Oxalate decarboxylase mutant and use thereof

Abstract

Provided is an oxalate decarboxylase mutant. The mutant has high enzyme activity and is stable in the gastrointestinal environment, wherein mutant 5 has optimal stability under pH 2 conditions and exhibits a reduction in enzyme activity by about 4.47% at 48 h. The mutant also has superior thermal stability and has a low enzyme activity reduction percentage at 40ºC, wherein mutant 3 has optimal stability. Animal experiments have shown that mutant 5 can effectively reduce absorption of exogenous oxalate in vivo. Further provided are a use of the oxalate decarboxylase mutant and an enzyme formulation thereof in the prevention and / or treatment of hyperoxaluria, urinary calculus caused by exogenous oxalate, hypocalcemia, joint pain, etc. The oxalate decarboxylase mutant and the enzyme formulation thereof are safe, have no toxic side effects, and are suitable for long-term use.

Description

An oxalate decarboxylase mutant and its application Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an oxalate decarboxylase mutant, its enzyme preparation, and its applications. Background Technology

[0002] Oxalic acid is a metabolic product of organisms, widely distributed in plants, animals, and fungi, and plays different functions in different organisms. Studies have found that more than 100 plants are rich in oxalic acid, especially spinach, amaranth, beets, purslane, taro, sweet potatoes, and rhubarb. Because oxalic acid can reduce the bioavailability of mineral elements, it is considered an antagonist of mineral absorption and utilization. The human body cannot degrade oxalic acid and usually excretes it through the excretory route. Free oxalic acid is absorbed in the stomach, small intestine, and colon, and excreted by the kidneys. The stomach is the first organ to mediate the absorption of exogenous oxalic acid; the basement membrane of gastric parietal cells has a large expression of SLC26A7, which can be transported via reverse transport of Cl... - and Oxlate - Mediating oxalate absorption. The small intestine is a crucial site for oxalate transport, mediating not only its absorption but also its secretion. The SLC26A6 transporter is a key carrier mediating oxalate secretion, highly expressed in the epithelial cells of the duodenum, jejunum, ileum, and renal tubules. It has been reported that 80-90% of endogenous oxalate and 50-80% of exogenous oxalate are filtered by the kidneys and excreted in urine. The amount of oxalate the human body can process is limited; long-term, repeated excessive intake of oxalate may induce a series of diseases such as hyperoxaluria, systemic oxalate toxicity, kidney stones, and hypocalcemia.

[0003] Large amounts of free oxalate in the blood are excreted through the kidneys in the form of urine. However, excess oxalate combines with calcium to form calcium oxalate, which deposits in the kidneys and eventually forms stones. Approximately 70-80% of kidney stones are caused by calcium oxalate. Furthermore, oxalate that is not filtered by the kidneys can accumulate in various tissues, such as bones, joints, and muscles, forming insoluble oxalate crystals. These crystals cause mechanical wear and tear on body tissues, leading to pain and other symptoms.

[0004] Once oxalic acid enters the body, it binds with minerals such as calcium, iron, magnesium, and zinc, reducing mineral absorption and leading to mineral deficiencies. Oxalic acid increases calcium oxalate saturation ten times more effectively than calcium. If the daily excretion of oxalic acid in urine increases by 10%, it's equivalent to a 100% increase in urinary calcium, resulting in rapid calcium loss and reduced bone mass.

[0005] In healthy individuals, the daily urinary oxalate excretion is 10-40 mg / 24h (0.1-0.45 mmol / 24h). A concentration exceeding 40-45 mg / 24h (0.45-0.5 mmol / 24h) is considered a clinical indicator of hyperoxaluria. Hyperoxaluria is classified into primary and secondary hyperoxaluria. Primary hyperoxaluria is a rare congenital glyoxylate metabolism error that leads to oxalate production in the liver, caused by an AGT mutation. Secondary hyperoxaluria is induced by factors such as a high-oxalate diet, intestinal hyperoxaluria, and oxalate-degrading microbial dysbiosis. Intestinal hyperoxaluria is caused by disease or gastrointestinal resection, leading to malabsorption syndrome. As the concentration of ionic oxalate in the lumen increases, soluble oxalate exists at a relatively high concentration in the colon and can passively diffuse into the bloodstream, where it is filtered and excreted by the kidneys, increasing the risk of kidney damage.

