Oxalate decarboxylase mutant, and method for obtaining same and use thereof

Abstract

Provided are an oxalate decarboxylase mutant, and a method for obtaining the same and the use thereof. Proposed is protein engineering modification on a pore channel leading from a manganese ion active center in the C-terminal cupin domain of oxalate decarboxylase to an external solvent. By means of narrowing the pore diameter (bottleneck pore diameter) at the narrowest part of the pore channel leading from the manganese ion center to the external solvent, the catalytic duration and specific enzyme activity of oxalate decarboxylase are further improved. By means of a series of screening and optimization processes, a plurality of oxalate decarboxylase mutants are obtained. These mutants exhibit a significantly reduced inactivation rate in the catalytic process compared with a wild-type protein. Some mutants show no significant inactivation within a 30-minute catalytic test period, thereby improving the effective activity of the oxalate decarboxylase and prolonging the catalytic duration. Experiments demonstrate that the mutants have a significant effect in reducing urinary oxalic acid excretion. In addition, the construction of a recombinant expression strain and the production process strategy for oxalate decarboxylase are further optimized, which can improve the enzyme activity of oxalate decarboxylase.

Description

An oxalate decarboxylase mutant, a method for obtaining the mutant, and its applications.

[0001] This application claims priority to an earlier application filed on January 27, 2025, with the China National Intellectual Property Administration, patent application number 202510126758.8, entitled "An Oxalate Decarboxylase Mutant and a Method and Application for Obtaining the Mutant". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of biotechnology and enzyme engineering technology, specifically relating to an oxalate decarboxylase mutant, a method for obtaining the mutant, and its application. Background Technology

[0003] Oxalic acid (also known as oxalic acid) is a final product in the human body that cannot be further metabolized. Most oxalic acid in the body is excreted through the kidneys in the form of urine, and a portion is secreted into the digestive tract through the intestinal wall and excreted in feces. Oxalic acid in the body mainly comes from two sources: production by the liver and absorption from dietary oxalate. Primary hyperoxaluria is caused by impaired oxalate metabolism in the liver; it is a rare genetic disease with an incidence of 1-3 per million people. Clinically, the vast majority of hyperoxaluria patients have it due to excessive dietary oxalate intake or abnormally high absorption rates, known as secondary hyperoxaluria. Clinically, hyperoxaluria is usually diagnosed when the 24-hour urinary oxalate excretion is >40 mg. Human blood and urine contain abundant calcium ions. When oxalate combines with calcium ions, it forms calcium oxalate, which has extremely low solubility. When calcium oxalate exceeds its saturation point, microcrystals precipitate out. These crystals in urine can then aggregate to form stones. When kidney function declines, preventing the kidneys from excreting oxalate in a timely manner, oxalate accumulates in bodily fluids such as blood, tissue fluid, or lymph. Calcium oxalate crystals formed when saturation exceeds this level can deposit in the heart, bone marrow, eyes, and muscle tissue, causing vascular calcification, oxidative stress, and chronic inflammation, ultimately damaging organ function. This condition is clinically known as systemic oxalosis. Calcium oxalate is the main component of urinary tract stones, accounting for over 80%. Hyperoxaluria is widely considered the leading risk factor for calcium oxalate stones. Furthermore, hyperoxaluria has been reported to be associated with various other diseases, such as cardiac conduction disorders, autism spectrum disorder (ASD), chronic kidney disease, nephritis, and accelerated kidney failure.

[0004] Currently in clinical practice, some small stones detected in the early stages (maximum diameter <0.6cm) can be treated by drinking plenty of water, taking diuretics, anti-inflammatory drugs, or ureter-dilating medications to help expel the stones, and combining this with exercise. However, the success rate of stone expulsion is not high, and there is a risk of the stone obstructing the ureter and causing hydronephrosis during the expulsion process. For larger urinary tract stones, the main clinical treatment is surgery, such as extracorporeal shock wave lithotripsy, minimally invasive stone removal or lithotripsy, and open surgery. Existing treatments not only cause trauma to the body (especially the kidneys), but also have problems such as high recurrence rates and high surgical costs (average 20,000-40,000 RMB per surgery). More seriously, patients with recurrent stones may repeatedly use the above treatments, causing repeated trauma and potentially leading to kidney failure. Patients with chronic kidney disease caused by frequent stone surgeries, or those who have progressed to end-stage renal disease (kidney failure), will experience a rapid deterioration in their condition due to the accumulation of toxins in their bodies, which cannot be effectively eliminated, leading to other serious secondary diseases. Patients with kidney failure require dialysis multiple times a week, which not only incurs high medical costs but also significantly impacts their quality of life. Most diseases secondary to hyperoxaluria are serious chronic conditions requiring long-term or lifelong treatment, consuming substantial medical resources and placing a heavy burden on my country's medical insurance payment system. Therefore, developing a drug to prevent and treat hyperoxaluria is urgently needed.

[0005] To date, although several investigational drugs have entered clinical trials, no drug has been approved for the treatment of secondary hyperoxaluria, representing a serious unmet clinical need. Oral oxalate-degrading enzyme therapy is a very promising approach for treating hyperoxaluria; however, currently available oral oxalate-degrading enzyme drugs in clinical trials are either acid-sensitive, leading to rapid inactivation and denaturation in the stomach, or have low enzyme activity, requiring very high doses. For example, ALLN-177 rapidly denatures and becomes inactive below pH 3.0, and its enzyme activity is only about 30 U / mg (Shenoy BC et al., crystallized oxalate decarboxylase and its usage, CN 103272225). The daily dose of ALLN-177 is as high as 37,500 enzyme activity units, approximately more than 1 gram of pure enzyme, resulting in very high production costs and a high post-market price. Furthermore, its effect on reducing urinary oxalate is not significant (Craig B. Langman et al., Am J Nephrol, 2016, 44:150-158). The two drugs already on the market for treating hyperoxaluria (Lumasiran and nedosiran) are clinically indicated for the treatment of type 1 primary hyperoxaluria, but are not suitable for the treatment of secondary hyperoxaluria. Moreover, their treatment costs are as high as $300,000 to $500,000 per year, and lifelong medication is required, which is unaffordable for the vast majority of patients.

[0006] Oxalate decarboxylase belongs to the Cupin protein superfamily and is a protein containing Mn2+ The homologous polymerases of the Cupin superfamily catalyze the conversion of oxalate to formic acid and CO2. The metal-binding sites of Cupin superfamily members are typically composed of conserved histidine residues, which act as metal coordination sites in the structure. In oxalate decarboxylases, there are two manganese ion-binding sites, located in the N-terminal and C-terminal cupin domains, respectively. Studies by Victoria et al. have shown that the N-terminal cupin domain is the catalytic active site of oxalate decarboxylases, while the C-terminal cupin domain is purely structural (Biochem.J.(2007)407, 397-406). In oxalate decarboxylases, Mn... 2+ It coordinates with three surrounding histidine (His) residues and one glutamic acid (Glu) residue, which constitute the coordination environment of the manganese ion. Mn 2+ The central tunnel leading to the external solvent is considered the pathway for substrates and products to enter and exit the active site in enzyme-catalyzed reactions. In cupin proteins, this tunnel may be composed of β-sheets and α-helices, which interconnect to form a β-barrel structure, providing a stable coordination environment for metal ions while also allowing the entry and exit of substrates and products. The cupin domain has a typical βαβ structure, where β-sheets and α-helices are interconnected. This structural feature provides a relatively closed environment for the active site while connecting it to the solvent environment through specific tunnels to facilitate catalytic reactions. Previous reports on the catalytic mechanism of oxalate decarboxylase proteins have mainly focused on the N-terminal cupin domain, with limited research on the C-terminal cupin domain, and no studies on the relationship between the tunnels of the C-terminal cupin domain and the persistence of enzyme activity have been found.

[0007] CB6301 oxalate decarboxylase is an oxalate decarboxylase derived from cyanobacterial species. After comprehensive optimization by the inventors' team, this oxalate decarboxylase exhibits significantly higher activity than oxalate decarboxylases derived from Bacillus subtilis (Yvrk, specific activity 30-60 U / mg, native enzymes of ALLN-177 and Oxazyme), reaching up to approximately 200 U / mg or higher. Furthermore, this enzyme possesses excellent acid resistance, with an optimal activity pH of 2.0-2.5, maintaining very good stability and activity even in gastric juice (approximately pH 1.5) during an empty stomach. However, our research team discovered a critical drawback in this oxalate decarboxylase: a short duration of sustained catalytic activity. Approximately 40% of the enzyme loses its catalytic activity within 2-5 minutes of the reaction, and over 90% loses its activity after 5-10 minutes. Therefore, improving and enhancing the sustained catalytic time of this oxalate decarboxylase while maintaining high enzyme activity is a key issue in solving the application of this enzyme in new drug development. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide an oxalate decarboxylase mutant. Compared to wild-type oxalate decarboxylase, the oxalate decarboxylase mutant obtained by the present invention exhibits a significantly reduced inactivation rate during the catalytic process, enhancing the activity of oxalate decarboxylase and prolonging the duration of sustained catalytic action. In in vitro and in vivo pharmacodynamic experiments, the mutant significantly reduces urinary oxalate excretion.

[0009] The inventors summarized and analyzed mutants with positive effects and found that the mutation points that prolong the catalytic time are mostly located in the C-terminal cupin domain of oxalate decarboxylase, and the pore radius of the manganese ions in the C-terminal cupin domain of oxalate decarboxylase to the external solvent is changed.

[0010] Based on the aforementioned rational design principles, the inventors have, for the first time, proposed a protein engineering modification of the pores connecting the manganese ion center of the C-terminal cupin domain of oxalate decarboxylase to the external solvent. By narrowing the bottleneck radius of these pores, the catalytic time and specific activity of oxalate decarboxylase are improved. The inventors extended this rational design / protein engineering strategy to other oxalate decarboxylases with similar characteristics, finding that it is applicable to oxalate decarboxylases from multiple species, achieving very good positive results in all cases. Furthermore, this invention also performed a reverse mutagenesis design on Bacillus subtilis oxalate decarboxylase (Yvrk) (widening the bottleneck radius of the pores connecting the manganese ion center of the C-terminal cupin domain to the external solvent). The results showed that the resulting Yvrk oxalate decarboxylase mutant exhibited a significantly shorter catalytic time compared to the wild-type Yvrk oxalate decarboxylase, demonstrating the universality of this protein engineering strategy.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] In a first aspect, the present invention provides a rational design method to improve the inactivation of oxalate decarboxylase during catalysis. This method reduces the radius of the narrowest point (bottleneck) of the pore leading from the manganese ion active center in the C-terminal cupin domain of the oxalate decarboxylase to the external solvent through protein engineering. The oxalate decarboxylase suitable for the rational design strategy of the present invention must include, but is not limited to, at least one of the following characteristics:

[0013] (1) Oxalate decarboxylase irreversibly and gradually loses its enzyme activity during the catalytic conversion of oxalate substrate to formic acid; (2) The manganese ion in the C-terminal cupin domain of oxalate decarboxylase has six coordination structures. In addition to the four coordination spaces occupied by the enzyme structure itself (3 histidine and 1 glutamic acid residues), the coordination space on the side of the 340th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is open and has the potential for coordination; (3) The bottleneck radius of the pore from the manganese ion active center in the C-terminal domain of oxalate decarboxylase to the solvent is greater than (4) The amino acid at position 340 of the amino acid sequence corresponding to SEQ ID NO: 1 of oxalate decarboxylase is an amino acid other than glutamic acid (Glu).

[0014] In one or more embodiments, the rational design method includes the following steps:

[0015] 1) The three-dimensional structure of wild-type oxalate decarboxylase was constructed using molecular modeling software;

[0016] 2) The pore path from the manganese ion active center in the C-terminal cupin domain of the three-dimensional structure of oxalate decarboxylase obtained in step 1) to the solvent was analyzed using molecular pore analysis software, and the bottleneck radius of the pore was also analyzed.

[0017] 3) Based on the analysis of amino acids along the pore, candidate mutation sites are selected. Taking advantage of the steric hindrance of the side chain of the newly replaced amino acid, the hydrogen bond, salt bond, and hydrophobic interaction formed between the newly replaced amino acid and other amino acids, mutation design is carried out on the amino acids near the pore of the manganese ion active center in the C-terminal domain of oxalate decarboxylase to the external solvent. It is predicted which mutations may reduce the radius of the narrowest part of the pore (bottleneck radius) of the manganese ion active center in the C-terminal cupin domain to the solvent, and candidate oxalate decarboxylase mutants are obtained.

[0018] 4) Using molecular modeling software, construct the three-dimensional structure of the oxalate decarboxylase mutant obtained in step 3);

[0019] 5) The bottleneck radius of the pores leading to the external solvent in the manganese ion active center in the C-terminal cupin domain of the candidate oxalate decarboxylase mutants in step 4) was analyzed using molecular pore analysis software. Mutants with a smaller bottleneck radius of pores compared to the wild-type oxalate decarboxylase measured in step 2) were retained.

[0020] 6) The candidate oxalate decarboxylase mutant identified in step 5) was obtained by site-directed amino acid mutation of the original oxalate decarboxylase, and the mutant was transformed into a host cell to obtain a strain containing the target gene. The mutant was then cultured, expressed, extracted, and purified to obtain the oxalate decarboxylase mutant.

[0021] 7) Verify whether the oxalate decarboxylase mutant obtained in step 6) improves the inactivation problem of oxalate decarboxylase during catalysis. For the screened beneficial mutants, further superimposed mutations can be performed to increase the advantages of the mutants.

[0022] In step 1), the molecular modeling software is selected from phyre2, Swiss model, RoseTTAfold, RaptorX, MODELLER, and AlphaFold software, with AlphaFold software being preferred.

[0023] In step 2), the molecular pore analysis software used is selected from, but not limited to, Caver, MOLE, Molaxis, Critical comparison, Hole, Chunnel, Porewalker, Hollow, and 3V software; Caver software is preferred.

