WO2021143725A1 - 黄嘌呤酰胺水解酶及其用途 - Google Patents

黄嘌呤酰胺水解酶及其用途 Download PDF

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WO2021143725A1
WO2021143725A1 PCT/CN2021/071501 CN2021071501W WO2021143725A1 WO 2021143725 A1 WO2021143725 A1 WO 2021143725A1 CN 2021071501 W CN2021071501 W CN 2021071501W WO 2021143725 A1 WO2021143725 A1 WO 2021143725A1
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xanthine
seq
amino acid
bacillus
amide hydrolase
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French (fr)
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张雁
李佩珊
仝杨
胡逸灵
刘大志
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Tianjin University
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Tianjin University
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Priority claimed from CN202010036500.6A external-priority patent/CN113186181A/zh
Priority claimed from CN202010036519.0A external-priority patent/CN113186180A/zh
Application filed by Tianjin University filed Critical Tianjin University
Priority to EP21741220.4A priority Critical patent/EP4092116A4/en
Priority to US17/793,014 priority patent/US20230064173A1/en
Priority to JP2022543080A priority patent/JP7546305B2/ja
Publication of WO2021143725A1 publication Critical patent/WO2021143725A1/zh
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Definitions

  • This application generally relates to the field of biomedicine technology; specifically, this application provides a new purine degradation pathway, enzymes participating in the pathway, and their uses, especially in the treatment of gout.
  • Gout is a type of arthritis caused by abnormal purine metabolism. It is characterized by recurring red, tender, burning, and swollen joints. The pain usually occurs quickly, reaching maximum intensity in less than 12 hours.
  • the cause of gout is the increase in the level of uric acid in the body, which causes the deposition of urate in the joints and kidneys, and uric acid is a product of purine metabolism.
  • the existing drugs for gout include colchicine, allopurinol, febuxostat and other non-steroidal anti-inflammatory drugs and glucocorticoids.
  • Colchicine inhibits the chemotaxis, adhesion and phagocytosis of neutrophils, so as to control local pain, swelling and inflammation in the joints.
  • Allopurinol and febuxostat are selective xanthine oxidase inhibitors that can treat gout by reducing the blood urate concentration, but the above drugs have relatively large side effects.
  • the present application provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has xanthine amide hydrolase activity.
  • polypeptide has a catalytic site defined as follows in spatial conformation:
  • the catalytic site comprises amino acid residues H59, H61, K151, H186, H242, and D316 that are close to each other in spatial conformation and refer to SEQ ID NO:1.
  • the catalytic site further comprises a divalent metal ion.
  • the polypeptide further comprises a binding site defined as follows in spatial conformation:
  • the binding site comprises amino acid residues I288, A289, P338 and G339 of SEQ ID NO:1 that are close to each other in spatial conformation.
  • the catalytic site is not more than 5 angstroms away from the binding site
  • the functional variant is a natural isozyme of the amino acid sequence shown in SEQ ID NO:1.
  • the functional variant is the insertion, substitution and/or of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO:1 or its natural isozyme. Resulting from missing.
  • the present application provides a nucleic acid molecule that encodes the polypeptide described in the first aspect.
  • the present application provides an expression cassette, which contains the nucleic acid molecule described in the second aspect.
  • the present application provides an expression vector, which comprises the nucleic acid molecule described in the second aspect or the expression cassette described in the third aspect.
  • the present application provides a host cell, which comprises the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, or the expression vector described in the fourth aspect.
  • the host cell can express and produce: a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has xanthine amide hydrolysis Enzyme activity.
  • polypeptide has a catalytic site defined as follows in spatial conformation:
  • the catalytic site comprises amino acid residues H59, H61, K151, H186, H242, and D316 that are close to each other in spatial conformation and refer to SEQ ID NO:1.