[0006] Oxalate decarboxylase is one of the main enzymes catalyzing the degradation of oxalate in plants and microorganisms. It is a manganese-dependent enzyme that catalyzes the decarboxylation of oxalate to produce formic acid and carbon dioxide. The entire process is simple, rapid, efficient, and requires no cofactors. This enzyme was first discovered by Shimazono in white-rot fungi, and later found in brown-rot fungi, Bacillus, and other fungi. The optimal pH range and cofactors for the enzyme reaction differ depending on its source. The optimal pH for oxalate decarboxylase from white-rot fungi is 2.5-3.0; from brown-rot fungi, it is 2.0-2.2; and from Bacillus subtilis, it is 3.5-4.0. The most studied oxalate decarboxylase is from Bacillus subtilis, which can induce its synthesis under low pH conditions. Ruchi Anand et al. identified the protein structure of oxalate decarboxylase from Bacillus subtilis using X-ray crystallography. Natural oxalate decarboxylase is a 32-point symmetrical hexamer belonging to the cupin superfamily. Each subunit of this enzyme contains two cupin folds, and each cupin domain contains a manganese ion binding site. There are four manganese ion binding residues (three histidines and one glutamate) and several highly conserved hydrophobic residues. Some studies have suggested that Glu333, the metal-binding site of the second cupin domain of oxalate decarboxylase, is a proton donor; its mutation to alanine reduces catalytic activity by 25-fold. However, other literature reports that the cupin I domain primarily affects enzyme activity, suggesting that Glu162 is a proton donor, while cupin II plays a crucial role in structural stability.

[0007] Oxalate decarboxylases are mainly produced by the following organisms: Bacillus subtilis, Flammulina velutipes, Ganoderma lucidum (Curtis) P. Karst., Boletus sp., Agaricus, Aspergillus niger, Pseudomonas, Synechocystis sp., Trametes hirsute, white-rot fungi (T. versicolor), and brown-rot fungi (Postia placenta). The oxalate decarboxylase used in this invention is an enzyme known in the art that catalyzes the oxidation of oxalate to carbon dioxide and formic acid according to the following reaction. It is produced by Synechocystis sp. (NCBI Reference Sequence: WP_011244027.1) and possesses oxalate carboxylase activity. Summary of the Invention

[0008] To address the above issues, this invention provides an oxalate decarboxylase mutant, which has at least one of the following mutations based on SEQ ID NO:2:

[0009] (1) The M at position 309 is mutated to V or Y;

[0010] (2) The Q at position 334 mutates to W;

[0011] (3) The M at position 357 mutates to K or A; or

[0012] (4) Add EGA at positions 349-351.

[0013] In this invention, as one embodiment, the mutant has any one of the following mutations:

[0014] (1) The M at position 309 is mutated to V or Y;

[0015] (2) The Q at position 334 mutates to W;

[0016] (3) The M at position 357 mutates to K or A; or

[0017] (4) Add EGA at positions 349-351.

[0018] In this invention, as one embodiment, the mutant has any of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:19.

[0019] Accordingly, the present invention also claims protection for the polynucleotide encoding the above-mentioned mutant. Based on the amino acid sequence provided by the present invention, those skilled in the art can readily obtain the corresponding nucleotide sequence and optimize it according to the codon characteristics of different organisms.

[0020] Accordingly, the present invention also claims protection for expression vectors containing the said polynucleotides.

[0021] Accordingly, the present invention also claims protection for host cells containing the expression vector or the polynucleotide integrated into its genome.