[0024] Secondly, the present invention provides an oxalate decarboxylase mutant, wherein the bottleneck radius of the pore leading to the solvent from the manganese ion active center in the C-terminal cupin domain of the oxalate decarboxylase mutant is smaller than that of its wild-type oxalate decarboxylase, resulting in a significantly reduced inactivation rate during the catalytic process compared to its wild-type oxalate decarboxylase, and a longer effective catalytic duration.

[0025] In one or more embodiments, the oxalate decarboxylase mutant is selected from any of the following:

[0026] (a) Its amino acid sequence is derived from the sequence shown in SEQ ID NO: 1 by mutation at one or more amino acid residue sites selected from the group consisting of: position 61, position 211, position 232, position 250, position 252, position 267, position 269, position 340, position 349, position 351, and / or position 384; and compared with the oxalate decarboxylase shown in SEQ ID NO: 1, the enzyme has a significantly reduced inactivation rate during catalysis and a prolonged effective catalytic duration;

[0027] or

[0028] (b) The oxalate decarboxylase mutant has 80%, preferably 90%, preferably 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence described in (a), and has the function of the oxalate decarboxylase mutant described in (a), wherein any amino residue or combination corresponding to position 61, 211, 232, 250, 252, 267, 269, 340, 349, 351, and / or 384 of the amino acid sequence shown in SEQ ID NO: 1 is the same as that in the amino acid sequence described in (a);

[0029] or

[0030] (c) The oxalate decarboxylase mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the oxalate decarboxylase mutant described in (a), wherein any amino residue or combination corresponding to position 61, 211, 232, 250, 252, 267, 269, 340, 349, 351, and / or 384 of the amino acid sequence shown in SEQ ID NO: 1 is the same as that in the amino acid sequence described in (a).

[0031] According to an embodiment of the present invention, the amino acid sequence of the oxalate decarboxylase mutant is mutated at one or more sites selected from the group consisting of the following amino acid residues:

[0032] At position 61, aspartic acid is replaced by tyrosine.

[0033] At position 211, glutamic acid is replaced by proline;

[0034] At position 232, tyrosine is replaced by phenylalanine;

[0035] At position 250, leucine is replaced by glutamic acid, tyrosine, tryptophan, methionine, or glutamine.

[0036] At position 252, leucine is replaced by valine and isoleucine.

[0037] At position 267, alanine is replaced by valine, isoleucine, methionine, leucine, or glutamine.

[0038] At position 340, isoleucine is replaced by leucine, phenylalanine, glutamic acid or methionine.

[0039] At position 349, serine is replaced by aspartic acid;

[0040] At position 351, aspartic acid is replaced by serine.

[0041] At position 384, isoleucine is replaced by methionine, phenylalanine, arginine, lysine, glutamine, tryptophan, or glutamic acid.

[0042] In a specific embodiment, the mutant of the oxalate decarboxylase is one containing I340L, A267V, A267I, I384M, I384F, L269V, I340L-I384M, I340L-I384F, I340L-S349D-D351S, I340L-S349D-D351S-L269A, A267I-L250M, A267I-L250I, A267I-L252V, A267I-L252I, A267I-I340L, L250Y, L250W, L250M, I384R, I340L-A267M, I340L-L250E-S349D-D351S, A26 Any one of the following mutations, or a combination containing any two or more mutations: 7M, I340L-L250E-A267M-I384F, A267L, A267I-L250M-I384F, A267I-L250M-I384R, A267I-I340A, A267I-I340V, A267I-L250M-L252V, A267I-L250M-L252I, A267I-L250M-I340A, A267I-L250M-I340V, L250Q, A267I-L250Q, A267V-L250Q, A267L-L250Q, I384K, I384Q, I384W, and I384E.

[0043] In a specific embodiment, the amino acid sequence of the oxalate decarboxylase mutant is shown in any one of SEQ ID NO: 3-25.

[0044] In one or more embodiments, the oxalate decarboxylase mutant is selected from any of the following:

[0045] (d) Its amino acid sequence is derived from the sequence shown in SEQ ID NO: 29-32 by mutation, and is selected from any of the following: A270I mutant and A270L mutant of SEQ ID NO: 29; G266I mutant, V339L mutant, and V339I mutant of SEQ ID NO: 30; A267I mutant, A267L mutant, and I340L mutant of SEQ ID NO: 31; S226L mutant, I299L mutant, and S226I mutant of SEQ ID NO: 32; and has a prolonged effective catalytic time compared with the oxalate decarboxylase shown in SEQ ID NO: 29-32;

[0046] or

[0047] (e) The oxalate decarboxylase mutant has 90%, preferably 95%, more preferably 98%, and more preferably 99% sequence identity with the amino acid sequence described in (d), and has the function of the oxalate decarboxylase mutant described in (d), wherein the amino residues corresponding to position 270 of the amino acid sequence shown in SEQ ID NO: 29, position 266 or 339 of the amino acid sequence shown in SEQ ID NO: 30, position 267 or 340 of the amino acid sequence shown in SEQ ID NO: 31, and position 226 or 299 of the amino acid sequence shown in SEQ ID NO: 32 are identical to those in the amino acid sequence described in (d);

[0048] or

[0049] (f) The oxalate decarboxylase mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (d), and has the function of the oxalate decarboxylase mutant described in (d), wherein the amino residues corresponding to position 270 of the amino acid sequence shown in SEQ ID NO: 29, position 266 or 339 of the amino acid sequence shown in SEQ ID NO: 30, position 267 or 340 of the amino acid sequence shown in SEQ ID NO: 31, and position 226 or 299 of the amino acid sequence shown in SEQ ID NO: 32 are the same as those in the amino acid sequence described in (d).

[0050] Thirdly, the present invention provides a polynucleotide encoding the oxalate decarboxylase mutant described in the second aspect.

[0051] Fourthly, the present invention provides an expression vector comprising the polynucleotide described in the third aspect of the present invention.

[0052] Fifthly, the present invention provides a host cell comprising the expression vector described in the fourth aspect of the present invention or a polynucleotide whose genome integrates the oxalate decarboxylase mutant described in the third aspect of the present invention.

[0053] In one or more embodiments, the host cell is a bacterium; preferably, the host cell is *Escherichia coli*, *Bacillus*, or *Corynebacterium*; more preferably, the host cell is *Escherichia coli*.

[0054] In a sixth aspect, the present invention provides a composition comprising the oxalate decarboxylase mutant described in the second aspect of the present invention.

[0055] In one or more embodiments, the composition further comprises an excipient.

[0056] According to embodiments of the present invention, the excipient may be a diluent, filler, binder, disintegrant, lubricant, solvent, or other food- or pharmaceutically acceptable excipient.

[0057] In specific embodiments, the composition may be a solution, suspension, emulsion, powder, lozenge, pill, syrup, lozenge, tablet, chewing gum, concentrate, capsule, or other food- or pharmaceutically acceptable dosage form.

[0058] In a seventh aspect, the present invention provides the use of the oxalate decarboxylase mutant described in the second aspect, the polynucleotide sequence described in the third aspect, the expression vector described in the fourth aspect, the host cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of medicaments for the prevention, treatment, or relief of hyperoxaluria / hyperoxalemia caused by oxalate abnormalities and diseases related to calcium oxalate stones. In one or more embodiments, the diseases related to calcium oxalate stones are selected from, but are not limited to, kidney stones, ureteral stones, bladder stones, urethral stones, nephrocalcinosis, renal colic, hematuria, nephritis and chronic kidney disease caused by calcium oxalate deposition, and renal failure.

[0059] In an eighth aspect, the present invention provides a method for preventing, treating or alleviating hyperoxaluria, hyperoxalemia and calcium oxalate-related diseases caused by abnormal oxalate levels, comprising administering to a subject the composition described in the sixth aspect of the present invention or the drug described in the seventh aspect of the present invention.

[0060] In one or more embodiments, the calcium oxalate stone-related diseases are selected from, but not limited to, kidney stones, ureteral stones, bladder stones, urethral stones, nephrocalcinosis, renal colic, hematuria, nephritis and chronic kidney disease caused by calcium oxalate deposition, and renal failure.

[0061] In a ninth aspect, the present invention provides a highly active oxalate decarboxylase, with a specific activity ≥70 U / mg protein at 37°C and 10 mM oxalate substrate; preferably, the specific activity is ≥80 U / mg protein, ≥90 U / mg protein, ≥100 U / mg protein, or ≥110 U / mg protein; more preferably, the specific activity is ≥120 U / mg protein.

[0062] The composition contains an average Mn ion content of oxalate decarboxylase of ≥1.0 ions / subunit, or a molar ratio of Mn ions to oxalate decarboxylase protein of ≥3:1.

[0063] In a specific implementation, the amino acid sequence of the highly active oxalate decarboxylase is as shown in SEQ ID NO: 2, and the oxalate decarboxylase, its mutants or homologs, or mutants of the above homologs; preferably, the variants or homologs have at least 80%, preferably 90%, more preferably 95%, most preferably 98% or 99% sequence identity with SEQ ID NO: 2, and have oxalate degrading enzyme activity.

[0064] In a specific implementation, the amino acid sequence of the highly active oxalate decarboxylase is as shown in any one of SEQ ID NO: 2-33, or is the A270I mutant or A270L mutant of SEQ ID NO: 29; the G266I mutant, V339L mutant, or V339I mutant of SEQ ID NO: 30; the A267I mutant, A267L mutant, or I340L mutant of SEQ ID NO: 31; or the S226L mutant, I299L mutant, or S226I mutant of SEQ ID NO: 32.

[0065] In a specific implementation, the manganese ion content of the highly active oxalate decarboxylase is ≥1.0 ions / subunit; preferably, ≥1.1 ions / subunit, ≥1.2 ions / subunit, ≥1.3 ions / subunit, ≥1.4 ions / subunit, or ≥1.5 ions / subunit; and / or, the molar ratio of Mn ions to oxalate decarboxylase protein is ≥3:1, preferably ≥3.3, ≥3.6, ≥3.9, ≥4.2, or ≥4.5.

[0066] In a specific implementation, the iron ion content of the highly active oxalate decarboxylase is ≤0.2 ions / subunit; preferably, ≤0.1 ions / subunit, more preferably ≤0.05 ions / subunit.

[0067] In a specific implementation, the nickel ion content of the highly active oxalate decarboxylase is ≤0.1 ions / subunit; preferably, ≤0.05 ions / subunit, more preferably ≤0.01 ions / subunit, and most preferably below the detection limit; and / or, the zinc ion content of the oxalate decarboxylase is ≤0.2 ions / subunit; preferably, ≤0.1 ions / subunit, more preferably ≤0.05 ions / subunit, and most preferably ≤0.02 ions / subunit.

[0068] In a specific implementation scheme, when the highly active oxalate decarboxylase is analyzed by anion exchange chromatography, the area of ​​the main peak preferentially eluted accounts for ≥45%. The conditions for the anion exchange chromatography analysis are: Proteomix SAX-NP5 (250mm × 4.6mm, 5μm); mobile phase A is 20mM CAPSO buffer containing 20mM NaCl, pH 9.8; mobile phase B is 20mM CAPSO buffer containing 300mM NaCl, pH 9.8; and the detection wavelength is 280nm.

[0069] In a tenth aspect, the present invention provides a method for preparing a highly active oxalate decarboxylase, wherein a sample containing oxalate decarboxylase is incubated in an alkaline buffer solution at pH 8.0-10.5 for at least 2 hours, preferably at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours, preferably at pH 8.0-10.0; the sample containing oxalate decarboxylase is a purified oxalate decarboxylase solution, an unpurified oxalate decarboxylase feed solution, or a cell lysate supernatant containing oxalate decarboxylase.

[0070] In a specific implementation scheme, the method for preparing oxalate decarboxylase includes a buffer solution containing, but not limited to, any one of carbonate, bicarbonate, arginine, Tris, lysine, histidine, or borate, or any combination of two or more of these components.

[0071] Eleventhly, the present invention provides an expression vector or combination thereof, the expression vector or combination comprising a polynucleotide encoding oxalate decarboxylase as shown in SEQ ID NO: 2-33, and a variant or homolog thereof, and further comprising a polynucleotide encoding the manganese ion transporter MntS; preferably, the polynucleotide encoding oxalate decarboxylase and the polynucleotide encoding the manganese ion transporter MntS are located in the same vector or different vectors.

[0072] In a specific implementation, the expression vector or combination, and the promoter of the manganese ion transporter gene, are inducible promoters or constitutive promoters.

[0073] In specific implementations, the expression vector or combination further comprises a polynucleotide encoding a molecular chaperone protein that helps with protein folding;

[0074] Preferably, the molecular chaperone protein is groEL-groES;

[0075] Preferably, the promoter of the molecular chaperone protein gene is an inducible promoter or a constitutive promoter.

[0076] In a twelfth aspect, the present invention provides a host cell comprising the plasmid expression vector or combination described in the eleventh aspect of the present invention; or having its genome integrated with a polynucleotide encoding oxalate decarboxylase as described in the first to eleventh aspects of the present invention and / or a polynucleotide encoding manganese ion transporter MntS and / or a molecular chaperone protein.

[0077] Preferably, the host cell contains two copies of a polynucleotide encoding the manganese ion transporter MntS.

[0078] Preferably, the host cell is a bacterium; more preferably, the host cell is Escherichia coli, Bacillus, or Corynebacterium; even more preferably, the host cell is Escherichia coli.

[0079] In a thirteenth aspect, the present invention provides a fermentation production method for highly active oxalate decarboxylase, characterized in that it includes the steps of culturing a recombinant host cell containing an oxalate decarboxylase encoding gene and expressing oxalate decarboxylase, wherein the nickel content in the culture medium used is ≤0.2mM; preferably, ≤0.1mM, ≤0.05mM; more preferably, it is nickel-free.

[0080] In a specific implementation scheme, the fermentation production method of the highly active oxalate decarboxylase further includes an induced fermentation step, characterized in that the induction pH range is 5.5–6.4; preferably, 5.8–6.2; more preferably, 5.9–6.1, and / or the inducer is lactose.

