Strain 44XB capable of producing urate oxidase and use thereof

Abstract

A strain 44XB capable of producing urate oxidase and a use thereof, relating to the technical field of microorganisms and medicines. A strain 44XB capable of producing urate oxidase is preserved in the Korean Collection for Type Cultures (KCTC), with the preservation number: KCTC 43631, the preservation date: 26 September 2023, the preservation address: 181 lpsin-gil. Jeongeup-si, Jeonbuk 56212, South Korea, and the classification name: Ornithinibacillus sp. A use of the strain 44XB in preparation of drugs for treating hyperuricemia and gout. The strain 44XB can produce highly active urate oxidase, and can be used for microecological therapy of hyperuricemia to reduce serum uric acid in hyperuricemia, and thus has the potential to be used in microecological treatment of hyperuricemia and gout.

Description

A urate oxidase-producing strain 44XB and its application Technical Field

[0001] The present invention relates to the field of microorganisms and medical technology, and in particular to a urate oxidase-producing strain 44XB and applications thereof. Background Art

[0002] The Bacillaceae family belongs to the domain Bacteria, phylum Firmicutes, class Bacilli, and orders Caryophanales. Its type species, Bacillus subtilis, was discovered and described in 1835 and officially named in 1872, serving as the type strain of the Bacillaceae family. To date, the Bacillaceae family comprises 118 published genera (https: / / lpsn.dsmz.de / family / bacillaceae). To address the heterogeneity of the Bacillus genus, numerous Bacillus species have been reclassified in recent years, significantly increasing the number of genera within the family. Members of the Bacillaceae family are widely distributed. Recently described genera—Radiobacillus deserti, Pseudoneobacillus rhizosphaerae, Mangrovibacillus cuniculi, Thermolongibacillus altinsuensis, and Aquibacillus halophilus—have been isolated from soil, plant rhizospheres, sediments, hot springs, and salt lakes, respectively. Most species in the Bacillaceae family are spore-forming and exhibit strong stress tolerance, with some exhibiting exceptional adaptability to cold, heat, and salinity. Furthermore, some Bacillaceae species are capable of producing enzymes such as alkaline phosphatase, protease, and amylase, making them valuable for industrial applications.

[0003] Urate oxidase is an enzyme with significant medical potential. As a key enzyme in the uric acid metabolic pathway, it catalyzes the conversion of low-solubility uric acid to the more soluble allantoin. Urate oxidase has important applications in enzymatic uric acid concentration determination, uric acid-lowering treatment of foods, and the treatment of hyperuricemia. Within the Bacillaceae family, Bacillus sp. TB-90 and Bacillus cereus SKIII have the ability to produce highly active urate oxidase. Although some strains have been reported to produce urate oxidase, no strain within the Bacillaceae family has been shown to alleviate hyperuricemia or reduce serum uric acid in hyperuricemic mice. Therefore, the present invention provides a urate oxidase-producing strain 44XB and its use. Summary of the Invention

[0004] The purpose of the present invention is to provide a urate oxidase-producing strain 44XB and its application, which can be used for microecological therapy of hyperuricemia, reduce serum uric acid in hyperuricemia, and has the potential to be used in microecological treatment of hyperuricemia and gout.

[0005] To achieve the above objectives, the present invention provides a urate oxidase-producing strain 44XB, which is deposited in the Korean Collection of Type Cultures (KCTC) with accession number KCTC 43631, deposited on September 26, 2023, at 181 lpsin-gil. Jeongeup-si, Jeonbuk 56212, South Korea, and classified as Ornithinibacillus sp.

[0006] Preferably, the 16S rRNA sequence of strain 44XB is shown as SEQ ID NO.1.

[0007] Preferably, the genome of strain 44XB contains six pucL genes encoding urate oxidase.

[0008] Preferably, the pucL gene sequence encoding uricase is shown as SEQ ID NOs. 2-7, and the amino acid sequences of the uricase encoded thereby are shown as SEQ ID NOs. 8-13, respectively.

[0009] The present invention also provides the use of the above-mentioned strain 44XB in preparing a drug for treating hyperuricemia.

[0010] The present invention also provides the use of the strain 44XB in preparing a medicine for treating gout.

[0011] The advantages and positive effects of the new genus of uricase-producing Bacillaceae and its application described in the present invention are:

[0012] 1. The strain 44XB provided by the present invention can produce highly active urate oxidase and can be used for microecological therapy of hyperuricemia to reduce serum uric acid in hyperuricemia.