[0022] By inserting the polynucleotide sequence into a suitable expression vector and further transfecting host cells, a recombinant expression strain for expressing the oxalate decarboxylase mutant can be obtained; the expression vector is preferably the Escherichia coli expression vector pET-28a(+), and the host cell is preferably Escherichia coli BL21(DE3) cells.

[0023] The present invention also provides an enzyme preparation containing the above-mentioned mutant.

[0024] In this invention, as one embodiment, the enzyme preparation is an oral preparation.

[0025] In this invention, as one embodiment, the enzyme preparation is a bacterial powder containing the above-mentioned mutant, a feed additive, feed, food additive, health product, special medical purpose formula food, or medicine.

[0026] The present invention also relates to the use of the above-mentioned mutants or the enzyme preparations in the prevention and / or treatment of secondary hyperoxaluria, urinary tract stones, hypocalcemia or joint pain caused by exogenous oxalate. Beneficial effects

[0027] 1. The oxalate decarboxylase mutants provided by this invention have high enzyme activity and are stable in the gastrointestinal environment. Mutants 4, 5, 10, 11 and 18 significantly enhance stability under extreme acidic conditions. Among them, mutant 5 has the best stability at pH 2, with enzyme activity decreasing by about 4.47% after 48 hours, ensuring that it has sufficient time to function in the stomach.

[0028] 2. The oxalate decarboxylase mutants provided by this invention have improved thermal stability to a certain extent. The enzyme activity reduction ratio is relatively low at 40℃. The thermal stability of mutants 3, 4 and 5 is better than that of mutant 1 in the prior art. The thermal stability of mutants 11 and 18 is comparable to that of mutant 1.

[0029] 3. Animal experiments have shown that mutant 5 of this invention can effectively reduce the absorption of exogenous oxalic acid in the body. The oxalate decarboxylase mutant and its enzyme preparation provided by this invention are safe and have no toxic side effects, and can be taken long-term. Attached Figure Description

[0030] Figure 1: Enzyme activity of each mutant;

[0031] Figure 2: Percentage of enzyme activity reduction in each mutant under different pH conditions;

[0032] Figure 3: Percentage of enzyme activity reduction in each mutant at 40℃;

[0033] Figure 4: Data analysis of the test substance's effectiveness in beagle dogs. Detailed Implementation

[0034] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0035] Those skilled in the art should understand that, unless otherwise specified, the reagents used in the following examples are all commercially available.

[0036] Example 1 Molecular Design

[0037] Wemol software was used to predict the B-factor, saturation mutation, and RMSF of wild-type oxalate decarboxylase (amino acid sequence shown in SEQ ID NO:1), and to predict the increase in stability. The B-factor threshold was set to 30, the stability increase threshold in saturation mutation was set to 15, and the RMSF threshold was set to 0.20. The changes in bond energy in the mutation site region were observed, and the site prediction analysis results are shown in Table 1. Based on the analysis of molecular structure and prediction results, and considering the characteristics of the E. coli expression system, based on the truncated signal peptide molecule SEQ ID NO:2, oxalate decarboxylase mutant 3-18 was designed as shown in Table 2.

[0038] SEQ ID NO: 1 (wild type, Synechocystis sp. (NCBI Reference Sequence:WP_011244027.1))MQKKSKFFLGLLGVITCFVLIGSFCLPSLAQTQTWRSLSNVVWGKDLPAFSYPFSKTPLVDYDGGVTKQVGTYNFPVS KGMAGVYMTLKPGAIRELHWHANAAEWAYVIEGRTRVTLTNPDGQVQIADVDQGGLWYFPRGWGHSIEGIGPGTAKFLLVFNDGTFSEGATFSITDWLSHTPIS WVQQNFGWSQDEVEKLPKKQVYISRYNPEVKPLDKTQSRNPKVSRIVLPYTHNLLAEKPRTSQAGNTLKLASAKEFPASFNMAGALLRLEPGAMRQLHWHPNA DEWQYVLNGSMDLAVFASEGKASMSRLQKGDVGYVPKGYGHALRNSSDQPLDVLIVFNDGDYQSIDLNDWIMSNPNTVLDDVFQLSPQLLDKLPKESEILIPRS

[0039] Note: The underlined 29 amino acids are the signal peptide sequence.