[0081] In the fourteenth aspect, the oxalate decarboxylase obtained by the production method or combination of the production methods described in any one of the tenth and thirteenth aspects of the present invention, wherein the oxalate decarboxylase has a specific activity of ≥70 U / mg protein at 37°C and 10 mM oxalate substrate, preferably ≥80 U / mg protein, ≥90 U / mg protein, ≥100 U / mg protein, ≥110 U / mg protein, or ≥120 U / mg protein.

[0082] By adopting the above technical solution, the present invention has achieved significant beneficial effects:

[0083] To address the challenge of rapid inactivation of oxalate decarboxylase during catalysis, leading to a short effective catalytic time, this invention proposes, for the first time, protein engineering modification of the manganese ion active center pore of the C-terminal cupin domain of oxalate decarboxylase. By narrowing the radius of the narrowest point of the pore leading from the manganese ion center to the solvent (the bottleneck radius), the effective catalytic time and specific activity of oxalate decarboxylase are improved. Through a series of variant screening and optimization, this invention yielded several mutants of oxalate decarboxylase, whose inactivation rate during catalysis was significantly lower than that of the wild-type protein. Some mutants showed no significant inactivation within a 30-minute test catalytic time, thus enhancing the activity of oxalate decarboxylase and prolonging the catalytic reaction time. In in vitro and in vivo pharmacodynamic experiments, the mutants significantly reduced urinary oxalate excretion.

[0084] This invention extends the rational design / protein engineering strategy explored to other oxalate decarboxylases with similar characteristics, finding that this strategy is applicable to oxalate decarboxylases from multiple species, achieving very good and beneficial results. Therefore, for the first time, a rational molecular design and protein engineering strategy for optimizing and improving the catalytic duration of oxalate decarboxylases is proposed.

[0085] The oxalate decarboxylase of this invention has significant advantages over Bacillus subtilis-derived oxalate decarboxylases (the original enzymes of drugs such as ALLN-177 and Oxazyme) currently undergoing clinical trials. Its specific activity is 5-10 times higher, and its gastric acid stability is 2-2.5 pH lower. The optimal enzyme activity pH is 2.0-2.5, which is 1.5-2 pH lower than ALLN-177 (optimal pH approximately 4.0-4.2). It has demonstrated significant advantages in both in vitro and in vivo animal pharmacodynamic studies. It is expected to significantly reduce the dosage of oxalate decarboxylase in clinical practice, leading to better treatment outcomes for patients while improving treatment experience and compliance. It also reduces the economic burden of treatment for patients with gallstones and alleviates pressure on medical insurance, thus having immense value for public health and society.

[0086] To address the problem of low oxalate decarboxylase activity, this invention has developed a stable production process for high-activity oxalate decarboxylase by modifying the production strain, optimizing the fermentation medium and fermentation control conditions, and improving post-treatment conditions. This high-activity oxalate decarboxylase exhibits a specific activity that is 50% or more higher than that of the enzyme before optimization. Attached Figure Description

[0087] Figure 1 shows a schematic diagram of the recombinant expression plasmid for oxalate decarboxylase (pSGEL-cb6301-D29).

[0088] Figure 2 shows the average enzyme activity of wild-type cb6301 oxalate decarboxylase at different catalytic times.

[0089] Figure 3 shows the amino acid sequence alignment of cb6301 and Yvrk oxalate decarboxylase.

[0090] Figure 4 shows a structural comparison of cb6301 and Yvrk oxalate decarboxylase (the color changes from dark blue to dark red from the N-terminus to the C-terminus);

[0091] A: Single subunit structure of cb6301 oxalate decarboxylase; B: Single subunit structure of Yvrk oxalate decarboxylase; C: Overlay diagram of cb6301 and Yvrk oxalate decarboxylase subunits (dashed boxes indicate N-terminal comparison); D: Multi-subunit structure of cb6301 oxalate decarboxylase; E: Multi-subunit structure of Yvrk oxalate decarboxylase.

[0092] Figure 5 shows the structure of the N-terminal Mn ion site of cb6301 oxalate decarboxylase (left) and the site conservation analysis diagram (right).

[0093] Figure 6 shows a comparison of the N-terminal cupin domain of cb6301 and Yvrk oxalate decarboxylase.

[0094] Figure 7 shows a comparison of the C-terminal cupin domains of cb6301 and Yvrk oxalate decarboxylase.

[0095] Figure 8 shows the structure of the C-terminal Mn ion site of cb6301 oxalate decarboxylase (left) and the site conservation analysis diagram (right).

[0096] Figure 9 shows a schematic diagram of the C-port bag structure of oxalate decarboxylase derived from Bacillus subtilis.

[0097] Figure 10 shows the structure and pore diagram of the C-terminal Mn ion site of cb6301 oxalate decarboxylase;

[0098] The narrowest radii of the blue and green channels are 1.38 and 1.38, respectively.

[0099] Figure 11. SDS-PAGE gel image of cb6301 oxalate decarboxylase mutant and its superimposed Y232F.

[0100] Figure 12 shows the correlation analysis between the enzyme activity persistence of the oxalate decarboxylase mutant and the tunnel pore size of the C-terminal domain.

[0101] Figure 13 shows a diagram of conserved amino acid composition around the Mn ion in the C-terminal cupin domain of oxalate decarboxylase derived from cyanobacteria.

[0102] Figure 14 shows a clustering network diagram of cb6301 oxalate decarboxylase sequence similarity (non-glutamic acid at position 340) screened from the UniProt database.

[0103] Figure 15. Sequence comparison of cb6301 oxalate decarboxylase with other similar oxalate decarboxylases.

[0104] Figure 16 shows the oxalate-lowering effect of cb6301 oxalate decarboxylase and its mutants.

[0105] The top image shows 24-hour urinary oxalate levels, and the bottom image shows 24-hour fecal oxalate levels.

[0106] Figure 17 shows a schematic diagram of the pET-28a-cb6301-D29 plasmid.

[0107] Figure 18 shows a schematic diagram of the pGEL-cb6301-D29 plasmid.

[0108] Figure 19 shows a schematic diagram of the pMMTS-cb6301-D29 plasmid.

[0109] Figure 20 shows a schematic diagram of the pET-28a-MntS-cb6301-D29 plasmid.

[0110] Figure 21 shows a comparison of the effects of different zinc and nickel ion concentrations in the culture medium on the expression and specific activity of oxalate decarboxylase.

[0111] A: Comparison of biomass; B: Comparison of enzyme activity per unit cell; C: Comparison of specific enzyme activities.

[0112] Figure 22 shows a typical detection spectrum for IEC detection of oxalate decarboxylase. Detailed Implementation

[0113] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0114] Definitions and explanations in this patent:

[0115] (1) Oxalate decarboxylase: Oxalate decarboxylase is an enzyme that converts oxalic acid into carbon dioxide and formic acid.

[0116] (2) The channel of manganese ion active center in the C-terminal cupin domain of oxalate decarboxylase to the external solvent: Taking cb6301 oxalate decarboxylase as an example, the channel of manganese ion center in the C-terminal cupin domain to the external solvent is the channel of manganese ion (Mn)-I340-A267-L250-solvent or Mn-I340-A267-L250-I384-solvent.

[0117] (3) Bottleneck radius: refers to the radius of the narrowest part of the tunnel.

[0118] (4) Enzyme activity unit (U): The amount of enzyme required to degrade 1 micromolar oxalate per minute at 37°C.

[0119] (5) Average enzyme activity: refers to the ratio of the total enzyme activity calculated from the amount of substrate converted by the enzyme over a specified period of time to the corresponding time.

[0120] (6) Specific enzyme activity: The number of enzyme activity units per unit weight (mg) of protein under specific conditions.

[0121] (7) Hyperoxaluria, hyperoxalemia and diseases related to calcium oxalate stones caused by abnormal oxalate: including but not limited to kidney stones, ureteral stones, bladder stones, urethral stones, nephrocalcinosis, renal colic, hematuria, nephritis and chronic kidney disease and renal failure caused by calcium oxalate deposition.

[0122] (8) Subject: usually refers to the person who is tested with the test product, and in this patent it mainly refers to the test person who is treated with the composition of the present invention.

[0123] (9) Application: refers to intervention and treatment according to a specific procedure. In this patent, it mainly refers to oral treatment.

[0124] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0125] Example 1: Gene acquisition, recombinant expression, and enzymatic characterization of oxalate decarboxylase

[0126] Based on the oxalate decarboxylase gene information in the NCBI database (Genbank: BAD79907.1), SignalP 6.0 software analysis revealed that amino acids 1-30 of the N-terminus of cb6301 oxalate decarboxylase (SEQ ID NO: 1) are the signal peptide, and amino acids 31-389 are the mature peptide. Based on the sequence (cb6301-D29, SEQ ID NO: 2) with the signal peptide removed and the start codon retained, primers for amplifying the target gene were designed (Table 1). Using pSGEL plasmid as a template, the MntS expression framework was amplified using the 22b-Ms-F / pSGEL-R primer combination. Using genomic DNA from cyanobacterial PCC6301 as a template, the cb6301-D29 target gene sequence was amplified using the cb6301-D29-F / cb6301-D29-R primer combination. The target gene sequence was combined with the MntS gene fragment for overlap PCR amplification to obtain the MntS+OxDC fusion sequence, and the purified fusion gene fragment was obtained by agarose gel extraction.

[0127] Table 1. Primers for amplification of the cb6301 oxalate decarboxylase gene

[0128] Using pSGEL expression vector DNA as a template, a linearized vector was obtained by PCR amplification of the whole plasmid using the pSGEL-F / pET22b-R2 primer combination. After digestion of the template plasmid with the restriction endonuclease DpnI, the linearized plasmid was recovered by agarose gel electrophoresis. A seamless cloning method was used to insert the fusion target gene fragment into the expression vector pSGEL, which was then transformed into E. coli DH5α competent cells. The cells were plated on LB agar plates containing kanamycin, and single colonies were picked to screen for positive clones. After confirmation by DNA sequencing, the positive bacterial culture was expanded and the plasmid was extracted to obtain the recombinant plasmid pSGEL-cb6301-D29. A schematic diagram of the recombinant plasmid is shown in Figure 1. The recombinant plasmid was heat-shocked into the expression host E. coli BL21(DE3) to obtain the recombinant expression strain BL21(DE3)(pSGEL-cb6301-D29).

[0129] The recombinant expression strain [E. coli BL21(DE3)(pSGEL-cb6301-D29)] was streaked onto LB-Agar agar plates containing 50 μg / ml kanamycin (Kan) and incubated overnight at 37°C inverted mode. Single colonies were picked from the streaked plates and activated in 50 mL of LB liquid medium containing 50 μg / ml Kan antibiotic, and incubated overnight at 37°C and 200 rpm. The activated bacterial culture was then transferred at a 1% ratio to JL liquid medium containing 50 μg / ml Kan antibiotic (Yeast Extract 0.5% (w / v), Tryptone 2% (w / v), KH2PO4 10 mM, (NH4)2SO4 25 mM, Mannitol 2% (w / v), Sodium Succinate 20 mM, MgSO4 0.5 mM), and incubated at 37°C and 200 rpm until OD500. 600nm =1.2-1.5, add 2% lactose and 5mM MnCl2 to a final concentration, and induce overnight at 200rpm for 25s. Centrifuge the inducing culture overnight to collect the cells, wash with sterile water, and then resuspend in 50mM arginine buffer (pH 9.5) to a cell density OD of 1.2-1.5. 600 =20, sonicated. Centrifuge to collect the supernatant. While stirring, slowly add 2 mol / L phosphate solution to adjust the pH to 3.0. Centrifuge again to collect the supernatant, and load it onto a CM Sepharose column for protein purification. Collect the target protein peak, desalt it using a 30 kDa ultrafiltration tube to obtain the oxalate decarboxylase sample for subsequent enzymatic property analysis.

[0130] The method for detecting the average enzyme activity of oxalate decarboxylase is as follows: 1.0 mL of 10 mM oxalate solution (containing 25 mM citrate buffer, pH 3.0) was preheated at 374°C for 10 minutes. Then, 20 μL of a solution containing oxalate decarboxylase (the enzyme protein concentration can be adjusted according to the enzyme activity) was added to initiate the reaction. After 10 minutes of reaction, 50 μL of 15% (v / v) sulfuric acid solution was added to inactivate the enzyme and terminate the reaction. The mixture was rapidly centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. The residual oxalate concentration was determined by HPLC. The HPLC test conditions were as follows: Sepax Carbomix H-NP 10 column; 0.015% (v / v) sulfuric acid solution; flow rate 0.6 mL / min; column temperature 55 s; detection wavelength 210 nm. Based on the residual oxalate concentration determined by HPLC, the oxalate consumption (or formic acid production) and average enzyme activity were calculated. One unit of enzyme activity (U) is defined as the amount of enzyme required to degrade one micromolar of oxalate per minute at 374°C.

[0131] The method for persistently detecting the activity of oxalate decarboxylase is as follows: 1.0 mL of 50 mM oxalate solution (containing 25 mM citrate buffer, pH 3.0) was preheated at 37°C for 10 minutes. Then, 20 μL of a solution containing oxalate decarboxylase (the enzyme protein concentration can be adjusted according to the enzyme activity) was added to initiate the reaction. After reacting for 2-60 minutes, 50 μL of 15% (v / v) sulfuric acid solution was added to inactivate the enzyme and terminate the reaction. The mixture was rapidly centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. The residual oxalate concentration was determined by HPLC. The HPLC test conditions were as follows: Sepax Carbomix H-NP 10 column; 0.015% (v / v) sulfuric acid solution; flow rate 0.6 mL / min; column temperature 55°C; detection wavelength 210 nm. The average enzyme activity was calculated based on the residual oxalate concentration or the formic acid concentration generated after the reaction, as determined by HPLC. One unit of enzyme activity (U) is defined as the amount of enzyme required to degrade one micromole of oxalate per minute at 37°C.

