg / ml

[0479] Draw the calibration curve according to the supplier's recommendations:

[0480] 1. Calculate the partition coefficient (K) using the following equation: av ):

[0481] K av =(v e -v o ) / (v c -v o )

[0482] where v e = elution volume, v c = geometric cylinder volume, v o = column void volume.

[0483] The column void volume was defined as the elution volume of the dextran blue standard.

[0484] The geometric column volume is calculated as follows:

[0485] v c =r 2 ×π×l

[0486] Where r is the radius of the column and l is the length of the column.

[0487] 2. The partition coefficients are plotted as logarithms (MW).

[0488] The SEC results showed that the Yvrk enzyme is an oligomer, i.e., a hexamer, with a retention time of 8.8 minutes. There are no additional peaks corresponding to higher-order aggregation or degradation. Similarly, Bce is also an oligomeric hexamer with a retention time of 8.7 minutes. However, Cb6301 is a trimer with a retention time of 9.6 minutes. These results all confirm the hypotheses proposed in the previous section "Amino Acid Sequence Analysis" and the next section (Non-denaturing Polyacrylamide Gel Electrophoresis Analysis).

[0489] 3. Non-denaturing polyacrylamide gel electrophoresis analysis

[0490] Native polyacrylamide gel electrophoresis separates enzymes based on a combination of molecular weight and pI. Therefore, if OxDC appears to be dissociated, this will result in a gel shift, meaning that the enzyme band will be further into the gel.

[0491] Fig. 9Native polyacrylamide gel electrophoresis of Cb6301, Bce and Yvrk at different pH is shown.

[0492] To prepare the test samples, 20 μl of CB6301 was added to 1980 μl of buffer at pH 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 and vortexed. 40 μl of Yvrk was added to 1960 μl of buffer at pH 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 and vortexed. 50 μl of Bee was added to 450 μl of buffer at pH 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 and vortexed. The concentration of all the above samples was 1 mg / ml. At the same time, samples with a concentration of 2 mg / ml were also prepared and loaded on the gel.

[0493] The above solutions were incubated at 37°C at 300 rpm. Then 15 μl of each solution was mixed with 5 15 μl of sample buffer. 20 μl of each sample was added to the respective gel wells. 10% non-denaturing polyacrylamide gel electrophoresis gel was run at 100 volts for 75 minutes.

[0494] Note: The pH of each sample was measured before loading on the gel. The actual measured pH values ​​are given in Fig.11 Legend for .

[0495] According to non-denaturing polyacrylamide gel electrophoresis ( Fig.11), the Cb6301 OxDC enzyme remained a trimer (confirmed by SEC and mass spectrometry) at all pH values ​​evaluated, i.e., 1.40-2.52 (no gel shift). The Yvrk enzyme existed as a hexamer at pH 4.07 (confirmed by SEC and mass spectrometry), as a mixture of hexamer and trimer / dimer / monomer (presented as a wider band) at pH 3.6, and as a mixture of trimer / dimer / monomer (presented as a wider band) at pH 3.02-3.05, as determined by gel shift. Finally, the Bce enzyme was a trimer / dimer / monomer mixture (bands were found to be more diffuse in the lanes) at all pH values ​​evaluated, i.e., 1.57-2.27, however, at pH 2.57, the enzyme was a hexamer. These results provide a convincing evidence that the pH profile of the OxDC enzymes is directly related to the dissociation of the protein quaternary structure. For example, the Cb6301 enzyme shows activity at pH between 1.5 and 5.0 (as a trimer under all pH conditions), the YvrK enzyme shows activity at pH 3.5-5.5 (as a hexamer under these pH conditions), and the Bce enzyme shows activity at pH 2.4-4.5 (as a hexamer under these pH conditions). Once the pH of the Yvrk enzyme drops below pH 3.5 and the Bce enzyme drops below 2.4, the protein quaternary structure dissociates, followed by a complete loss of activity. The quaternary unfolding process and loss of activity are irreversible.

[0496] Example 9:

[0497] Stability of OxDC in the presence of different chemicals at 40°C:

[0498] Several different chemicals were added to the Bce or Cb6301_D29 purified OxDC enzyme solutions obtained as in Example 3 at a final concentration of 2 mM. After thorough mixing, these mixtures in 50 mM arginine at pH 9.5 were incubated at 40°C without shaking (standing) for 6 days. The enzyme itself was incubated at 40°C and 4°C as a control without the addition of chemicals. The enzyme activity was tested at pH 3 according to the procedure described in Example 1, with reaction times of 8 minutes, 17 minutes, 41 minutes and 106 minutes, respectively.