[0013] 2. The strain 44XB provided by the present invention can produce urate oxidase with high activity and has the potential to be used in the microecological treatment of hyperuricemia and gout.

[0014] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG1 is a plate image of the strain 44XB of the present invention cultured for 48 h;

[0016] FIG2 is a scanning electron micrograph of strain 44XB of the present invention after culturing for 12 h;

[0017] FIG3 is a Gram staining result diagram of strain 44XB of the present invention;

[0018] FIG4 is a diagram showing the spore Gram staining results of strain 44XB of the present invention;

[0019] Figure 5 shows the polar lipid profile of strain 44XB of the present invention; A is the glycolipid profile, B is the amino lipid profile, C is the total lipid profile, and D is the phospholipid profile; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; APL, unidentified aminophospholipid;

[0020] Figure 6 is an analysis of respiratory quinone components of strain 44XB of the present invention, wherein A is a UPLC chromatogram, B is a mass spectrogram, and C is the main respiratory quinone MK-7;

[0021] FIG7 is a 16S rRNA neighbor-joining phylogenetic tree of the present invention;

[0022] FIG8 is a whole genome phylogenetic tree of the present invention;

[0023] Figure 9 shows that the strain 44XB of the present invention reduces the serum uric acid level in hyperuricemia mice. , p < 0.001; , p < 0.01; , p<0.05. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0025] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0026] Unless otherwise stated, the instruments, reagents and kits used in the present invention are all commercially available.

[0027] Preservation Information

[0028] Strain 44XB was deposited in the Korean Collection of Type Cultures KCTC with accession number KCTC 43631 on September 26, 2023 at 181 lpsin-gil. Jeongeup-si, Jeonbuk 56212, South Korea, and was classified as Ornithinibacillus sp.

[0029] Example 1 Isolation and identification of strain 44XB

[0030] 1. Isolation and Screening of Strain 44XB

[0031] Strain 44XB was collected and isolated from seagull feces in Qingdao City.

[0032] The isolation procedure was as follows: seagull feces were collected and inoculated into a uric acid culture medium (uric acid, 5 g / L; yeast extract, 0.5 g / L; NaCl, 0.5 g / L; MgSO₄, 0.5 g / L; K₂HPO₄, 2 g / L; KH₂PO₄, 0.5 g / L; pH = 7.5) for enrichment and incubation at 37°C for 48 hours. The fermentation broth was diluted and plated onto a uric acid culture medium supplemented with 15 g / L agar powder and incubated at 37°C for 96 hours. Single colonies producing clear zones were selected and purified multiple times to obtain pure bacteria. Images of the plates after 48 hours of incubation are shown in Figure 1.

[0033] 2. Multi-phase classification and identification of bacterial species

[0034] (1) Phenotypic characteristics. The growth of strain 44XB was observed at different culture temperatures, salinities, and pH values. The growth temperature range and the optimal growth temperature range were tested at 4°C, 10°C, 20°C, 30°C, 37°C, 42°C, and 50°C, respectively. The growth salinity detection range was 0%, 1%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, and 20% NaCl gradient (w / v). The growth pH detection range was 4-10, with 1 pH value detected in each gradient. The strain was inoculated into uric acid culture medium, placed in an anaerobic gas production bag and indicator, and cultured in an anaerobic jar for 7 days to detect anaerobic growth. The bacterial morphology was observed using a scanning electron microscope (Figure 2). Gram staining (Figure 3) and spore staining (Figure 4) were performed using a Gram staining kit and a spore staining kit.

[0035] As shown in Figure 3, the strain is purple in Gram stain, indicating it is a Gram-positive bacterium. As shown in Figure 4, spore staining shows endospore formation, with spores appearing green and the bacteria light red.

[0036] Strain 44XB grows at 20-50°C, with optimal growth at 37-42°C. It grows at NaCl concentrations of 0-9%, with optimal growth at 0-3%. It grows at a pH of 6-9, with optimal growth at 7-8. Strain 44XB cannot grow under anaerobic conditions. Strain 44XB is Gram-positive, and malachite green staining reveals light green spores under a light microscope.