  • the catalytic site further comprises a divalent metal ion.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a yeast cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia, Lactobacillus, Bifidobacterium, Bacteroides and Firmicutes
  • the present application provides a pharmaceutical composition or health food, which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • a pharmaceutical composition or health food which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • the carrier or the host cell described in the fifth aspect and a pharmaceutically acceptable carrier or excipient which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • the carrier or the host cell described in the fifth aspect and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition or health food is used for the prevention and/or intervention and/or treatment of gout.
  • the present application provides the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, the expression vector described in the fourth aspect, and the host described in the fifth aspect Use of the cell or the pharmaceutical composition or health food of the sixth aspect in degrading purines.
  • the degradation of purines occurs in vitro.
  • this application provides the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, the expression vector described in the fourth aspect, and the host described in the fifth aspect Use of the cell or the pharmaceutical composition or health food of the sixth aspect in the preparation of a medicament for the prevention, intervention and/or treatment of gout.
  • this application provides a method for preventing, intervening and/or treating gout, which comprises administering the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, and the method of the third aspect to an individual in need
  • Figure 1 shows the human body's metabolic pathways that degrade various purines and the EC numbers of enzymes that catalyze each step of the reaction.
  • Figure 2 shows the metabolic reaction of xanthine oxidase to degrade xanthine to produce uric acid and its catalytic mechanism involving complex Mo-containing prosthetic groups.
  • Figure 3 shows the mechanism of allopurinol in the treatment of gout by inhibiting xanthine oxidase to prevent the production of uric acid.
  • Figure 4 shows the reaction formula (1) and the entire metabolic pathway for xanthine amide hydrolase to degrade xanthine under anaerobic conditions.
  • Figure 5 shows the electrophoresis results of the purified xanthine amide hydrolase, where lane M represents the molecular weight indicator, lane T is the bacterial lysate expressing xanthine amide hydrolase, and lane U represents the bacterial lysate after the Co affinity column , Lanes E 1 , E 2 , and E 3 represent 1, 2 and 4 ⁇ g xanthine amide hydrolase purified and eluted by a cobalt affinity column, respectively.
  • Figure 6 shows the LC-MS (liquid mass spectrometry) test results of the enzymatic activity of xanthine amide hydrolase catalyzing the hydrolysis and opening of xanthine, where A is the LC-MS test result of the negative control without enzyme, and B is the test group LC-MS detection results.
  • Figure 7 is a diagram of the active center of the xanthine amide hydrolase structure obtained by computer simulation by the PHYRE2 server (PHYRE2Protein Fold Recognition Server).
  • the catalytic site contains 4 histidines and a carboxylated lysine that are close to each other in spatial conformation.
  • Figure 8 is a phylogenetic tree diagram of all xanthine amide hydrolase enzymes in UniRef50_Q3AEA1. Gray markers are selected strains from each tree branch to represent Bacillus firmus, Clostridium cylindrosporum DSM 605 (Clostridium cylindrosporum DSM 605), Clostridium purinilyticum, Carbydothermus hydrogenoformans, strain ATCC BAA- 161/DSM 6008/Z-2901) and Paenibacillus donghaensis, the amino acid sequences of xanthine amide hydrolase encoded by them are A0A366K523, A0A0J8G334, A0A0L0W692, Q3AEA1 and A0A2Z2KEH1, respectively, in Figure 9 for sequence comparison.
  • Figure 9 shows five different species of Bacillus firmus, Clostridium cylindrosporum DSM 605 (Clostridium cylindrosporum DSM 605), Clostridium purinilyticum, and Carbydothermus hydrogenoformans (Carbydothermus hydrogenoformans, strain ATCC BAA-161/DSM 6008/ Z-2901) and Paenibacillus donghaensis, the encoded accession numbers are A0A366K523, A0A0J8G334, A0A0L0W692, Q3AEA1 and A0A2Z2KEH1 xanthine amide hydrolase sequence comparison results, wherein the catalytic site contains two divalent metals (such as Zn 2+ or Mn 2+, etc.) and four histidine (H), one lysine (K) and one aspartic acid (D) residues (solid box) coordinated with the gray background; the binding site contains The background that binds to xanthine is gray with
  • SEQ ID NO: 1 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus firmus (CGMCC1.2010).