[0040] Table 1. Results of Site Prediction Analysis

[0041] Table 2 Oxalate decarboxylase mutants Note: D-29 and D-27 represent 29 and 27 amino acids of the signal peptide truncated, respectively;

[0042] The amino acid numbering of the mutants described in this invention is based on SEQ ID NO:1. Taking SEQ ID NO:2 as an example, the second amino acid after 29 truncated signal peptide amino acids is numbered 31, and the subsequent amino acids are numbered sequentially.

[0043] Example 2 Molecular Construction and Expression

[0044] The nucleotide sequences of SEQ ID NO:1-SEQ ID NO:19 from Example 1 were reverse translated to obtain nucleotide sequences. Based on codon degeneracy and E. coli codon bias, nucleotide sequences of 19 molecules were optimized and collectively referred to as the OXDC gene. The expression framework was designed as araBAD promoter-MntH-rrnB terminator-linking sequence-T7 / Lac promoter-OXDC gene-T7 terminator. Following the guidelines of *Molecular Cloning: A Laboratory Manual*, XhoI and SphI restriction sites were designed, and the aforementioned expression framework gene sequence was constructed into the pET28a(+) vector, named plasmid 1. The MntH gene sequence was synthesized, and XhoI and NdeI restriction sites were designed. The MntH gene sequence was constructed into plasmid 1, which was also digested with these restriction sites and named pET28a(+)--MntH. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells via chemical transformation, added to LB liquid medium, and cultured at 37°C and 150 rpm for 8 h as the primary seed cell.

[0045] Oxalate decarboxylase and its mutants were expressed using a general high-density fermentation process for *E. coli*. Frozen seed culture was inoculated into primary culture medium and cultured on a shaker; 5 mL of primary seed culture was inoculated into secondary seed culture medium and cultured on a shaker; 20 mL of secondary seed culture was inoculated into the reactor for further culture, and expression was induced and regulated using IPTG and manganese chloride. Basic parameters are as follows:

[0046] Initial fermentation volume: 0.4L;

[0047] Growing temperature: 37℃;

[0048] Induction temperature: 28℃;

[0049] Speed ​​control: Initial speed 300 rpm, speed is automatically controlled according to dissolved oxygen, maximum speed 1200 rpm;

[0050] Ventilation control: The initial air flow rate is 0.5 L / min. After the rotation speed reaches the upper limit, the ventilation rate is adjusted according to the dissolved oxygen level, with an upper limit of 5 L / min. The tank pressure is kept constant at 0.3 MPa. Feeding: Feeding begins 3.5 hours after fermentation.

[0051] Induction control: Induction begins when OD reaches approximately 80, with a one-time addition of IPTG at a final concentration of 0.3 mM / L and manganese chloride tetrahydrate at a concentration of 10 mM / L.

[0052] Sampling control: Take samples at 0 and 17 hours after induction to detect density and pH, and retain 1 mL of the sample;

[0053] Conditions for induction: 17-24 hours; dissolved oxygen control: 40% during the growth period, and 30% after induction.

[0054] Analysis of enzyme activity and yield data in Table 3 and Figure 1 revealed that mutants 6, 7, 8, 9, 12, 15, 16, and 17 had excessively low specific enzyme activities and were not further investigated. Mutants 2, 3, 4, 5, 10, 11, 13, 14, and 18 had specific enzyme activities no lower than mutant 1. Mutants 1 and 2 showed differences in enzyme activity, purity, and yield. Mutant 1 exhibited approximately twice the enzyme activity, molecular purity, and yield of mutant 2, indicating that truncation of the signal peptide affects protein expression yield. Since the *E. coli* expression system lacks organelles and typically does not require signal peptide localization, excess signal peptides cannot be recognized and cleaved by cells, potentially affecting proper protein folding. Therefore, signal peptides are usually truncated for protein expression. Based on the expression results, it can be inferred that for signal peptides, the more amino acids truncated, the higher the protein expression level and the better the enzyme activity.