[0499] like Fig.12 As shown in Figure 5, the enzyme activity of Bce more than doubled when incubated at 40 °C. Furthermore, the activity increased by nearly 20% in the presence of magnesium sulfate and by 20% in the presence of ZnSO when compared to the control sample of Bce enzyme at 40 °C. 4 The activity was significantly reduced in the presence of EDTA.

[0500] like Fig.13As shown, unlike the Bce enzyme, the enzyme activity of Cb6301_D29 remained essentially unchanged after incubation at 40°C. In the presence of magnesium sulfate or manganese sulfate, the activity increased by more than 80%. However, they still showed a loss of activity during the reaction.

[0501] Example 10:

[0502] Enzyme Kinetics:

[0503] The enzyme kinetics of OxDC from four enzymes, namely Bce, Bcl, Cb6301_D29 and A8, were measured and compared with the known kinetic data of YvrK. Reaction buffers (100 mM citrate buffer, pH 3) with different concentrations of oxalate (0.024-12.5 mM) were prepared. The A8 enzyme was measured at four independent pH values: namely, 5.0, 4.0, 3.5 and 3.0. These buffers were used to test the oxalate degradation enzyme activity of different OxDC enzymes by monitoring oxalate degradation and formate generation. The OxDC enzyme was added to the oxalate reaction buffer to start the reaction and incubated at 37°C for 5 minutes with shaking at 1100 revolutions per minute. 2.5N H2SO4 was added to terminate the reaction and the content of formate was analyzed by HPLC as described in Example 1. The reaction without oxalate was counted as a negative control. The initial reaction rate during the first 5 minutes when the substrate concentration was between 0.024-12.5 mM was determined using the same procedure as described in Example 1. The initial reaction rate of Cb6301_D29 at different oxalate concentrations is as follows Fig.14 shown.

[0504] In order to determine the kinetic parameter k cat and K m , the reaction rate v0 at different substrate concentrations [S] is fitted to the Michaelis-Menten equation in KaleidaGraph software:

[0505] v 0 =k cat *[E] t *[S] / (Km+[S])

[0506] in:

[0507] ·v 0 is the initial reaction rate during a short reaction time (5 min) as determined by HPLC.

[0508] ·k cat is the turnover number of the enzyme,

[0509] ·K M is the Michaelis constant,

[0510] ·[E] tis the total concentration of OxDC enzyme,

[0511] [S] is the initial concentration of the substrate, oxalate.

[0512] The results are compared in Table 5. The KMs of the Bce, Bcl, Cb6301_D29, and A8 (0.32 mM, 0.2 mM, and 0.08 mM, respectively) enzymes at pH 3.0 were much lower than those of the YvrK enzyme at pH 4.2, indicating that they have a stronger affinity for the substrate, oxalate. m It also showed that Bce, Bcl, Cb6301, and A8 enzymes were able to effectively degrade oxalate to lower levels than YvrK enzyme. We attempted to determine the K of YvrK enzyme at pH 3.0. m , but now the enzyme does show any activity at this pH. The fact that the YvrK enzyme shows no significant / sustained activity below pH 3.3 limits its use as an enzyme for removing oxalate from the human gastric environment, whereby the pH of a fed stomach is between pH 1.0-4.5.

[0513] A8 enzyme K m The results were measured at four pH values: 5.0, 4.0, 3.5 and 3.0. Fig.15 As shown in m It decreases with increasing acidity of pH. Monophosphonate oxalate (acid dissociation constant = 3.81 and acid dissociation constant = 1.25) binds to the unprotonated glutamic acid in the active site. The unprotonated glutamic acid in the undestroyed active site is more likely to remain unprotonated than the equivalent residue in the destroyed active site (e.g., the active site of the destroyed hexamer). Therefore, when the pH is reduced from 6 to about 3, the proportion of monophosphonate oxalate compared to unprotonated oxalate will be maximized. Therefore, this will increase the binding of oxalate to the undestroyed active site, resulting in a lower Km and higher catalytic efficiency.

[0514] Table 5: Comparison of enzyme kinetic constants of different OxDC enzymes.

[0515]

[0516] Source: Ellen W. Moomaw et al., Biochemistry. 2009; 48(26): 6116-6125.

[0517] Example 11:

[0518] Creating the ideal pH microclimate for enzymes during drying and formulation:

[0519] Freeze drying:

[0520] Bce enzyme was freeze dried in a 5% w / v trehalose formulation in deionized water. Initially, the shelf temperature and pressure were -30°C and 50-150 mTorr, respectively. After 18 hours, the temperature was raised (0.1°C per minute) to 4°C and maintained until the end of the treatment.