[0132] The optimal pH for determining the enzyme activity of oxalate decarboxylase is as follows: Prepare oxalate-containing test reaction solutions (final oxalate concentration 5 mM) with pH values ​​ranging from 1.5 to 7.0. After preheating 1 mL of the reaction solution at 37°C for 10 minutes, add 20 μL (the enzyme protein concentration can be adjusted appropriately according to enzyme activity) of oxalate decarboxylase solution to initiate the reaction. After 5 minutes of reaction, add 50 μL of 15% (v / v) sulfuric acid solution to terminate the reaction. Centrifuge quickly and collect the supernatant. Filter through a 0.22 μm filter membrane and determine the residual oxalate concentration using HPLC. Based on the residual oxalate concentration or formic acid concentration generated after the reaction as determined by HPLC, calculate the specific activity of oxalate decarboxylase at different pH values ​​(at 37°C). The pH at which the maximum specific activity is observed is defined as the optimal pH, and the enzyme activity at the optimal pH is defined as the optimal specific activity. Test the oxalate decarboxylase activity at the optimal pH, setting the reaction time to 2 minutes. The maximum enzyme activity measured is defined as the initial specific activity.

[0133] Using the above-mentioned method for testing the persistence of enzyme activity, the average specific enzyme activity of CB6301 oxalate decarboxylase at different catalytic reaction times was tested. The results are shown in Table 2, and the average enzyme activity is shown in Figure 2. Simultaneously, based on the amount of formic acid generated at different reaction times, the average enzyme activity for different time periods was calculated (for example: in this enzyme activity persistence test, the concentration of CB6301 oxalate decarboxylase was 0.2 mg / mL. As shown in Table 2, CB6301 oxalate decarboxylase generated 1.44 mM of formic acid in the first 2 minutes of the reaction, so the average specific enzyme activity of oxalate decarboxylase = (1.44 mM × 1.07 mL) / (0.2 mg / mL × 0.02 mL × 2 min) = 192.6 U / mg; in the first 5 minutes, 2.85 mM of formic acid was generated, so the amount of formic acid generated in the 2-5 minute time period was 2.85 - 1.44 = 1.41 mM, and the average enzyme activity in the 2-5 minute time period was calculated to be 125.7 U / mg, and so on).

[0134] Table 2. Enzyme activity of CB6301 oxalate decarboxylase at different reaction times Note: *When the oxalate decarboxylase activity result is negative, it is uniformly marked as 0.

[0135] The results (Table 2 and Figure 2) showed that the activity of CB6301 oxalate decarboxylase decreased to below 70% of its initial activity after 2-5 min of reaction, and to below 20% of its initial activity after 5-10 min of reaction. At 60 min and 120 min of reaction, the amount of formic acid produced did not increase compared to 30 min of reaction, indicating that the oxalate decarboxylase was completely inactivated at 30 min.

[0136] Example 2: Protein engineering to enhance the structural stability between oxalate decarboxylase subunits

[0137] As described in Example 1, the inventors discovered during their research that the activity of cb6301 oxalate decarboxylase decreased sharply with prolonged catalytic time, accompanied by the simultaneous formation of flocculent protein precipitation. Furthermore, previous studies by our research group showed that cb6301 oxalate decarboxylase is a homologous trisubunit polymerase, as determined by both size exclusion chromatography-HPLC and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS). This differs from the previously reported homologous hexadecimaltose decarboxylase of Bacillus subtilis (Anand, R. (2002) Biochemistry 41: 7659-7669). Based on these two findings, the inventors analyzed that the unstable trisubunit structure of this enzyme may lead to subunit depolymerization due to high-frequency molecular vibrations during catalysis. Therefore, the plan is to improve the intersubunit interactions of this enzyme through protein engineering.

[0138] Since the oxalate decarboxylase (Yvrk) from Bacillus subtilis is well-studied, and several research groups have resolved its protein crystal structure (Anand, R. (2002) Biochemistry 41: 7659-7669; Just, VJ, (2004) J Biol Chem 279: 19867), this invention uses AlphaFold 3 software to model the 3D protein structure of cb6301 oxalate decarboxylase using the mature peptide sequence (SEQ ID NO: 2). The constructed cb6301 oxalate decarboxylase protein model is then compared with the crystal structure of Bacillus subtilis (PDB: 1UW8). Analysis showed that cb6301 oxalate decarboxylase is similar to 1UW8 in the main structural domain of its single subunit, but lacks the "claw" hydrophobic hinge region at the N-terminus for inter-subunit interactions, which is presumably the main reason for its failure to form a hexamer (Figures 3 and 4). Therefore, by adding the N-terminal "claw" hydrophobic hinge region, it is hoped that the protein's stability can be increased and the protein's inactivation and denaturation during catalysis can be improved.

[0139] By chimerizing the N-terminal sequence (SEQ ID NO: 26) of oxalate decarboxylase (Yvrk) from Bacillus subtilis and the N-terminal sequences (SEQ ID NO: 27 and 28) of oxalate decarboxylase from Agrocybe aegerita, and performing recombinant expression, it was verified whether this could improve the short catalytic time of cb6301 oxalate decarboxylase. The recombinant expression method is the same as described in Example 1.

[0140] Method for detecting the persistence of enzyme activity in screening mutants: The collected bacterial sludge expressing the recombinant oxalate decarboxylase mutant was resuspended in arginine buffer solution to OD. 600=20. After ultrasonic disruption, centrifuge and collect the supernatant. Dilute with sterile water to an appropriate ratio for detection. Add 1.0 mL of 10 mM oxalate solution (containing 25 mM citrate buffer, pH 3.0) to 37°C for 10 minutes. Then add 20 μL (the enzyme protein concentration can be adjusted according to enzyme activity) of oxalate decarboxylase solution, mix well, and incubate at 37°C and 800 rpm for a constant temperature shaker to start the reaction. After reacting for 5 minutes and 30 minutes respectively, add 50 μL of 15% (v / v) sulfuric acid solution to inactivate the enzyme and terminate the reaction. Centrifuge quickly and collect the supernatant. Filter through a 0.22 μm filter membrane and use an oxalate assay kit to determine the residual oxalate concentration and calculate the average enzyme activity (U / L). The enzyme activity of the cb6301 oxalate decarboxylase mutant was assessed by comparing the average enzyme activity ratio after 30 minutes and after 5 minutes of reaction. This was used to evaluate whether the enzyme activity of the cb6301 oxalate decarboxylase mutant was improved compared to that of the wild-type cb6301 oxalate decarboxylase.

[0141] Table 3. Results of enzyme activity persistence in mutants with chimeric N-terminal sequences Note: U / L refers to OD. 600 The bacterial resuspension with a concentration of 20 was disrupted by sonication, and the enzyme activity of all total proteins in the supernatant was measured by centrifugation.

[0142] Meanwhile, the inventors also specifically mutated the hydrophobic region between the subunits of cb6301 oxalate decarboxylase to enhance the interaction between subunits, in order to increase the stability of the enzyme between subunits during catalysis. The specific results are shown in Table 4 below.

[0143] Table 4. Results of enzyme activity persistence in mutants that enhance the interaction between subunits of the CB6301 oxalate decarboxylase protein. Note: U / L refers to OD. 600 The bacterial resuspension with a concentration of 20 was disrupted by sonication, and the enzyme activity of all total proteins in the supernatant was measured by centrifugation.

[0144] The results (Tables 3 and 4) showed that while the cb6301 oxalate decarboxylase protein exhibited oxalate decarboxylase activity after being chimeric with the N-terminal hinge region peptides of Bacillus subtilis oxalate decarboxylase (Yvrk) and Agrocybe aegypti oxalate decarboxylase (A2), no significant sustained improvement in enzyme activity was observed. Among the mutants designed with a rational strategy to enhance inter-subunit stability, the D61Y and Y232F mutants showed some improvement, but the effect was weak. Unexpectedly, we found that the E211P and Y232F mutants increased protein expression levels and the proportion of soluble protein, resulting in a significant increase in enzyme activity in the cell lysate, suggesting that enhancing enzyme stability is helpful.

[0145] Example 3: Analysis and verification of the active pocket region of oxalate decarboxylase through protein engineering

[0146] The study by Victoria et al. on oxalate decarboxylase (Yvrk) from Bacillus subtilis (Victoria, et al.; Biochem. J. (2007) 407, 397406) showed that the N-terminal cupin domain of oxalate decarboxylase is the active catalytic region of the enzyme, while the C-terminal cupin domain is a non-catalytic, purely structural region. Furthermore, the inventors conducted a sustained enzyme activity assay on the Yvrk oxalate decarboxylase, and the results showed that the enzyme did not exhibit inactivation during catalysis. Therefore, the inventors used AlphaFold 3 software to model the 3D protein structure of oxalate decarboxylase using the mature peptide sequence (SEQ ID NO: 2) of cb6301 oxalate decarboxylase. The model was compared with the crystal structure of Yvrk oxalate decarboxylase from Bacillus subtilis (PDB: 1UW8). Each single subunit of the cb6301 oxalate decarboxylase protein has two Mn metal ion binding regions, with the N-terminal cupin domain surrounding the Mn ions. The residues within the range were not significantly different from those of Bacillus subtilis oxalate decarboxylase (Figure 5). Simultaneously, the conservation of catalytically relevant residues at the N-terminal Mn ion binding pocket of all oxalate decarboxylase proteins in the Unipro family (ID IPR017774) retrieved from the Unipro database was analyzed using weblogo3 software. All residues were found to be conserved (Figure 5), consistent with its role as a catalytic center. The inventors further compared the N-terminal cupin domain of cb6301 oxalate decarboxylase with that of Yvrk oxalate decarboxylase, finding a significant difference in the edge of the Mn ion active pocket at the N-terminus of cb6301 oxalate decarboxylase compared to Yvrk (Figure 6). Therefore, the inventors designed a mutation in the amino acids at the edge of the Mn ion active pocket at the N-terminus of cb6301 oxalate decarboxylase to investigate whether it improved enzyme activity.

[0147] Table 5. Results of enzyme activity persistence in mutants at the edge of the N-terminal active pocket. Note: U / L refers to OD. 600 The bacterial resuspension with a concentration of 20 was disrupted by sonication, and the enzyme activity of all total proteins in the supernatant was measured by centrifugation.

[0148] The results (Table 5) show that mutants designed based on amino acids at the edge of the N-terminal active pocket did not achieve good improvement.

[0149] Because various mutation strategies were attempted at the N-terminus without significant results, the inventors conducted a detailed analysis of the C-terminal domain of the oxalate decarboxylase. They found that the S349 and D351 sites in the Mn ion active pocket region of the C-terminus of cb6301 oxalate decarboxylase were opposite to the corresponding sites (D342 and S344) of Yvrk oxalate decarboxylase. Furthermore, they discovered that the I340 site of cb6301 oxalate decarboxylase also differed from the corresponding E333 site of Yvrk oxalate decarboxylase (Figure 7). Therefore, mutations were designed targeting these three sites to investigate whether they improved the sustainability of enzyme activity.

[0150] Table 6. Results of enzyme activity persistence in mutants with C-terminal active pockets Note: U / L refers to OD. 600 =20 bacterial resuspension, after ultrasonic disruption, the enzyme activity of all total proteins in the supernatant after centrifugation was measured. *Data below the detection limit is inaccurate, and efficacy evaluation was not performed.

[0151] The results (Table 6) showed that the D351S and S349D-D351S mutants reduced the activity of cb6301 oxalate decarboxylase without significantly improving enzyme activity persistence. Surprisingly, the I340E mutant significantly improved enzyme activity persistence, but enzyme activity and protein expression were significantly reduced, and protein solubility was very poor. The I340E-S349D-D351S combined mutant showed further reduction in enzyme activity, almost complete inactivation. In this example, the Mn at the C-terminus of cb6301 oxalate decarboxylase... 2+ The I340E mutant in the vicinity unexpectedly enhanced the persistence of enzyme activity, a finding that strongly suggests that the loss of enzyme activity of cb6301 oxalate decarboxylase may be related to the C-terminal Mn ion domain.

[0152] Example 4: Characterization of the C-terminal domain of cb6301 oxalate decarboxylase

[0153] In Example 3, the inventors discovered that the C-terminal Mn of cb6301 oxalate decarboxylase 2+ The nearby I340E mutation resulted in a sustained increase in enzyme activity, suggesting that the loss of activity in cb6301 oxalate decarboxylase may be related to the C-terminal Mn ion domain. Therefore, the inventors further analyzed the C-terminal cupin domain and found that the C-terminal Mn ion domain of cb6301 oxalate decarboxylase... Aside from the four fixed coordinating residues H280, H282, E287, and H326, the coordination space on the side of amino acid position 340 is open (unoccupied), possessing the potential for coordination. This is significantly different from oxalate decarboxylases (Yvrk) derived from Bacillus subtilis. Furthermore, in the family distribution (all protein sequences of the family with InterPro number IPR017774 in the Unipro database), the C-terminal Mn ion of cb6301 oxalate decarboxylase is located around... Among the residues, the I340 site is widely distributed as glutamate E in the oxalate decarboxylase family (Figure 8).

[0154] The structure of Yvrk oxalate decarboxylase (PDB: 1UW8) from Bacillus subtilis (Figure 9) shows that the C-terminal Mn ion coordinates with H273, H275, H319, and E280, occupying four of the octahedral six-coordinate configurations of the Mn ion. At the two remaining coordinate positions, E at position 333 (corresponding to position I340 of cb6301 oxalate decarboxylase) forms a salt bridge with R270, occupying one of the remaining coordinate spaces of the Mn ion, thus limiting the C-terminal Mn ion to a maximum of five coordination positions. Simultaneously, Yvrk oxalate decarboxylase also relies on the interaction network between E333, Y340, and R270 to block the pathway for the Mn ion within the pocket to reach the outside. As shown in Figure 10, the side chain at site I340 of cb6301 oxalate decarboxylase cannot occupy the vacant coordination space of the C-terminal Mn ion, nor can it attract the side chains of R277 and Y347 through non-bonded interactions to block the pores of Mn ions in the C-terminal pocket leading to the external solution environment. Thus, compared to oxalate decarboxylase (Yvrk) from Bacillus subtilis, the pores of Mn ions in the C-terminal cupin domain of cb6301 oxalate decarboxylase leading to the solvent are opened, and the closed state of the C-terminal Mn ions is altered (Figure 10). Based on this finding, an attempt was made to engineer the C-terminal cupin domain of cb6301 oxalate decarboxylase to restore the closed state of the pores of C-terminal Mn ions leading to the external solution environment by reducing the pore size.