[0037] (2) Physiological and biochemical characteristics. Oxidase and catalase tests were performed using standard methods. Enzyme production and other physiological and biochemical parameters of the strain were tested using API 20NE and API ZYM kits. The results are shown in Tables 1 and 2. Carbon source utilization of the strain was tested using the Biolog GENIII kit. The results are shown in Table 3.

[0038] Table 1 API 20NE test results of strain 44XB and 5 reference strains

[0039]

[0040] Among them, strains: 1, strain 44XB; 2, Ornithinibacillus salinisoli CGMCC 1.15809 T ; 3, Ornithinibacillus contaminans DSM 22953; 4, Ornithinibacillus bavariensis DSM 15681; 5, Oceanobacillus halotolerans CGMCC 1.17002; 6, Oceanobacillus iheyensis JCM 11309. +, positive; -, negative; w, weakly positive.

[0041] Table 2 API ZYM test results of strain 44XB and five reference strains

[0042]

[0043] The strains are: 1, strain 44XB; 2, Ornithinibacillus salinisoli CGMCC 1.15809; 3, Ornithinibacillus contaminans DSM 22953; 4, Ornithinibacillus bavariensis DSM 15681; 5, Oceanobacillus halotolerans CGMCC 1.17002; 6, Oceanobacillus iheyensis JCM 11309. +, positive; -, negative; w, weakly positive.

[0044] Table 3 Biolog GENIII test results of strain 44XB and 5 reference strains

[0045] WellBiolog GENIII substrate123456A1Negative Control------A2Dextrin-++w-+A3D-Maltose-++w++A4D-Trehalose-+++++A5D-Cellobiose-+++++A6Gentiobiose-ww+++A7Sucrose-ww+++A8D-Turanose-www++A9Stachyose-www++A10Positive Control++++++A11pH 6w+++++A12pH 5w-----B1D-Raffinose-w-w-+B2α-D-Lactose-w-w++B3D-Melibiose-ww+++B4β-Methyl-D-Glucoside-w+w++B5D-Salicin-++ww+B6N-Acetyl-D-Glucosamine-+++++B7N-Acetyl-β-D-Mannosamine-+w+++B8N-Acetyl-D-Galactosamine----++B9N-Acetylneuraminic Acid---w-+B101% NaCl-+++++B114% NaCl-++-++B128% NaCl-+w-+wC1α-D-Glucose-+++w+C2D-Mannose--+-w+C3D-Fructose-+++++C4D-Galactose-w++++C53-Methyl Glucose--w+++C6D-Fucose-ww+++C7L-Fucosew-w+++C8L-Rhamnose--w+++C9Inosine--++++C101% Sodium Lactatew-w--+C11Fusidic Acid----+-C12D-Serinew++++-D1D-Sorbitol----w+D2D-Mannitol--++++D3D-Arabitolw-++++D4myo-Inositol---w-+D5Glycerol-+++-+D6D-Glucose-6-PO4--w+w+D7D-Fructose-6-PO4--w+-+D8D-Aspartic Acid--w+++D9D-Serine-w-+-+D10Troleandomycin-++++wD11RifamycinSV---++-D12Minocycline-www+-E1Gelatin-++--+E2Glycyl-L-Proline--w+w+E3L-Alanine-+++++E4L-Arginine-ww--+E5L-Aspartic Acidw-w+-+E6L-Glutamic Acidw-w+w+E7L-Histidinew-++++E8L-Pyroglutamic Acidw-w+w+E9L-Serine-+w+w+E10Lincomycin-w+++-E11Guanidine HCl---w--E12Niaproof 4------F1Pectin-----+F2D-Galacturonic Acid--w+-+F3L-Galactonic Acid Lactone--w+w+F4D-Gluconic Acid-----+F5D-Glucuronic Acid---ww+F6Glucuronamidew--w++F7Mucic Acid--ww-+F8Quinic Acidw-w+w+F9D-Saccharic Acid--w+-+F10Vancomycin----+-F11Tetrazolium Violet--w-+-F12Tetrazolium Blue------G1p-Hydroxy-Phenylacetic Acid---w-+G2Methyl Pyruvatew----wG3D-Lactic Acid Methyl Ester-w++w+G4L-Lactic Acid-w-w-+G5Citric Acidw----+G6α-Keto-Glutaric Acid-w++++G7D-Malic Acid--w+-wG8L-Malic Acid-ww+++G9Bromo-Succinic Acid-----+G10Nalidixic Acid-+++++G11Lithium Chloridew+++++G12Potassium Telluritew++--wH1Tween 40-+++++H2γ-Amino-Butyric Acid---w-+H3α-Hydroxy-Butyric Acid---w-+H4β-Hydroxy-D, LButyric Acid--w+-+H5α-Keto-Butyric Acid-+--w+H6AcetoaceticAcidw+++++H7Propionic Acidwwwww+H8Acetic Acid-+++++H9Formic Acid---w-wH10Aztreonam-+++++H11Sodium Butyrate-+++++H12Sodium Bromate-------