  • SEQ ID NO: 2 shows the nucleotide sequence of the upstream primer used for PCR amplification of the nucleic acid molecule encoding xanthine amide hydrolase.
  • SEQ ID NO: 3 shows the nucleotide sequence of the downstream primer used for PCR amplification of the nucleic acid molecule encoding xanthine amide hydrolase.
  • SEQ ID NO: 4 shows the amino acid sequence of the xanthine amide hydrolase expressed by Clostridium purinilytica with the accession number A0A0L0W692.
  • SEQ ID NO: 5 shows the amino acid sequence of the xanthine amide hydrolase with the accession number A0A364K317 expressed by Micrococcus flavus FBKL4.011 (Thermoflavimicrobium sp. FBKL4.011).
  • SEQ ID NO: 6 shows the amino acid sequence of the xanthine amide hydrolase expressed by the salt-tolerant marine filamentous bacteria (Marininema halotolerans) with the accession number A0A1I6SUY8.
  • SEQ ID NO: 7 shows the amino acid sequence of the xanthine amide hydrolase expressed by Clostridiaceae bacterium with the accession number A0A3D2NSM2.
  • SEQ ID NO: 8 shows the amino acid sequence of the xanthine amide hydrolase with the accession number E5WNF6 expressed by Bacillus 2_A_57_CT2 (Bacillus sp.2_A_57_CT2).
  • SEQ ID NO: 9 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus FSL H8-0259 (Paenibacillus sp.FSL H8-0259) with the accession number A0A1R1CK18.
  • SEQ ID NO: 10 shows the amino acid sequence of the xanthine amide hydrolase expressed by Thermoflavimicrobium dichotomicum with the accession number A0A1I3U261.
  • SEQ ID NO: 11 shows the amino acid sequence of the xanthine amide hydrolase expressed by Fictibacillus enclensis with the accession number A0A0V8JCN2.
  • SEQ ID NO: 12 shows the amino acid sequence of the xanthine amide hydrolase expressed by the marine filamentous bacteria (Marininema mesophilum) with the accession number A0A1H2QVP9.
  • SEQ ID NO: 13 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus FSL R5-0912 (Paenibacillus sp.FSL R5-0912) with the accession number A0A089K5P4.
  • SEQ ID NO: 14 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus typhae with the accession number A0A1G9DGW2.
  • SEQ ID NO: 15 shows the amino acid sequence of the xanthine amide hydrolase expressed by Tissierella praeacuta DSM18095 with the accession number A0A1M4UHZ6.
  • SEQ ID NO: 16 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus cl95 (Bacillus sp. cl95) with the accession number A0A1I6C7J4.
  • SEQ ID NO: 17 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bradyrhizobium japonicum with the accession number A0A0A3XEE6.
  • SEQ ID NO: 18 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus firmus with the accession number A0A380XTT6.
  • SEQ ID NO: 19 shows the amino acid sequence of the xanthine amide hydrolase expressed by Acidaminobacter hydrogenoformans DSM 2784 with the accession number A0A1G5S5P6.
  • SEQ ID NO: 20 shows the amino acid sequence of the xanthine amide hydrolase expressed by the thermo-iron-reducing bacteria TR13 (Caloranaerobacter sp. TR13) with the accession number A0A0P8Z9T7.
  • SEQ ID NO: 21 shows the amino acid sequence of xanthine amide hydrolase expressed by Tissierella sp. P1 with the accession number A0A265Q2B2.
  • SEQ ID NO: 22 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus 3-2-2 (Bacillus sp. 3-2-2) with the accession number A0A429Y566.
  • SEQ ID NO: 23 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus donghaensis with the accession number A0A2Z2KEH1.
  • SEQ ID NO: 24 shows the amino acid sequence of the xanthine amide hydrolase with the accession number K0AWA5 expressed by Gottschalkia acidurici (strain ATCC7906/DSM 604/BCRC 14475/CIP 104303/NCIMB 10678/9a).