[0155] Example 5: Mutation strategy of cb6301 oxalate decarboxylase and verification of mutant expression

[0156] Based on the analysis in Example 4, this example designs mutations in the amino acids near the pores of the Mn ion in the C-terminal cupin domain of cb6301 oxalate decarboxylase, which connect to the external solvent. Wet experiments are used to verify whether narrowing the pore size of the Mn active pocket leading to the outside is beneficial for extending the catalytic duration of the cb6301 oxalate decarboxylase protein. Analysis revealed that the main amino acid sites involved are L250, L252, A267, L269, I340, S349, D351, and I384. The pore radius is reduced by lengthening the side chain and increasing the interaction with surrounding amino acids through salt bridges or hydrogen bonds. The recombinant expression method of the mutant is the same as described in Example 1. The enzyme activity persistence analysis of the successfully expressed mutant is performed using the same method as described in Example 2. A 30% increase in the average enzyme activity ratio of the cb6301 oxalate decarboxylase mutant compared to wild-type cb6301 is considered a significant extension of the enzyme activity reaction time.

[0157] Table 7. Results of enzyme activity persistence in mutants with narrowed C-terminal cupin domain pores. Note: U / L refers to OD. 600 =20 bacterial resuspension, after ultrasonic disruption, the enzyme activity of all total proteins in the supernatant after centrifugation was measured. / : The detection result was below the detection limit and could not be evaluated.

[0158] The results (Table 7) show that mutants that narrowed the pore size of the C-terminal cupin domain through amino acid mutations, except for L250E and some mutants that were not successfully expressed, all had a significant effect on prolonging catalytic activity.

[0159] Example 6: The effect of combining two strategies

[0160] In Example 2, the applicant discovered that D61Y and Y232F had a certain prolonging effect on the enzyme activity persistence of cb6301 oxalate decarboxylase. Unexpectedly, E211P and Y232F were found to significantly promote the expression level of cb6301 oxalate decarboxylase (enzyme activity in cell lysate per unit OD was significantly increased). Therefore, given that the mutant Y232F can improve enzyme activity persistence while also promoting expression level, this example combines the better mutant from Example 5 with Y232F for superposition mutation to examine whether it yields a superior effect. The recombinant expression method is the same as described in Example 1, and the enzyme activity persistence analysis method is the same as in Example 2.

[0161] Table 8. Results of enzyme activity persistence in mutants with channel narrowing strategy and superimposed Y232F. Note: U / L refers to OD. 600The bacterial resuspension with a concentration of 20 was disrupted by sonication, and the enzyme activity of all total proteins in the supernatant was measured by centrifugation.

[0162] The results (Table 8) show that the mutants found in Example 5 that significantly prolonged enzyme activity catalytic time all showed a significant increase in enzyme activity after being superimposed with Y232F. Meanwhile, the SDS-PAGE gel results in Figure 11 show that the proportion of soluble protein in the supernatant of the cb6301 oxalate decarboxylase mutant increased significantly after being superimposed with Y232F, which is consistent with the trend of increased enzyme activity, indicating that the two strategies can be combined to achieve better results.

[0163] Furthermore, pore size analysis of the mutant with Y232F superimposed showed that the superimposed Y232F did not significantly change the pore radius of the C-terminal cupin domain of the original mutant, which is consistent with the result that there was no significant difference in enzyme activity persistence between the Y232F superimposed mutant and the original mutant.

[0164] Example 7: Comparison of enzymatic properties between the cb6301 oxalate decarboxylase mutant and the original cb6301 protein

[0165] Based on the mutant enzyme activity detection results in Examples 5 and 6, six mutants with significantly prolonged catalytic time were selected. After recombinant expression and protein purification, the optimal enzyme activity pH and K were determined. m V max and k cat Enzymatic properties were analyzed, and SEC-HPLC analysis was performed simultaneously. The effect of the mutant on the protein structure was determined by the retention time. The detection method was the same as in Example 1.

[0166] Table 9. Comparison of enzymatic properties of mutants with significantly improved enzyme catalytic time

[0167] The results (Table 9) showed that the retention times of the six cb6301 oxalate decarboxylase mutants on SEC-HPLC were consistent with those of the wild-type cb6301 oxalate decarboxylase protein, indicating that the mutants did not alter the enzyme structure (homotrimer). However, some mutants showed K... m The value decreased significantly, V max It significantly improved enzyme kinetic properties.

[0168] Example 8: Relationship between pore size changes and enzyme activity persistence in the cb6301 oxalate decarboxylase mutant

[0169] In this embodiment, the mutants obtained by narrowing the pore size of the C-terminal cupin domain in Example 5 were selected. The mutants with enzyme activity greater than 10000 U / L were selected. The pore radius of the C-terminal cupin domain of the oxalate decarboxylase mutant was analyzed using Caver software, and the correlation between the pore radius and the enzyme activity persistence of the mutant was analyzed. The results are shown in Table 10 and Figure 12 below.

[0170] Table 10. Relationship between pore size changes and enzyme activity persistence in oxalate decarboxylase mutants.

[0171] The results (Figure 12) showed that the enzyme activity persistence (average enzyme activity ratio over 30 min / 5 min) of the cb6301 oxalate decarboxylase mutant was significantly correlated with the pore size of the C-terminal domain analyzed by Caver software (p < 0.001). Smaller pore sizes resulted in better enzyme activity persistence in the mutant. This result effectively validated the analysis and prediction of the C-terminal domain of cb6301 oxalate decarboxylase in Example 3 and also confirmed the correctness of the rational design / protein engineering strategy proposed in this invention.

[0172] Example 9. Sequence Similarity Network Analysis of Oxalate Decarboxylases with Similar Characteristics to cb6301 Oxalate Decarboxylase

[0173] In the above embodiments (Examples 4, 5, and 8), the inventors discovered for the first time that the openness of the pores leading to the external solvent from the Mn ion center of the C-terminal cupin domain of oxalate decarboxylase may be an important rule and rational design strategy for improving the catalytic persistence of oxalate decarboxylases with similar characteristics to cb6301 oxalate decarboxylase. To confirm the universality of this strategy, the present invention conducted an observation and analysis after comparing the cb6301 oxalate decarboxylase sequence with the oxalate decarboxylase family (InterPro number: IPR017774) in the UniProt database (Figure 8 of Example 4). The amino acid corresponding to position 340 of cb6301 oxalate decarboxylase has the largest proportion in the entire oxalate decarboxylase family (E, the oxalate decarboxylase of Bacillus subtilis from Yvrk is E). However, in known oxalate decarboxylases from cyanobacteria, the amino acid distribution corresponding to position 340 is mainly valine (V), isoleucine (I), and leucine (L) (Figure 13). Therefore, this invention selected three oxalate decarboxylase sequences (SEQ ID NO: 29-31) from cyanobacteria for verification. Simultaneously, all oxalate decarboxylase sequences were retrieved from the entire UniProt database for sequence alignment, and sequences not containing glutamate E at position 340 were selected. Sequence similarity network analysis was then performed. When the Alignment Score was 127, the cluster percent identity was in the range of 58-62%, and the sequence similarity network at this point is shown in Figure 14. Based on different classes, two representative sequences (SEQ ID NO: 32-33) were randomly selected from the larger clusters for expression testing.

[0174] Example 10. Validation of the applicability of the mutation strategy to other oxalate decarboxylases

[0175] This embodiment uses five oxalate decarboxylases from other species selected in Example 9 to verify the applicability of the rational design strategy (see Table 11). The identity of these five oxalate decarboxylase sequences with the cb6301 oxalate decarboxylase protein sequence ranged from 39.2% to 86.1%, and the overall identity of the six protein sequence groups was only 27.7% (Figure 15). Following the analytical strategies of Examples 4 and 5, protein modeling was performed on the five oxalate decarboxylases in Table 11, and the pore radius of their C-terminal cupin domains was analyzed. The results showed that the pore radius of these oxalate decarboxylases was larger than that of the Yvrk oxalate decarboxylase. (Table 11).

[0176] Five other oxalate decarboxylases from selected species were mutated along the pore pathway using the protein engineering strategy employed for CB6301 oxalate decarboxylase. This reduced the bottleneck radius of the pore connecting the Mn ion active center of the C-terminal cupin domain to the external solution environment. Simultaneously, Bacillus subtilis oxalate decarboxylase (Yvrk) and Agrocybe aegypti oxalate decarboxylase (A2), which had previously been confirmed to have no persistent enzyme activity defects in wet assays, were subjected to reverse mutations (increasing the pore radius of their C-terminal cupin domain) to verify whether this would lead to a persistent decrease in enzyme activity. The recombinant expression of the mutants was performed as described in Example 1, and the enzyme activity persistence test method was as described in Example 2.

[0177] Table 11. Species origin, protein sequence information and pore size analysis results of multi-species oxalate decarboxylases

[0178] Table 12. Results of enzyme activity persistence in forward and reverse mutants of multi-species recombinant oxalate decarboxylase Note: U / L refers to OD. 600 The bacterial resuspension with a concentration of 20 was disrupted by sonication, and the enzyme activity of all total proteins in the supernatant was measured by centrifugation.

[0179] The results (Table 12) show that the mutation strategy of this invention is also applicable to the modification of other oxalate decarboxylases with inactivation characteristics during catalysis. The five oxalate decarboxylases in this embodiment come from a wide range of species, including cyanobacteria, Bacteroides, and Planctomycetes, with protein sequence homology ranging from 39.2% to 86.1%. After applying the protein engineering strategy of this patent, all achieved good enzyme activity persistence. Therefore, this rational design strategy has good universality for oxalate decarboxylases with similar properties from all species. Simultaneously, this invention performed reverse mutations (increasing the pore size of the C-terminal cupin domain) on Bacillus subtilis Yvrk oxalate decarboxylase and A2 oxalate decarboxylase Agaricus edulis. It was found that by widening the pore radius of the C-terminal cupin domain, the enzyme catalytic time of the mutant was significantly reduced compared to the wild type, thus verifying the causal relationship between the pore size of the C-terminal cupin domain and the persistence of oxalate decarboxylase activity, and supporting the reliability of the extended application of the protein engineering strategy described in this patent. In other words, the standardized protein structure analysis and protein engineering strategy established in this invention can effectively guide the mutational modification of oxalate decarboxylase.

[0180] The inventors of this invention modeled and calculated the pore radius of oxalate decarboxylases and their mutants from different species using computational simulation, hoping to obtain a clear pore radius threshold. However, the inventors found that there are significant differences between oxalate decarboxylases from different species. Since the analysis and calculation of this design method are based on protein structures obtained through computer-aided modeling, there may be differences in fine structure between computer-aided modeling and the actual protein structure. Therefore, it is not yet possible to obtain a clear bottleneck radius threshold for the pores.

[0181] Based on the successful selection experience of 5 oxalate decarboxylases from different species in this embodiment, the inventors searched for similar sequences in the UniProt database and screened out sequences with similar characteristics to the oxalate decarboxylase used in this patent. The inventors believe that these sequences are suitable for the rational design / protein engineering strategy described in this patent. The specific sequence information is shown in Table 13 below.

[0182] Table 13. Sequence information similar to the oxalate decarboxylase in this patent embodiment from the UniProt database (UniProt protein ID)

[0183] Example 11. In vitro oxalate-lowering effect test of cb6301 oxalate decarboxylase mutant

[0184] This embodiment demonstrates the oxalate degradation capacity of a recombinant oxalate decarboxylase mutant in simulated gastric juice. The artificial gastric juice (containing 10 g / L porcine pepsin) was prepared according to the method outlined in the Chinese Pharmacopoeia (2020 edition). Sodium oxalate was added to the prepared artificial gastric juice to a final concentration of 30 mM. The mixture was then dispensed into three portions, each adjusted to pH 2.0 and pH 3.0 respectively. Take 1 ml of artificial gastric fluid containing oxalic acid into each 1.5 ml EP tube. Under constant temperature and stirring at 37°C, add 100 U of cb6301 oxalate decarboxylase, cb6301 oxalate decarboxylase mutant A (I340L), oxalate decarboxylase mutant B (I384M), cb6301 oxalate decarboxylase mutant C (I340L-Y232F), oxalate decarboxylase mutant D (I384M-Y232F), oxalate decarboxylase mutant E (A267I), and oxalate decarboxylase mutant F (I340L-I384M-Y232F). Add an equal volume of pure water to the blank control group. Incubate the reaction slowly at 37°C with stirring. The reaction was terminated by adding 50 μl of 2.5 M sulfuric acid at 10 min, 30 min and 60 min respectively. After centrifugation at 12000g for 10 min, the supernatant was taken to determine the oxalic acid content. The oxalic acid content of the blank control group was used as a control to evaluate the degradation ability of oxalic acid by 7 oxalate decarboxylase samples in artificial gastric juice.

[0185] Table 14. In vitro oxalate-lowering results of CB6301 oxalate decarboxylase and its composition in different food chyme.

[0186] The results (Table 14) showed that all six CB6301 oxalate decarboxylase mutants were significantly superior to the CB6301 oxalate decarboxylase group, especially at the 30-minute and 60-minute time points. The CB6301 oxalate decarboxylase mutant group also showed a better sustained oxalate-reducing effect, while the total amount of oxalate degraded in the CB6301 oxalate decarboxylase group at 30 minutes did not increase significantly compared to 10 minutes. This suggests that in addition to significantly improving the sustained enzyme activity, the CB6301 oxalate decarboxylase mutant also has better stability in gastric juice.