[0521] Emulsion:

[0522] Poly(lactide-co-glycolide) PLGA (lactide: glycolide 85:15, Mn85,000-100,000) with acidic endcapping was dissolved in dichloromethane at a rate of 21% w / v. Freeze-dried OxDC was mixed with the PLGA solution at a rate of 1.3% w / v and mixed for 30 seconds using a hand-held tissue homogenizer at approximately 18,000 revolutions per minute. 1.5 mL of a 2% polyvinyl alcohol solution (Mw 9,000-1,0000) was added immediately after homogenization, and the sample was vortexed for 30 seconds. The resulting emulsion was added dropwise to 100 mL of a 0.5% polyvinyl alcohol solution and stirred for 14 hours. The resulting microbeads were collected by centrifugation, then resuspended in DI water for repeated washing, and finally, collected by centrifugation. After the last wash, the microbeads were resuspended in 5 mL of DI water. The microbeads were stored at a temperature of 4°C before spray drying.

[0523] Spray Drying:

[0524] The bead suspension (3 mL) was mixed with RL30D acrylic resin (1.9 mL) and trehalose (0.5 g), and DI water was added until the total volume was 100 mL. Spray drying was performed in a Buchi B-191 with an inlet temperature of 100°C and an outlet temperature of about 58-65°C, respectively. The feed rate was set to 10% (about 2 mL per minute) and the gas sparge (N 2 ) The flow rate and pressure were 20 liters per minute and 70 pounds per square inch, respectively. The yield of dry powder was 68% (g).

[0525] Activity test:

[0526] Activity was determined as described in Example 1, with the reaction pH set to 4, 5, 6, 7, 7.3, and 7.8 when using citrate and phosphate buffers, respectively. Fig.13 and 14 As shown, when the Bce enzyme was formulated with PLGA, an acidic microclimate was achieved as oxalate degradation activity was observed in pH neutral environments (pH 6, 7, 7.3 and 8). The unformulated Bce enzyme was not active at these pH values. Therefore, a microclimate pH value has been achieved so that the Bce enzyme remains active within the particles.

[0527] Example 12:

[0528] Beagle Principle Evidence-Based Research:

[0529] Six beagles were given a high oxalate diet (2.73 mmol oxalate per day) to induce hyperoxaluria. Hyperoxaluria occurred immediately, with an average animal excreting 0.8 mmol oxalate per 24 hours. After approximately 48 hours of a high oxalate diet (2 meals per day), the level stabilized. Four different enzymes in this hyperoxalate beagle model were evaluated orally (gavage) along with the enzymes for each meal. The enzymes were soluble in 50 mM arginine at pH 9.5, and before gavage, the enzymes were mixed with the carrier in a ratio of about 6:96 between the enzyme solution and the carrier. The carrier used was citric acid with a pH of 3. Urine was collected in a container containing sulfuric acid to ensure that the acidic pH of the urine was always maintained. Oxalate in urine was measured in a 12-hour urine sample using an oxalate assay kit (5910D) from Trinity Biotech, and creatinine in urine was measured using the direct creatinine LiquiColor program 0421 from StanBio.

[0530] Prior to administration, a pre-screening study was performed on the pH and distribution in the stomach of eight animals (beagles). The distribution study was based on the pH of the stomach in the fed state within the range of pH 2.0-4.5 (similar to the human fed state). Six animals were selected for the study and Bce, Yvrk, A0, Cb6301_D29 were administered at different doses by oral gavage or by mixing in oxalate-free food.

[0531] result:

[0532] All animals had hyperoxaluria with the high oxalate diet, increasing from a baseline oxalate excretion of 0.16 mmol oxalate per 24 hours to 0.8 mmol oxalate per 24 hours (high oxalate diet phase). Total creatinine excretion averaged about 2 mmol per 24 hours throughout the study. Following oral gavage dosing with the vehicle, the A0, Cb6301_D29, and Bce test articles showed significant reductions in urinary oxalate. A0 and Cb6301_D29 showed the highest reductions in mean urine oxalate levels of 60% and 40% per animal (high: 85%), see Figure 18-19 Bce showed an average decrease of 23%, with greater variability between animals, see Fig. 20 The Yvrk enzyme did not show a significant decrease, see Fig.21. These results suggest that only enzymes with a pH activity profile that spans acidic conditions (e.g., pH 1.5-4.5) can be effective in vivo. For example, A0 / A8 and Cb6301, which have a pH profile of approximately 1.5-5.0, showed significant reductions in urinary oxalate compared to Bce (pH 2.4-4.5, 24%) and Yvrk (pH 3.5-5.5, no significant reduction). Results were better when the test articles were administered in a citric acid vehicle than when mixed with an oxalate-free diet (results not shown here). In the study measurements, gastric pH values ​​averaged around pH 4 during the first hour postprandially (results not shown herein); however, all animals exhibited extreme high and extreme low pH peaks.