[0055] Table 3. Data on the expression of different molecules

[0056] Example 3 Purification

[0057] The bacterial cells expressing mutants 2, 3, 4, 5, 10, 11, 13, 14, and 18 from Example 2 were resuspended in phosphate buffer and homogenized 2-3 times. The crude extract was centrifuged at 10,000 rpm at 4°C, and the supernatant was collected. A Uni Gel 80Q column was packed, connected to the chromatography system, and the two column volumes were equilibrated using phosphate buffer. Sample loading was prepared, with a loading volume not exceeding 2 column volumes of fermentation supernatant. After loading, the volume was equilibrated with 3 column volumes of phosphate buffer, followed by gradient elution with sodium chloride buffer at a flow rate not exceeding 3 column volumes. The elution was monitored using a UV detector at 230 nm. Collection began when the UV signal at 230 nm reached 500 mAu. After collection, the sample was cleaned with 0.1 M hydrochloric acid and 1 M sodium hydroxide solution, regenerated with 1 M sodium chloride solution, and finally rinsed with purified water until the conductivity was below 0.1 mS / cm.

[0058] Example 4 Activity Detection

[0059] Enzyme activity was determined by high performance liquid chromatography (HPLC) according to General Chapter 0512 of the Chinese Pharmacopoeia, Part IV.

[0060] Sample preparation: Take 1 mL of 40 mM oxalate-200 mM citrate (artificial gastric juice) buffer solution, add 10 μL of sample, react in a 30℃ water bath for 10 min, remove, add 50 μL of 2.5 M sulfuric acid to inactivate the enzyme, and filter through a microporous membrane.

[0061] Chromatographic column: Welch Ultimate XB-C18; column temperature: 35℃; injection volume: 5μL; detection wavelength: 210nm; phase A: 0.1% phosphoric acid solution; phase B: pure methanol.

[0062] Enzyme activity definition: One unit of enzyme activity (U) is defined as the amount of enzyme required to degrade 1 micromolar oxalate or generate 1 micromolar formic acid per minute under the sample treatment conditions.

[0063] Example 5: Activity characteristics under different pH conditions

[0064] The purified molecules obtained in Example 3 were diluted with buffer solutions of different pH ranges until their enzyme activities were close, including but not limited to glycine-hydrochloric acid buffer solution, citric acid and citrate buffer solution, phosphate buffer solution, Tris buffer solution, sodium carbonate buffer solution, etc. The pH was adjusted to pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, and pH 11 using hydrochloric acid aqueous solution or sodium hydroxide aqueous solution. Samples were taken for enzyme activity detection at 0 h, and the detection method was the same as in Example 4. The remaining samples were subjected to enzyme activity determination at 48 h, following the method in Example 4.

[0065] Table 4. Enzyme activity data of each molecule under different pH conditions (unit: U / mL)

[0066] Exogenous oxalate dissociates in the stomach and is absorbed via SLC26A family transporters; therefore, the stability of oxalate decarboxylase molecules under gastric juice conditions is the primary factor for their function. Table 4 and Figure 2 show that mutant 1's enzyme activity is affected by pH 2, decreasing by approximately 30%, thus impacting degradation efficiency. Mutants 4, 5, 10, 11, 13, and 18 significantly enhanced stability under extremely acidic conditions, with mutant 5 exhibiting the best stability at pH 2, showing a reduction in enzyme activity of approximately 4.47% after 48 hours, ensuring sufficient time for it to function in the stomach.

[0067] Example 6: Activity characteristics under high temperature conditions

[0068] Molecular high-temperature stability is a crucial factor influencing process design, storage, and transportation conditions. Products with better high-temperature stability can reduce production costs and facilitate storage for users. This example investigated the stability of various mutants at 40°C.