[0187] Example 12. Effect of secondary hyperoxaluria rat model on urinary oxalate reduction and preliminary safety assessment

[0188] In this embodiment, two cb6301 oxalate decarboxylase mutants (I340L-Y232F and I384M-Y232F) were selected as representatives to investigate the oxalate-lowering effect of cb6301 oxalate decarboxylase and its mutants in a secondary hyperoxaluria SD rat model, and the safety evaluation after repeated administration was also investigated.

[0189] Thirty SD rats, half male and half female, aged 6-8 weeks and weighing 140-180g, were purchased from the Hubei Provincial Center for Disease Control and Prevention. After a 3-5 day acclimatization period, the rats were placed in metabolic cages, and 24-hour urine samples were collected from baseline into urine collection containers pre-acidified with 0.5 mL of 6M hydrochloric acid per container. The total 24-hour urinary oxalate content was measured. After baseline urine and fecal collection, the 30 rats were randomly divided into 5 groups (6 rats / group, half male and half female): a blank control group, a model control group, a cb6301 group, and two cb6301 mutant groups. Except for the blank control group, the other four groups were fed a high-oxalate diet (containing approximately 20 mg of oxalate per meal, twice a day, fed at 8:00 and 17:00) to model hyperoxaluria. Urine was collected on the last day of the modeling period (day 7) to measure urinary oxalate and evaluate the modeling effect. Based on a high-oxalate diet model, mice in the test group were given CB6301 oxalate decarboxylase and two corresponding CB6301 oxalate decarboxylase mutant test substances (dose: 150 U / time, twice a day, administered at 8:30 and 18:00 with meals) for an intervention (7 days). On day 7, urine was collected into a urine collection container pre-filled with hydrochloric acid to measure the total 24-hour urinary oxalate. After the intervention, a 7-day recovery period was observed, and urine was collected again on day 7 into a urine collection container pre-filled with hydrochloric acid to measure the total 24-hour urinary oxalate. Simultaneously, 24-hour fecal samples were collected from the metabolic cages, weighed, and frozen at -20°C for later analysis. An appropriate amount of fecal sample was weighed and added to an appropriate volume of 1M hydrochloric acid, adjusted to 0.1 g / ml, allowing the feces to fully swell, and the pH was adjusted to <1.0. The supernatant was collected by centrifugation for fecal oxalate determination. Urinary oxalate and fecal oxalate were determined using an oxalate assay kit (oxalate oxidase method).

[0190] The results (Figure 16) showed that, compared with the hyperoxaluria model group, both CB6301 oxalate decarboxylase and its mutants significantly reduced oxalate excretion in urine. At the same dose, the CB6301 oxalate decarboxylase mutant had a better oxalate-lowering effect than CB6301 oxalate decarboxylase, indicating that the sustained improvement in enzyme activity of the CB6301 oxalate decarboxylase mutant can also enhance the oxalate-lowering effect in vivo. After oral administration of CB6301 oxalate decarboxylase and its mutants, the fecal oxalate levels in both the hyperoxaluria model group and the oxalate decarboxylase intervention group decreased significantly. Furthermore, the fecal oxalate levels in the CB6301 oxalate decarboxylase mutant intervention group were lower than those in the CB6301 oxalate decarboxylase intervention group, indicating that oral administration of oxalate decarboxylase can degrade the vast majority of dietary oxalate and significantly reduce oxalate absorption in the digestive tract.

[0191] Furthermore, after oral administration of CB6301 oxalate decarboxylase and its two mutants for 7 days, no toxic side effects (indicators such as mental state, food intake, weight, and activity level) were observed in the test animals. This indicates that oral administration of CB6301 oxalate decarboxylase and its mutants is quite safe and is a very promising candidate drug molecule for the treatment of hyperoxaluria and its secondary diseases.

[0192] Example 13: Effect of recombinant strain construction strategy on the enzyme activity of oxalate decarboxylase

[0193] Besides improving the catalytic time of the enzyme by protein engineering cb6301-D29, the inventors unexpectedly discovered that the construction of the recombinant expression strain had a significant impact on the enzyme activity of oxalate decarboxylase. This embodiment provides the effect of the construction strategy of the recombinant oxalate decarboxylase expression system on its enzyme activity.

[0194] The construction of the recombinant expression strain E. coli BL21(DE3)(pSGEL-cb6301-D29) (containing a single copy of MntS and the molecular chaperone groEL-groES) is described in Example 1. The target gene sequence of oxalate decarboxylase was amplified using the cb6301-F / cb6301-R primer combination, and the purified target gene fragment was recovered by agarose gel electrophoresis. Using expression vectors pET-28a, pGEL, pMMTS, and pET-28a-MntS plasmid DNA as templates, and primer combinations PET28-F / PET28-R, pSGEL-F / pET22b-WF-R, pMMTS-F / pMMTS-R, and PET28-R / pSGEL-R (Table 15), the whole plasmid was amplified by PCR to obtain linearized vectors. After digestion of the template plasmid with the restriction endonuclease DpnI, the linearized plasmid was recovered by agarose gel electrophoresis. Using a seamless cloning method, the target gene fragment was inserted into the expression vectors pET-28a, pGEL, pMMTS, and pET-28a-MntS, respectively. These vectors were then transformed into DH5α competent cells, plated on LB agar plates containing kanamycin, and single colonies were picked to screen for positive clones. After confirmation by DNA sequencing, the positive bacterial cultures were expanded and plasmids were extracted to obtain recombinant plasmids pET-28a-cb6301-D29, pGEL-cb6301-D29, pMMTS-cb6301-D29, and pET-28a-MntS-cb6301-D29. A schematic diagram of the recombinant plasmids is shown in Figures 17-20. The recombinant plasmid was heat-shocked and transformed into the expression host E. coli BL21(DE3) to obtain the recombinant expression strains E. coli BL21(DE3)(pET-28a-cb6301-D29), E. coli BL21(DE3)(pGEL-cb6301-D29)(containing the molecular chaperone groEL-groES), E. coli BL21(DE3)(pMMTS-cb6301-D29)(containing two copies of MntS and the molecular chaperone groEL-groES), and E. coli BL21(DE3)(pET-28a-MntS-cb6301-D29)(containing a single copy of MntS.

[0195] Table 15. Primer information for oxalate decarboxylase plasmid construction

[0196] Five recombinant E. coli strains [BL21(DE3)(pET-28a-cb6301-D29)], [BL21(DE3)(pGEL-cb6301-D29)], [BL21(DE3)(pSGEL-cb6301-D29)], [BL21(DE3)(pMMTS-cb6301-D29)], and [BL21(DE3)(pET28a-MntS-cb6301-D29)] were streaked onto LB agar plates containing 50 μg / ml Kan and incubated overnight at 37°C with the plates inverted. Single colonies were picked from the streaked plates and activated in 50 mL of LB liquid medium containing 50 μg / ml Kan antibiotic, and incubated overnight at 37°C and 200 rpm. The activated bacterial culture was transferred at a ratio of 1% to shake-flask fermentation medium containing 50 μg / ml Kan antibiotic and incubated at 37°C and 200 rpm until OD reached. 600nm =1.2-1.5, add 2% lactose and 5mM MnCl2 to a final concentration, and induce overnight at 25℃ and 200rpm. Centrifuge the inducing culture overnight to collect the cells, wash with sterile water, and then resuspend in 50mM arginine buffer (pH 9.5) to a cell density OD of 1.2-1.5. 600 =20, sonicate to disrupt cells. Centrifuge and collect the supernatant. While stirring, slowly add 2 mol / L phosphate solution to adjust the pH to 3.0. Centrifuge and collect the supernatant, then load it onto a CM Sepharose chromatography column for protein purification. Collect the target protein peak, desalt it using a 30 kDa ultrafiltration tube to obtain the oxalate decarboxylase sample for subsequent enzymatic property analysis. The analytical method is the same as described in Example 1.

[0197] Table 16. Enzyme activity data of oxalate decarboxylase expressed by different bacterial species

[0198] The results (Table 16) showed that compared with the oxalate decarboxylase expressed by the original OxDc strain (pET-28a-cb6301-D29) (51.4 U / mg), the specific activity of the enzyme in the sample containing the molecular chaperone groEL-groES increased to 76.6 U / mg, with an expression level increase of approximately 100%. Furthermore, even with the same amount of MntS, the expression level of the recombinant strain containing the molecular chaperone also increased by approximately 100%, indicating that the molecular chaperone groEL-groES is crucial for increasing yield. Based on this, the specific activity of the enzyme was significantly increased after adding different amounts of MntS, from 76.6 U / mg to 103.8 U / mg (with one MntS) and 109.3 U / mg (with two MntS), respectively. This suggests that the addition of MntS is beneficial for enhancing the specific activity of the enzyme, and its mechanism of action may be related to promoting manganese ion supply or stabilizing the enzyme's active site.

[0199] The oxalate decarboxylases with different specific activities obtained above were replaced with pure water via a PD10 desalting column, and the protein concentration was adjusted to 1 mg / ml. The manganese ion content was determined by inductively coupled plasma-mass spectrometry (ICP-MS) to analyze the effect of the manganese ion content corresponding to each subunit on the enzyme activity of oxalate decarboxylase.

[0200] Table 17. Detection data of manganese ion content of each subunit of oxalate decarboxylase with different specific activities.

[0201] The results (Table 17) showed that after co-expression of MntS, the specific activity of oxalate decarboxylase in the recombinant expression strain increased synchronously with the manganese ion content bound to its subunits. Compared with the sample without MntS, the specific activity of the MntS-co-expressed system increased from 51.4 U / mg to 109.3 U / mg, and the manganese ion content corresponding to one subunit also increased from 0.765 to 1.418. These results indicate that the introduction of MntS simultaneously improved both the manganese ion binding level and the enzyme's catalytic activity, further suggesting that it may enhance the function of oxalate decarboxylase by promoting manganese ion supply. This also demonstrates that the recombinant expression strain (pSGEL-cb6301-D29) has a significant advantage in efficiently expressing high-activity oxalate decarboxylase.

[0202] Example 14: Screening and Development of Culture Medium Components with High Enzyme Activity

[0203] Based on the pSGEL-cb6301-D29 recombinant expression strain constructed in Example 1, the fermentation process was further optimized. During fermenter expression, the specific activity of oxalate decarboxylase was found to be significantly lower than that in shake flasks. The inventors speculate that this may be related to the inorganic salt medium used in the fermenter. Therefore, the effects of metal salts in the medium on the expression and activity of oxalate decarboxylase were investigated.

[0204] The recombinant expression strain [E. coli BL21(DE3)(pSGEL-cb6301-D29)] was streaked onto LB agar plates containing 50 μg / ml Kan and incubated overnight in an inverted incubator at 37°C. Single colonies were picked from the streaked plates and transferred to 70 mL of LB liquid medium containing 30 μg / ml Kan antibiotic for primary seed culture overnight at 37°C and 200 rpm. The culture medium was transferred at a ratio of 0.5-3% to 70 ml of sterile inorganic salt medium containing 30 μg / ml Kan antibiotic for secondary seed culture at 37°C and 200 rpm. Finally, the culture medium was transferred at a ratio of 0.5-3% to sterile 10 L fermenter medium (fermentation medium composition is shown in Tables 18 and 19). In the fermentation medium (control group), the zinc ion concentration was reduced to 20% of the original concentration (referred to as low-zinc medium), the zinc and nickel ion concentrations were both reduced to 20% of the original concentration (referred to as low-zinc-nickel medium), and the zinc ion concentration was reduced to 20% of the original concentration + the nickel ion concentration was reduced to 0% of the original concentration (referred to as low-zinc-zero-nickel medium). At OD... 600 When the concentration reaches 20-50, add 3-5% lactose to a final concentration, induce expression at 25℃, and express at pH 6.0 for approximately 25-28 hours. Centrifuge to collect the bacterial sludge. After collection and placement in the tank, the bacterial cells undergo post-processing and protein chromatography purification. The enzyme specific activity analysis method is the same as in Example 1.

[0205] Table 18. Fermentation medium composition

[0206] Table 19. Trace Element Mother Liquor Formula

[0207] The results (Figure 21) showed that, compared with the control group in the fermentation medium, reducing the zinc ion concentration in the medium to 20% of the basal level resulted in a decrease in the enzyme activity per cell (OD). 600 =20) increased by 46%, and the specific activity of the enzyme increased by 47%. Based on the low-zinc medium, further reducing the nickel ion concentration to 20% showed no significant effect on the enzyme activity per cell compared to the low-zinc medium group, but the specific activity increased by 8%. Based on the low-zinc-nickel medium, further reducing the nickel ion concentration from 20% to 0% showed no significant effect on the enzyme activity per cell compared to the low-zinc-nickel medium, but the specific activity decreased slightly (approximately 16%). The above multi-group data analysis shows that reducing the zinc ion concentration from the original concentration to 20% significantly increased the specific activity of the target protein enzyme, further increasing the enzyme activity per cell. Maintaining the zinc ion concentration at the original 20%, reducing the nickel ion concentration from the original concentration to 20% was beneficial for further increasing the specific activity of the target protein enzyme. Further reducing the nickel ion concentration from 20% to 0% was beneficial for increasing the expression level of the target protein, thus maintaining the enzyme activity per cell essentially unchanged.

[0208] Example 15: Determination of metal ion content in oxalate decarboxylases with different enzyme activities

[0209] The oxalate decarboxylases with different specific activities obtained in Example 14 were replaced with pure water using a PD10 desalting column, and the protein concentration was adjusted to 1 mg / ml. The metal ion content was determined by ICP-MS to analyze the effect of the metal ion content corresponding to each subunit on the enzyme activity of oxalate decarboxylase.

[0210] Table 20. Detection data of metal ion content of each subunit of oxalate decarboxylase with different specific activities.

[0211] The results (Table 20) showed that after reducing the zinc and nickel content in the fermentation medium, the zinc and nickel ion content corresponding to each subunit of the recombinant oxalate decarboxylase decreased significantly, while the manganese ion content increased significantly, which corresponds to the increase in enzyme activity. These results suggest that zinc and nickel ions compete with manganese ions in oxalate decarboxylase, thus affecting enzyme activity.