  • SEQ ID NO: 25 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus fortis with the accession number A0A443IKX3.
  • SEQ ID NO: 26 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus oceanisediminis with the accession number A0A2V2ZNB0.
  • SEQ ID NO: 27 shows the amino acid sequence of the xanthine amide hydrolase expressed by Virgibacillus profundi with the accession number A0A2A2IEE5.
  • SEQ ID NO: 28 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus sp. FSL R7-0331 (Paenibacillus sp.FSL R7-0331) with the accession number A0A089MDW3.
  • SEQ ID NO: 29 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus FJAT-21945 (Bacillus sp. FJAT-21945) with the accession number A0A0M0X8C9.
  • SEQ ID NO: 30 shows the amino acid sequence of xanthine amide hydrolase expressed by Tissierella praeacuta with the accession number A0A3F3S6I2.
  • SEQ ID NO: 31 shows the amino acid sequence of xanthine amide hydrolase expressed by Bacillus oceanisediminis with the accession number A0A1S1YDG4.
  • SEQ ID NO: 32 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus OV194 (Bacillus sp. OV194) with the accession number A0A1I1X526.
  • SEQ ID NO: 33 shows the amino acid sequence of the xanthine amide hydrolase expressed by Anaeromicrobium sediminis with the accession number A0A267MJF0.
  • SEQ ID NO: 34 shows the amino acid sequence of the xanthine amide hydrolase whose accession number is A0A1M5RDL0 expressed by Thermosyntropha lipolytica (Thermosyntropha lipolytica) DSM 11003.
  • SEQ ID NO: 35 shows the amino acid sequence of the xanthine amide hydrolase expressed by Alkaliphilus peptidifermentans DSM 18978 with the accession number A0A1G5KXH0.
  • SEQ ID NO: 36 shows the amino acid sequence of the xanthine amide hydrolase expressed by Aneurinibacillus migulanus with the accession number A0A1G8Q7H9.
  • SEQ ID NO: 37 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus IHB B 3415 (Paenibacillus sp. IHB B 3415) with the accession number A0A0B2F3Z2.
  • SEQ ID NO: 38 shows the amino acid sequence of the xanthine amide hydrolase expressed by Marinisporobacter balticus with the accession number A0A4R2KME8.
  • SEQ ID NO: 39 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus terrae with the accession number A0A429X0W8.
  • SEQ ID NO: 40 shows the amino acid sequence of the xanthine amide hydrolase expressed by Tindallia californiensis with the accession number A0A1H3Q6T8.
  • SEQ ID NO: 41 shows the amino acid sequence of the xanthine amide hydrolase with the accession number K0B360 expressed by Clostridium acidurici (strain ATCC7906/DSM 604/BCRC 14475/CIP 104303/NCIMB 10678/9a).
  • SEQ ID NO: 42 shows the amino acid sequence of the xanthine amide hydrolase expressed by Romboutsia lituseburensis DSM 797 with the accession number A0A1G9M635.
  • SEQ ID NO: 43 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paraclostridium bifermentans ATCC 19299 with the accession number T4V5T0.
  • SEQ ID NO: 44 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus praedii with the accession number A0A4R1ATL6.
  • SEQ ID NO: 45 shows the amino acid sequence of the xanthine amide hydrolase expressed by Carbydothermus islandicus with the accession number A0A1L8D5S0.
  • SEQ ID NO: 46 shows the amino acid sequence of the xanthine amide hydrolase expressed by Caloramator australicus RC3 with the accession number I7KTT3.
  • SEQ ID NO: 47 shows the amino acid sequence of the xanthine amide hydrolase with the accession number T4VRB8 expressed by Paraclostridium bifermentans ATCC 638.
  • SEQ ID NO: 48 shows the amino acid sequence of the xanthine amide hydrolase expressed by Tepidimicrobium xylanilyticum with the accession number A0A1H2RLU1.
  • SEQ ID NO: 49 shows the amino acid sequence of the xanthine amide hydrolase expressed by Aneurinibacillus migulanus with the accession number A0A0K2WJ73.