[0069] The molecule obtained in Example 3 was diluted with phosphate buffer to near its enzyme activity and incubated at 40°C. Enzyme activity was measured on days 3, 6, and 10. Sample preparation: 1 mL of 40 mM oxalate-200 mM citrate (artificial gastric juice) buffer was added to 10 μL of sample. The mixture was incubated in a 30°C water bath for 10 min. After inactivation, 50 μL of 2.5 M sulfuric acid was added, and the mixture was filtered through a microporous membrane. Samples were then injected sequentially for detection. Detection conditions were as follows:

[0070] Column: Welch Ultimate XB-C18

[0071] Column temperature: 35℃

[0072] Injection volume: 5 μL

[0073] Detection wavelength: 210nm

[0074] Phase A: 0.1% phosphoric acid solution; Phase B: pure methanol.

[0075] Table 5. Enzyme activity data of each molecule at 40℃ (unit: U / mL)

[0076] The test results are shown in Table 5 and Figure 3. Mutants 3, 4 and 5 have better high-temperature stability than mutant 1, with mutant 3 having the best stability. Mutants 11 and 18 have stability comparable to mutant 1.

[0077] Example 7 Animal Experiment

[0078] Beagles fed a high-oxalate diet were used as an animal model to study secondary hyperoxaluria induced by exogenous oxalate intake. In this study, eight beagles were randomly divided into four groups: a blank control group, a model control group, experimental group 1, and experimental group 2, with two dogs in each group. The model control group, experimental group 1, and experimental group 2 were fed 1-1.5% dietary oxalate, resulting in a more than five-fold increase in urinary oxalate levels. Simultaneously, experimental group 1 and experimental group 2 were orally administered oxalate decarboxylase mutant 1 and mutant 5, respectively, twice daily at 5000 U per meal. Urine was collected in a container containing hydrochloric acid to prevent the conversion of ascorbic acid in the urine to oxalate. The oxalate content in the urine samples was detected using an oxalate detection kit (Sigma-Aldrich).

[0079] Table 6 Analysis of Animal Urinary Oxalate Level Data

[0080] Both the model control group and the experimental group showed elevated urinary oxalate levels after ingesting exogenous oxalate. The results in Table 6 and Figure 4 indicate that after experimental groups 1 and 2 were given test mutant 1 and mutant 5, respectively, the urinary oxalate levels decreased to or were lower than those of the blank control group, demonstrating that test mutant 5 can effectively reduce the absorption of exogenous oxalate in vivo.

[0081] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be protected by the present invention.

Claims

1. An oxalate decarboxylase mutant, characterized in that, The mutant has at least one of the following mutations based on SEQ ID NO:2: (1) The M at position 309 is mutated to V or Y; (2) The Q at position 334 mutates to W; (3) The M at position 357 mutates to K or A; or (4) Add EGA at positions 349-351.

2. The mutant according to claim 1, characterized in that, It has any of the following mutations: (1) The M at position 309 is mutated to V or Y; (2) The Q at position 334 mutates to W; (3) The M at position 357 mutates to K or A; or (4) Add EGA at positions 349-351.

3. The mutant according to claim 1, characterized in that, The mutant has any of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

19.

4. A polynucleotide encoding the mutant of any one of claims 1-3.

5. An expression vector containing the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or the polynucleotide of claim 5 is integrated into its genome.

7. An enzyme preparation containing the mutant according to any one of claims 1-3.

8. The enzyme preparation according to claim 7, characterized in that, The enzyme preparation is an oral preparation.

9. The enzyme preparation according to claim 7, characterized in that, The enzyme preparation is a bacterial powder, feed additive, feed, food additive, health product, special medical purpose formula food or medicine containing the mutant of any one of claims 1-3.

10. The use of the mutant of any one of claims 1-3 or the enzyme preparation of any one of claims 7-9 in the prevention and / or treatment of secondary hyperoxaluria, urinary calculi caused by exogenous oxalate, hypocalcemia or joint pain.

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