[0212] Based on this, the inventors further conducted a multi-metal ion detection and analysis on oxalate decarboxylases with different enzyme activities during the above-mentioned process development.

[0213] Table 21. Detection data of multiple metal ions in each subunit of oxalate decarboxylase with different enzyme specific activities.

[0214] The results (Table 21) showed that with increasing specific activity, the manganese ion content per subunit of oxalate decarboxylase increased, while the iron and nickel ion content decreased. For oxalate decarboxylases with a specific activity greater than 70 U / mg, the manganese ion content per subunit was generally greater than or equal to 1.0 ions / subunit; the iron ion content was less than or equal to 0.2 ions / subunit, and the nickel ion content was less than or equal to 0.1 ions / subunit. These results further confirm the crucial role of manganese ions in the function of oxalate decarboxylase, while iron and nickel ions may have an inhibitory effect on its activity.

[0215] In addition, while investigating the stability of oxalate decarboxylase at different pH levels, researchers unexpectedly discovered that the activity of the purified oxalate decarboxylase increased by 20-30% when stored at pH 9.0. This phenomenon again attracted the researchers' attention, and based on this finding, they conducted further research on the storage conditions of the purified oxalate decarboxylase.

[0216] Example 16: Effect of pH adjustment on oxalate decarboxylase

[0217] During the study, it was observed that the activity of oxalate decarboxylase purified samples adjusted to alkaline conditions increased during storage. Based on this, this embodiment aims to conduct a more in-depth study of this phenomenon.

[0218] Weigh the purified oxalate decarboxylase powder and place it in an EP tube. Add 5 ml of 50 mM arginine solution to dissolve it (pH approximately 9.0-10.5). Dispense the solution into four 2 ml EP tubes, 1.8 ml per tube. One tube was stored overnight at 2-8°C as the control group, one tube was stored overnight at room temperature as experimental group 1, one tube was incubated overnight in a 40°C water bath as experimental group 2, and one tube was stored at 50°C as experimental group 3. After incubation, perform an enzyme specific activity test, using the same method as described in Example 1.

[0219] Table 22. Changes in the specific activity of purified oxalate decarboxylase under different storage conditions

[0220] The results (Table 22) showed that the activity of the purified oxalate decarboxylase was significantly improved in arginine buffer at a pH of approximately 9.0-10.5.

[0221] Given the presence of guanidinium groups on the side chains of arginine molecules, researchers analyzed whether the oxalate decarboxylase molecule might have undergone renaturation in the arginine buffer solution, thereby enhancing its activity. To verify this hypothesis, the inventors conducted incubation studies using alkaline buffer salt systems with different compositions and buffer systems of the same composition at different pH values.

[0222] To determine whether the buffer salt composition was the cause, the inventors incubated pH 10.0 buffer solutions with different buffer salt compositions at 40°C for 24 hours to evaluate their effect on enhancing the activity of oxalate decarboxylase. The test buffers included buffers with slow refolding components (such as 50 mM Tris-HCl, 50 mM Tris-HCl buffer containing 50 mM urea), buffers containing basic amino acid components (50 mM lysine, 50 mM histidine, 50 mM arginine), and buffers without amino components (50 mM sodium bicarbonate, 50 mM borate), as detailed in Table 23.

[0223] Table 23. Results of the increase in specific activity of purified oxalate decarboxylase under different buffer salt conditions (pH 9.5)

[0224] The results (Table 23) show that, under the same pH 10 conditions, different buffer components (slow refolding component, basic amino acid component, and amino-free component) all showed consistent enhancement effects on oxalate decarboxylase activity compared to the control group (arginine buffer), with some groups even outperforming the arginine buffer. Therefore, the arginine component in the buffer is not the key factor in enhancing enzyme activity; pH ​​is likely the crucial factor.

[0225] To determine whether pH was the cause, this study investigated the effect of dissolving oxalate decarboxylase samples in 50 mM Tris buffer at pH 6-10 and incubating at 40°C for 24 h on enhancing the enzyme activity of oxalate decarboxylase.

[0226] Table 24. Specific activity enhancement of purified oxalate decarboxylase under Tris buffer conditions at different pH values.

[0227] The results (Table 24) showed that the enzyme activity of oxalate decarboxylase increased with increasing pH when incubated in 50 mM Tris-HCl at pH 6.0-10.0, with the best enzyme activity enhancement at pH 9.5.

[0228] In summary, the key factor for improving the activity of purified oxalate decarboxylase is pH. Under conditions of pH 8.0-10.0 and storage at 15-50℃ for more than 24 hours, the enzyme activity shows a significant increase.

[0229] Samples were selected before and after pH adjustment to enhance enzyme activity. The manganese ion content was determined using the ICP-MS manganese ion determination method described in Example 14, and the manganese ion content was calculated down to each subunit.

[0230] Table 25. Manganese ion content of each subunit in samples before and after pH adjustment to enhance enzyme activity.

[0231] The results (Table 25) show that the manganese ion content of each subunit of oxalate decarboxylase did not change significantly before and after pH adjustment. This indicates that the enzyme activity of oxalate decarboxylase is affected by factors other than manganese ions.

[0232] Example 17. Characterization of highly active oxalate decarboxylase

[0233] In the above embodiments, high-activity oxalate decarboxylase was successfully obtained by optimizing the strain, fermentation conditions, and post-treatment conditions of the oxalate decarboxylase. Results from multiple embodiments showed that manganese ion content is an important factor affecting the enzyme activity of oxalate decarboxylase. However, Example 16 also showed that, in addition to manganese ions, other factors affect enzyme activity. Before and after alkaline pH treatment, the manganese ion content of the oxalate decarboxylase did not change significantly, but the enzyme activity was significantly improved. In this embodiment, oxalate decarboxylases with different manganese ion contents and those with the same level of manganese ion content (low, medium, and high specific activity) were selected, as well as oxalate decarboxylases before and after pH adjustment to improve enzyme activity, for further characterization of charge isomers. Specific chromatographic and elution conditions are shown in Table 26 below, and typical chromatograms are shown in Figure 22.

[0234] Table 26. Chromatographic conditions and gradient elution program for IEC-HPLC analysis of oxalate decarboxylase

[0235] (1) Ion exchange chromatography-high performance liquid chromatography (IEC-HPLC) analysis of oxalate decarboxylase with different enzyme activities at different manganese ion contents

[0236] The oxalate decarboxylase sample was diluted to 1 mg / ml with 20 mM CAPSO (containing 20 mM NaCl, pH 9.8), centrifuged at 10,000 rpm for 3 min, and the supernatant was collected for HPLC analysis.

[0237] Table 27. Peak area ratio of oxalate decarboxylases with different enzyme activities and different manganese ion contents according to IEC-HPLC. Note: Alkali peaks are those that precede the main peak; acid peaks are those that follow the main peak.

[0238] The results (Table 27) show that, under different manganese ion contents, the enzyme activity of oxalate decarboxylase samples with different activities showed a positive correlation with the proportion of the main peak in the IEC-HPLC chromatogram. The sample with an enzyme specific activity of 48.2 U / mg had a main peak proportion of 46.4%, which increased to 53.5% when the enzyme specific activity increased to 73.9 U / mg. The sample with the highest enzyme specific activity (105.0 U / mg) also had the highest main peak proportion, reaching 56.6%.

[0239] (2) Ion exchange chromatography-high performance liquid chromatography (IEC-HPLC) analysis of oxalate decarboxylases with different enzyme activities and the same Mn content

[0240] The oxalate decarboxylase sample was diluted to 1 mg / ml with 20 mM CAPSO (containing 20 mM NaCl, pH 9.8), centrifuged at 10,000 rpm for 3 min, and the supernatant was collected for HPLC analysis.

[0241] Table 28. Peak area ratio of IEC-HPLC detection for oxalate decarboxylases with different specific activities and the same manganese ion content. Note: Alkali peaks are those that precede the main peak; acid peaks are those that follow the main peak.

[0242] The results (Table 28) show that, under conditions of essentially the same manganese ion content, the specific activities of the three oxalate decarboxylases with different activities showed a positive correlation with the proportion of the main peak in the IEC-HPLC chromatogram. The main peak proportion of the sample with a specific activity of 64.3 U / mg was 44.2%, which increased to 49.1% when the specific activity was increased to 86.0 U / mg. The sample with the highest specific activity (105.0 U / mg) also had the highest main peak proportion, reaching 56.6%.

[0243] (3) Ion exchange chromatography-high performance liquid chromatography (IEC-HPLC) analysis of oxalate decarboxylase before and after pH adjustment to enhance enzyme activity

[0244] Select oxalate decarboxylase samples before and after pH adjustment to enhance enzyme activity, dilute to 1 mg / ml with 20 mM CAPSO (containing 20 mM NaCl, pH 9.8), centrifuge at 10000 rpm for 3 min, and take the supernatant for HPLC analysis.

[0245] Table 29. IEC-HPLC peak area percentage of oxalate decarboxylase before and after pH adjustment to increase enzyme activity. Note: Alkali peaks are those that precede the main peak; acid peaks are those that follow the main peak.

[0246] The results (Table 29) showed that the enzyme specific activity and the proportion of the main peak in the IEC-HPLC chromatogram were positively correlated before and after pH adjustment to increase enzyme activity. The proportion of the main peak in the sample with an enzyme specific activity of 82.0 U / mg was 48.2%, and the proportion of the main peak increased to 54.0% when the enzyme specific activity was increased to 98.4 U / mg.

[0247] The results above (Tables 27, 28, and 29) collectively demonstrate a significant positive correlation between the specific activity of oxalate decarboxylase and the proportion of its main peak in IEC-HPLC. This correlation was validated in experimental groups with different and the same manganese ion content, as well as in pH-adjusted experimental groups, indicating that the distribution of charge isomers may be a key structural factor affecting enzyme activity independently of manganese ion content and subject to pH adjustment.

[0248] The inventors also conducted tests on the construction of recombinant expression strains, fermentation culture processes, and pH-enhancing enzyme activity of the OxDc-D29 mutant and other oxalate decarboxylases (SEQ ID NO: 3-33) in this patent, and found that they also exhibited similar enzyme activity-enhancing effects. Due to space limitations, specific data are not listed here.

[0249] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rational design method for improving the inactivation of oxalate decarboxylase during catalytic processes, characterized in that, The bottleneck radius of the pore connecting the manganese ion active center in the C-terminal cupin domain of oxalate decarboxylase to the external solvent is reduced relative to wild-type oxalate decarboxylase using protein engineering methods; the oxalate decarboxylase suitable for the rational design strategy of this invention must include, but is not limited to, at least one of the following characteristics: (1) Oxalate decarboxylase irreversibly loses its enzyme activity during the catalytic conversion of oxalate substrate to formic acid; (2) The manganese ion in the C-terminal cupin domain of oxalate decarboxylase has six coordination structures. In addition to the four coordination spaces occupied by the enzyme structure itself (3 histidine and 1 glutamic acid residues), the coordination space on the side of amino acid position 340 of the amino acid sequence corresponding to SEQ ID NO: 1 is unoccupied and has the potential for coordination; (3) The bottleneck radius of the pore from the manganese ion active center in the C-terminal cupin domain of oxalate decarboxylase to the solvent is greater than (4) The amino acid at position 340 of the amino acid sequence corresponding to SEQ ID NO: 1 of oxalate decarboxylase is an amino acid other than glutamic acid.

2. The rational design method according to claim 1, characterized in that, The rational design method specifically includes the following steps: 1) The three-dimensional structure of wild-type oxalate decarboxylase was constructed using molecular modeling software; 2) The pore path from the manganese ion active center in the C-terminal cupin domain of the three-dimensional structure of oxalate decarboxylase obtained in step 1) to the solvent was analyzed using molecular pore analysis software, and the bottleneck radius of the pore was also analyzed. 3) Based on the analysis of amino acids along the pore, candidate mutation sites are selected. Taking advantage of the steric hindrance of the side chain of the newly replaced amino acid, the hydrogen bond, salt bond, and hydrophobic interaction formed between the newly replaced amino acid and other amino acids, mutation design is carried out on the amino acids near the pore of the manganese ion active center in the C-terminal cupin domain of oxalate decarboxylase to the external solvent (including but not limited to the replacement, addition, or deletion of amino acids). It is predicted which mutations may reduce the bottleneck radius of the pore of the manganese ion active center in the C-terminal cupin domain to the solvent, and candidate oxalate decarboxylase mutants are obtained. 4) Using molecular modeling software, construct the three-dimensional structure of the oxalate decarboxylase mutant obtained in step 3); 5) The bottleneck radius of the pores leading to the external solvent in the manganese ion active center in the C-terminal cupin domain of the candidate oxalate decarboxylase mutants in step 4) was analyzed using molecular pore analysis software. Mutants with a smaller bottleneck radius of pores compared to the wild-type oxalate decarboxylase measured in step 2) were retained. 6) The candidate oxalate decarboxylase mutant identified in step 5) was obtained by site-directed amino acid mutation of the original oxalate decarboxylase, and the mutant was transformed into a host cell to obtain a strain containing the target gene. The mutant was then cultured, expressed, extracted, and purified to obtain the oxalate decarboxylase mutant. 7) Verify whether the oxalate decarboxylase mutant obtained in step 6) improves the inactivation problem of oxalate decarboxylase during catalysis. For the screened beneficial mutants, further superimposed mutations can be performed to increase the advantages of the mutants. Preferably, the molecular modeling software mentioned in step 1) is selected from, but not limited to, phyre2, Swissmodel, RoseTTAfold, RaptorX, MODELLER, and AlphaFold software; more preferably, AlphaFold software. Preferably, step 2) uses molecular pore analysis software selected from, but not limited to, Caver, MOLE, Molaxis, Critical Comparison, Hole, Chunnel, Porewalker, Hollow, and 3V software; more preferably, Caver software.