[0046] The strains are: 1, strain 44XB; 2, Ornithinibacillus salinisoli CGMCC 1.15809; 3, Ornithinibacillus contaminans DSM 22953; 4, Ornithinibacillus bavariensis DSM 15681; 5, Oceanobacillus halotolerans CGMCC 1.17002; 6, Oceanobacillus iheyensis JCM 11309. +, positive; -, negative; w, weakly positive.

[0047] As shown in Tables 1, 2, and 3, strain 44XB was positive for oxidase and catalase. No positive results were observed in the API 20NE assay. In the API ZYM assay, the strain was positive for lipase (C8), alkaline phosphatase, and acid phosphatase. No substrates were found to be strongly utilized in the Biolog Gen III assay.

[0048] (3) Chemical characteristics. After freeze-drying the cells, polar lipids were extracted and separated, and analyzed using two-dimensional thin-layer chromatography. The results are shown in Figure 5. Whole-cell fatty acids were extracted from the strain, and the fatty acid composition was analyzed by gas chromatography using the MIDI Microbial Identification System. The results are shown in Table 4. Respiratory quinones were identified by UPLC-MS, and the results are shown in Figure 6.

[0049] Table 4 Fatty acid differences between strain 44XB and five reference strains

[0050]

[0051] Strains: 1, strain 44XB; 2, Ornithinibacillus salinisoli CGMCC1.15809; 3, Ornithinibacillus contaminans DSM 22953; 4, Ornithinibacillus bavariensis DSM 15681; 5, Oceanobacillus halotolerans CGMCC 1.17002; 6, Oceanobacillus iheyensis JCM 11309. Fatty acids with a content greater than 10% are considered major fatty acids and are indicated in bold. TR: trace amount (<1%)

[0052] The major whole-cell fatty acid (>10%) is anteiso-C 15:0 ,iso-C 15:0 ,iso-C 14:0 , C 16:0 and iso-C 16:0 , which was different from the reference strain.

[0053] Overall, the polar lipids of strain 44XB are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and three unknown aminophospholipids. The major respiratory quinone is MK-7, and the major whole-cell fatty acids are anteiso-C15:0, iso-C15:0, iso-C14:0, C16:0, and iso-C16:0.

[0054] (4) Genomic characterization. The 16S rRNA sequence of the strain was amplified by PCR using the standard primer pair 27F (SEQ ID NO. 14) and 1492R (SEQ ID NO. 15) and sequenced using the Sanger method. Sequences were aligned on the EzBiocloud server, and a phylogenetic tree was constructed using MEGA version X software (Figure 7). Whole-genome sequencing was performed using DNBSEQ sequencing and Nanopore.

[0055] The phylogenetic tree was reconstructed using the UBCG pipeline (Figure 8). The dDDH, ANI, AAI, and POCP values ​​of the strain and its related strains were calculated using the Genome-to-Genome Digital Calculator (GGDC), the Orthologous Average Nucleotide Identity Tool (OAT), the CompareM, and the POCP software packages, respectively.

[0056] The 16S rRNA sequence of strain 44XB obtained by Sanger sequencing is shown in SEQ ID NO.1.

[0057] Strain 44XB shared the highest 16S rRNA sequence similarities with Ornithinibacillus salinisoli LCB256, Ornithinibacillus contaminans CCUG 53201, Virgibacillus marseillensis Marseille-P3610, and Oceanobacillus profundus CL-MP28, at 96.36%, 96.17%, 95.75%, and 95.57%, respectively, which were far below the generally accepted interspecies 16S rRNA sequence similarity threshold of 98.65%, indicating that strain 44XB is a new species.