[0533] Example 13:

[0534] Insoluble oxalate degradation

[0535] The three OxDC enzymes, Bce, Yvrk and Cb6301, were evaluated for oxalate degradation activity at different molar ratios of oxalate to calcium at their respective pH activity maxima (Bce tested at pH 3.0, Cb6301 tested at pH 2.5 and Yvrk tested at pH 4.0). The activity reactions were performed as described in Example 1, however, calcium ions were included to obtain the following molar ratios of oxalate to calcium, 1:1, 1:2, 1:3, 1:4, 1:5. The percentage of formate generated was equal to the amount of oxalate degraded and was normalized to the 1:1 condition, then the results were analyzed for Fig. 22 (Bce), Fig.23 (Cb6301) and Fig.24 (Yvrk) ratio of oxalate:calcium in 100 μl of 10 μl ...

[0536] Cb6301 was more efficient in degrading insoluble oxalate, then Bce or Yvrk ( Figure 22-24) (more formate was produced at all ratios of Ca to Ox investigated). This can be attributed to the Cb6301 enzyme having a lower Km and higher catalytic efficiency than the Bce enzyme or the Yvrk enzyme. The Yvrk enzyme is the least efficient enzyme in degrading insoluble oxalate. This may be related to two factors: (1) the Yvrk enzyme has a high Km (mM level) and low catalytic efficiency, and (2) the pH activity profile is not conducive to solubilizing insoluble oxalate. Insoluble oxalate becomes more accessible under acidic conditions, and higher levels of insoluble oxalate can be dissolved under more acidic conditions. Since the Yvrk enzyme is only active at pH 3.5 and above, the enzyme is ineffective in removing oxalate (either soluble or insoluble oxalate).

[0537] Example 14:

[0538] Degradation of oxalate in different foods:

[0539] To evaluate the effectiveness of OxDC enzymes in degrading oxalate in human foods, several foods were evaluated. These foods are as follows:

[0540] 1) Ready-to-drink tea

[0541] 2) Beer

[0542] 3) Fruit juice

[0543] For each reaction, 0.990 mL of food and 0.010 mL of 80 U / L OxDC were mixed together and allowed to shake at 1000 rpm for 60 minutes at 37°C. The reaction was then quenched (terminated) by adding 0.1 mL of 2.5 N H2SO4. The remaining oxalate and the generated formate concentrations were analyzed by ion exclusion HPLC, see Example 1. The percentage of oxalate degradation was calculated using the following formula:

[0544] Oxalate degradation percentage = formate concentration / (oxalate concentration + formate concentration) × 100%

[0545] As shown in Table 6, OxDC degraded most of the oxalate in the food.

[0546] Table 6: Oxalate degradation in foods and beverages

[0547]

Claims

1. A composition comprising at least one oxalate degrading enzyme, wherein a single subunit of the at least one oxalate degrading enzyme is the amino acid sequence shown in Sequence ID No. 3 and is a trimer at pH 3.

0.

2. The composition of claim 1, wherein the at least one oxalate degrading enzyme has oxalate degrading activity at pH 2.0 and above. 3 . The composition of claim 1 , wherein the at least one oxalate degrading enzyme has a Km for oxalate of 1 mM or less at pH 2.

5.

4. The composition of claim 1, wherein the at least one oxalate degrading enzyme has a catalytic efficiency of at least 7000 turnovers / M / s.

5. The composition of claim 1, wherein the at least one oxalate degrading enzyme has oxalate decarboxylase activity.

6. The composition of claim 1, wherein the at least one oxalate degrading enzyme degrades insoluble oxalate and soluble oxalate to reduce total oxalate.

7. The composition of claim 1, further comprising a stabilizing compound that maintains active oxalate degrading enzyme.

8. The composition of claim 7, wherein one of the stabilizing compounds is a vitamin.

9. The composition of claim 1, wherein the at least one oxalate degrading enzyme is immobilized.

10. The composition of claim 9, wherein the catalytic activity is maintained at temperatures exceeding 60 degrees Celsius.

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