3. An oxalate decarboxylase mutant, characterized in that, The bottleneck radius of the pore leading to the solvent in the C-terminal cupin domain of the oxalate decarboxylase mutant is smaller than that of the wild-type oxalate decarboxylase, resulting in a significant decrease in the inactivation rate of the enzyme during catalysis and a longer effective catalytic duration.

4. The oxalate decarboxylase mutant according to claim 3, characterized in that, The oxalate decarboxylase mutant is selected from any one of the following: (a) Its amino acid sequence is derived from the sequence shown in SEQ ID NO: 1 by mutation at one or more amino acid residue sites selected from the group consisting of: position 61, position 211, position 232, position 250, position 252, position 267, position 269, position 340, position 349, position 351, and / or position 384; and compared with the oxalate decarboxylase shown in SEQ ID NO: 1, the enzyme has a significantly reduced inactivation rate during catalysis and a prolonged effective catalytic time; or (b) The oxalate decarboxylase mutant has 80%, preferably 90%, preferably 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence described in (a), and has the function of the oxalate decarboxylase mutant described in (a), wherein any amino residue or combination thereof corresponding to position 61, 211, 232, 250, 252, 267, 269, 340, 349, 351, and / or position 384 of the amino acid sequence shown in SEQ ID NO: 1 is identical to that in the amino acid sequence described in (a); or (c) The oxalate decarboxylase mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the oxalate decarboxylase mutant described in (a), wherein any amino residue or combination thereof corresponding to position 61, 211, 232, 250, 252, 267, 269, 340, 349, 351, and / or 384 of the amino acid sequence shown in SEQ ID NO: 1 is the same as that in the amino acid sequence described in (a).

5. The oxalate decarboxylase mutant according to claim 4, characterized in that, The amino acid sequence of the oxalate decarboxylase mutant is mutated at one or more sites selected from the group below to the amino acid residues shown below: At position 61, aspartic acid is replaced by tyrosine. At position 211, glutamic acid is replaced by proline; At position 232, tyrosine is replaced by phenylalanine; At position 250, leucine is replaced by glutamic acid, tyrosine, tryptophan, methionine, or glutamine. At position 252, leucine is replaced by valine and isoleucine. At position 267, alanine is replaced by valine, isoleucine, methionine, leucine, or glutamine. At position 340, isoleucine is replaced by leucine, phenylalanine, glutamic acid or methionine. At position 349, serine is replaced by aspartic acid; At position 351, aspartic acid is replaced by serine. At position 384, isoleucine is replaced by methionine, phenylalanine, arginine, lysine, glutamine, tryptophan, or glutamic acid.

6. The oxalate decarboxylase mutant according to claim 5, characterized in that, The mutants of the oxalate decarboxylase are those containing I340L, A267V, A267I, I384M, I384F, L269V, I340L-I384M, I340L-I384F, I340L-S349D-D351S, I340L-S349D-D351S-L269A, A267I-L250M, A267I-L250I, A267I-L252V, A267I-L252I, A267I-I340L, L250Y, L250W, L250M, I384R, I340L-A267M, I340L-L250E-S349D-D351S, A267M, I3 Any one of the following mutations, or a combination containing any two or more mutations: 40L-L250E-A267M-I384F, A267L, A267I-L250M-I384F, A267I-L250M-I384R, A267I-I340A, A267I-I340V, A267I-L250M-L252V, A267I-L250M-L252I, A267I-L250M-I340A, A267I-L250M-I340V, L250Q, A267I-L250Q, A267V-L250Q, A267L-L250Q, I384K, I384Q, I384W, and I384E.

7. The oxalate decarboxylase mutant according to claim 5, characterized in that, The amino acid sequence of the oxalate decarboxylase mutant is shown in any one of SEQ ID NO: 3-25.

8. The oxalate decarboxylase mutant according to claim 3, characterized in that, The oxalate decarboxylase mutant is selected from any one of the following: (d) Its amino acid sequence is derived from the sequence shown in SEQ ID NO: 29-32 by mutation, and is selected from any of the following: A270I mutant and A270L mutant of SEQ ID NO: 29; G266I mutant, V339L mutant, and V339I mutant of SEQ ID NO: 30; A267I mutant, A267L mutant, and I340L mutant of SEQ ID NO: 31; S226L mutant, I299L mutant, and S226I mutant of SEQ ID NO: 32; and has a prolonged effective catalytic time compared with the oxalate decarboxylase shown in SEQ ID NO: 29-32; or (e) The oxalate decarboxylase mutant has 90%, preferably 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence described in (d), and has the function of the oxalate decarboxylase mutant described in (d), wherein the amino residues corresponding to the amino acid sequence shown in SEQ ID NO: 29 at position 270, the amino acid sequence shown in SEQ ID NO: 30 at position 266 or 339, the amino acid sequence shown in SEQ ID NO: 31 at position 267 or 340, and the amino acid sequence shown in SEQ ID NO: 32 at position 226 or 299 are the same as those in the amino acid sequence described in (d); or (f) The oxalate decarboxylase mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (d), and has the function of the oxalate decarboxylase mutant described in (d), wherein the amino residues corresponding to position 270 of the amino acid sequence shown in SEQ ID NO: 29, position 266 or 339 of the amino acid sequence shown in SEQ ID NO: 30, position 267 or 340 of the amino acid sequence shown in SEQ ID NO: 31, and position 226 or 299 of the amino acid sequence shown in SEQ ID NO: 32 are the same as those in the amino acid sequence described in (d).

9. A polynucleotide encoding an oxalate decarboxylase mutant according to any one of claims 3-8.

10. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 9.

11. A host cell comprising the expression vector of claim 10 or the genome thereof having integrated a polynucleotide encoding an oxalate decarboxylase mutant as described in any one of claims 3-8; Preferably, the host cell is a bacterium; more preferably, the host cell is Escherichia coli, Bacillus, or Corynebacterium; even more preferably, the host cell is Escherichia coli.

12. A composition comprising the oxalate decarboxylase mutant according to any one of claims 3-8; Preferably, the composition further comprises an excipient; More preferably, the excipient may be a diluent, filler, binder, disintegrant, lubricant, solvent, or other food- or pharmaceutically acceptable excipient; Preferably, the composition is a solution, suspension, emulsion, powder, lozenge, pill, syrup, lozenge, tablet, chewing gum, concentrate, capsule, or other food- or pharmaceutically acceptable dosage form.

13. The use of the oxalate decarboxylase mutant according to any one of claims 3-8, the polynucleotide according to claim 9, the expression vector according to claim 10, the host cell according to claim 11, or the composition according to claim 12 in the preparation of a medicament for the prevention, treatment, or relief of hyperoxaluria, hyperoxalemia, and diseases related to calcium oxalate stones caused by oxalate abnormalities. Preferably, the diseases related to calcium oxalate stones are selected from, but not limited to, kidney stones, ureteral stones, bladder stones, urethral stones, nephrocalcinosis, renal colic, hematuria, nephritis and chronic kidney disease caused by calcium oxalate deposition, and renal failure.

14. A method for preventing, treating, or alleviating hyperoxaluria, hyperoxalemia, and calcium oxalate-related diseases caused by abnormal oxalate levels, comprising administering to a subject the composition of claim 12 or the drug of claim 13. Preferably, the diseases related to calcium oxalate stones are selected from, but not limited to, kidney stones, ureteral stones, bladder stones, urethral stones, nephrocalcinosis, renal colic, hematuria, nephritis and chronic kidney disease caused by calcium oxalate deposition, and renal failure.

15. A highly active oxalate decarboxylase, characterized in that, At 37°C and with a 10 mM oxalic acid substrate, the enzyme specific activity is ≥70 U / mg protein; preferably, the enzyme specific activity is ≥80 U / mg protein, ≥90 U / mg protein, ≥100 U / mg protein, or ≥110 U / mg protein; more preferably, the enzyme specific activity is ≥120 U / mg protein.

16. The oxalate decarboxylase according to claim 15, characterized in that, The amino acid sequence of the oxalate decarboxylase is as shown in SEQ ID NO: 2, and its mutants or homologs, or mutants of the above homologs; preferably, the mutants or homologs have at least 80%, preferably 90%, more preferably 95%, most preferably 98% or 99% sequence identity with SEQ ID NO: 2, and have oxalate degrading enzyme activity.

17. The oxalate decarboxylase according to claim 15, characterized in that, The amino acid sequence of the oxalate decarboxylase is as shown in any one of SEQ ID NO: 2-33, or is the A270I mutant or A270L mutant of SEQ ID NO: 29; the G266I mutant, V339L mutant, or V339I mutant of SEQ ID NO: 30; the A267I mutant, A267L mutant, or I340L mutant of SEQ ID NO: 31; or the S226L mutant, I299L mutant, or S226I mutant of SEQ ID NO:

32.

18. The oxalate decarboxylase according to claim 15, characterized in that, The manganese ion content of the oxalate decarboxylase is ≥1.0 ions / subunit; preferably, ≥1.1 ions / subunit, ≥1.2 ions / subunit, ≥1.3 ions / subunit, ≥1.4 ions / subunit or ≥1.5 ions / subunit; and / or, the molar ratio of Mn ions to oxalate decarboxylase protein is ≥3:1, preferably ≥3.3, ≥3.6, ≥3.9, ≥4.2 or ≥4.

5.

19. The oxalate decarboxylase according to claim 15, characterized in that, The iron ion content of the oxalate decarboxylase is ≤0.2 ions / subunit; preferably, ≤0.1 ions / subunit, more preferably ≤0.05 ions / subunit.

20. The oxalate decarboxylase according to claim 15, characterized in that, The nickel ion content of the oxalate decarboxylase is ≤0.1 ions / subunit; preferably, ≤0.05 ions / subunit, more preferably ≤0.01 ions / subunit, and most preferably below the detection limit; and / or, the zinc ion content of the oxalate decarboxylase is ≤0.2 ions / subunit; preferably, ≤0.1 ions / subunit, more preferably ≤0.05 ions / subunit, and most preferably ≤0.02 ions / subunit.

21. The oxalate decarboxylase according to claim 15, characterized in that, When the oxalate decarboxylase was analyzed by anion exchange chromatography, the area of ​​the preferentially eluted main peak was ≥45%. The anion exchange chromatography conditions were as follows: Proteomix SAX-NP5 (250mm × 4.6mm, 5μm); mobile phase A was 20mM CAPSO buffer containing 20mM NaCl, pH 9.8; mobile phase B was 20mM CAPSO buffer containing 300mM NaCl, pH 9.8; and the detection wavelength was 280nm.

22. A method for preparing a highly active oxalate decarboxylase, characterized in that, The sample containing oxalate decarboxylase is incubated in an alkaline buffer solution at pH 8.0-10.5 for at least 2 hours, preferably at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. Preferably, the pH is 8.0-10.

0. The sample containing oxalate decarboxylase is a purified oxalate decarboxylase solution, an unpurified oxalate decarboxylase feed solution, or a cell lysate supernatant containing oxalate decarboxylase.

23. The method for preparing oxalate decarboxylase according to claim 22, characterized in that, The buffer solution contains, but is not limited to, any one of the following components: carbonate, bicarbonate, arginine, Tris, lysine, histidine, or borate, or any combination of two or more of these components.

24. An expression carrier or combination, characterized in that, The expression vector or combination comprises a polynucleotide encoding the oxalate decarboxylase shown in SEQ ID NO: 2, its mutants or homologs, or mutants of homologs, and further comprises a polynucleotide encoding the manganese ion transporter MntS; preferably, the polynucleotide encoding the oxalate decarboxylase and the polynucleotide encoding the manganese ion transporter MntS are located in the same vector or different vectors; preferably, the amino acid sequence of the oxalate decarboxylase is as shown in any one of SEQ ID NO: 2-33, or is the A270I mutant or A270L mutant of SEQ ID NO: 29; the G266I mutant, V339L mutant, or V339I mutant of SEQ ID NO: 30; the A267I mutant, A267L mutant, or I340L mutant of SEQ ID NO: 31; or the S226L mutant, I299L mutant, or S226I mutant of SEQ ID NO:

32.

25. The expression vector or combination according to claim 24, wherein the promoter of the manganese ion transporter gene is an inducible promoter or a constitutive promoter.

26. The expression vector or combination according to claim 24, characterized in that, The expression vector or combination further contains a polynucleotide encoding a molecular chaperone protein that helps with protein folding; Preferably, the molecular chaperone protein is groEL-groES; Preferably, the promoter of the molecular chaperone protein gene is an inducible promoter or a constitutive promoter.

27. A host cell comprising a plasmid expression vector or combination thereof as described in any one of claims 10, 24-26; or having integrated into its genome a polynucleotide encoding an oxalate decarboxylase as described in any one of claims 1-26 and / or a manganese ion transport protein MntS and / or a molecular chaperone protein. Preferably, the host cell contains two copies of a polynucleotide encoding the manganese ion transporter MntS; Preferably, the host cell is a bacterium; more preferably, the host cell is Escherichia coli, Bacillus, or Corynebacterium; even more preferably, the host cell is Escherichia coli.

28. A fermentation method for producing a highly active oxalate decarboxylase, characterized in that, The method includes the steps of culturing recombinant host cells containing a gene encoding oxalate decarboxylase and expressing oxalate decarboxylase, wherein the nickel content in the culture medium is ≤0.2mM; preferably, ≤0.1mM, ≤0.05mM; more preferably, nickel-free.

29. The fermentation production method of the highly active oxalate decarboxylase according to claim 28 further includes an induced fermentation step, characterized in that, The induction pH range is 5.5–6.4; preferably, 5.8–6.2; more preferably, 5.9–6.1, and / or the inducer is lactose.

30. An oxalate decarboxylase obtained by any one of the production methods or combinations thereof according to claims 22-23, 28-29, wherein the oxalate decarboxylase has a specific activity ≥70 U / mg protein at 37°C and 10 mM oxalate substrate, preferably ≥80 U / mg protein, ≥90 U / mg protein, ≥100 U / mg protein, ≥110 U / mg protein, or ≥120 U / mg protein.

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