[0058] In the whole-genome phylogenetic tree (Figure 8), strain 44XB formed a highly supported, independent clade, indicating its unique evolutionary status, distinct from any existing genus. The maximum dDDH and ANI values ​​of 31.50% and 67.69% were significantly lower than the generally accepted species delineation thresholds of 70% and 95-96%. The maximum AAI value of 65.59% and the maximum POCP value of 50.92% further confirmed the taxonomic status of strain 44XB as a new genus, consistent with the conclusions of the whole-genome phylogenetic tree.

[0059] The phenotypic differences between strain 44XB and five reference strains were compared below, and the results are shown in Table 5.

[0060] Table 5 Phenotypic differences between strain 44XB and five reference strains

[0061]

[0062] Strains: 1, 44XB; 2, Ornithinibacillus salinisoli CGMCC 1.15809; 3, Ornithinibacillus contaminans DSM 22953; 4, Ornithinibacillus bavariensis DSM 15681; 5, Oceanobacillus halotolerans CGMCC 1.17002; 6, Oceanobacillus iheyensis JCM 11309. +, positive; -, negative; w, weakly positive.

[0063] As shown in Table 5 , based on the phylogenetic tree, strain 44XB differed from similar strains in some phenotypic characteristics, chemical characteristics, genome size, and CG mol%. Based on the above conclusions, 44XB was considered to be a new genus under the Bacillaceae family.

[0064] Example 2: Strain 44XB is capable of producing urate oxidase

[0065] 1. Detect urate oxidase activity using a uricase activity assay kit. After 10 h of culture, harvest the cells, wash with sterile phosphate buffer, and extract uricase according to the kit instructions. Detect enzyme activity using ultrasonication (5 s on, 7 s off) at 300 W.

[0066] The crude enzyme activity of urate oxidase produced by strain 44XB can reach 0.98 U / mL, and the crude specific activity can reach 5.14 U / mg protein.

[0067] 2. After whole-genome sequencing of strain 44XB, gene annotation was performed using the KEGG database. It was found that the genome of strain 44XB contains six copies of the pucL gene encoding urate oxidase. The nucleotide sequences of these genes are shown in SEQ ID NOs. 2-7, and the amino acid sequences of the urate oxidases they encode are shown in SEQ ID NOs. 8-13, respectively.

[0068] Example 3: Bacterial strain 44XB can reduce serum uric acid levels in hyperuricemic mice

[0069] 1. Hypoxanthine and potassium oxalate were used to establish a hyperuricemia mouse model. The treatment group was given 10 9 The mice were treated with colony-forming units of strain 44XB for 28 consecutive days. After the last experiment, the mice were killed and serum was collected. Serum uric acid levels were measured using an automatic biochemical analyzer.

[0070] The results, as shown in Figure 9, showed that the serum uric acid level in the model group was significantly elevated compared to the control group, reaching 576.6 µmol / L. Treatment with strain 44XB significantly reduced the serum uric acid level in the model mice, from 576.6 µmol / L to 352.9 µmol / L, with a statistically significant difference (p < 0.01). This demonstrates that strain 44XB can reduce serum uric acid in hyperuricemic mice and has broad application value.

[0071] Therefore, the present invention adopts the above-mentioned urate oxidase-producing strain 44XB and its application, which can be used for microecological therapy of hyperuricemia, reduce serum uric acid in hyperuricemia, and has the potential to be used in microecological treatment of hyperuricemia and gout.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A urate oxidase-producing strain 44XB, characterized in that: It is deposited in the Korean Type Culture Collection KCTC, with the deposit number KCTC 43631, the deposit date September 26, 2023, the deposit address 181 lpsin-gil. Jeongeup-si, Jeonbuk 56212, South Korea, and the classification name Ornithinibacillus sp.

2. The strain 44XB according to claim 1, characterized in that: The 16S rRNA sequence of strain 44XB is shown in SEQ ID NO.

1.

3. The strain 44XB according to claim 1, characterized in that: The genome of strain 44XB contains six pucL genes encoding urate oxidase.

4. The strain 44XB according to claim 3, characterized in that: The pucL gene sequences encoding uricase oxidase are shown in SEQ ID NOs. 2-7, and the amino acid sequences of the uricase oxidase encoded by the pucL gene are shown in SEQ ID NOs. 8-13, respectively.

5. Use of the strain 44XB according to any one of claims 1 to 4 in the preparation of a drug for treating hyperuricemia.

6. Use of the strain 44XB according to any one of claims 1 to 4 in the preparation of a drug for treating gout.

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