  • SEQ ID NO: 50 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus bacterium with the accession number A0A3D0EBN7.
  • SEQ ID NO: 51 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus notoginsengisoli with the accession number A0A417YW08.
  • SEQ ID NO: 52 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus oceanisediminis 2691 (Bacillus oceanisediminis 2691) with the accession number A0A160MBB4.
  • SEQ ID NO: 53 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus FSL R7-0273 (Paenibacillus sp.FSL R7-0273) with the accession number A0A089LXU3.
  • SEQ ID NO: 54 shows the amino acid sequence of the xanthine amide hydrolase with the accession number M1ZGS5 expressed by [Clostridium]ultunense Esp.
  • SEQ ID NO: 55 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus freudenreichii with the accession number A0A448FF64.
  • SEQ ID NO: 56 shows the amino acid sequence of the xanthine amide hydrolase expressed by Caloramator fervidus with the accession number A0A1H5RUL9.
  • SEQ ID NO: 57 shows the amino acid sequence of xanthine amide hydrolase expressed by Bacillus oceanisediminis with the accession number A0A4R8GVS7.
  • SEQ ID NO: 58 shows the amino acid sequence of the xanthine amide hydrolase expressed by Soehngenia saccharolytica with the accession number A0A4T9ZWH0.
  • SEQ ID NO: 59 shows the amino acid sequence of the xanthine amide hydrolase with the accession number W7LB72 expressed by Bacillus firmus DS1 (Bacillus firmus DS1).
  • SEQ ID NO: 60 shows the amino acid sequence of the xanthine amide hydrolase expressed by Thermotalea metallivorans with the accession number A0A140L3I5.
  • SEQ ID NO: 61 shows the amino acid sequence of the xanthine amide hydrolase expressed by Ornithinibacillus halophilus with the accession number A0A1M5FNK3.
  • SEQ ID NO: 62 shows the amino acid sequence of the xanthine amide hydrolase whose accession number is A0A1M5LQE8 expressed by Thermosyntropha lipolytica (Thermosyntropha lipolytica) DSM 11003.
  • SEQ ID NO: 63 shows the amino acid sequence of the xanthine amide hydrolase expressed by Fictibacillus sp. S7 with the accession number A0A4Q2HRQ1.
  • SEQ ID NO: 64 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus mesonae with the accession number A0A3Q9QZ31.
  • SEQ ID NO: 65 shows the amino acid sequence of the xanthine amide hydrolase expressed by Clostridium purinilytica with the accession number A0A0L0W688.
  • SEQ ID NO: 66 shows the amino acid sequence of the xanthine amide hydrolase expressed by Tindallia magadiensis with the accession number A0A1I3ETA9.
  • SEQ ID NO: 67 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus borealis with the accession number A0A089LHF8.
  • SEQ ID NO: 68 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paraclostridium benzoelyticum with the accession number A0A0M3DN30.
  • SEQ ID NO: 69 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus solani with the accession number A0A0Q3QMR7.
  • SEQ ID NO: 70 shows the amino acid sequence of the xanthine amide hydrolase expressed by Thermohalobacter berrensis with the accession number A0A419T2I0.
  • SEQ ID NO: 71 shows the amino acid sequence of the xanthine amide hydrolase expressed by Acinetobacter RIT592 (Acinetobacter sp. RIT592) with the accession number A0A369PBX6.
  • SEQ ID NO: 72 shows the amino acid sequence of the xanthine amide hydrolase expressed by Maledivibacter halophilus with the accession number A0A1T5JUM9.
  • SEQ ID NO: 73 shows the amino acid sequence of the xanthine amide hydrolase expressed by Tissierella creatinini with the accession number A0A4T9WHZ3.
  • SEQ ID NO: 74 shows the amino acid sequence of the xanthine amide hydrolase expressed by the compost metagenome (compost metagenome) with the accession number A0A3R1HSK2.
  • SEQ ID NO: 75 shows the amino acid sequence of the xanthine amide hydrolase expressed by Natronincola peptidivorans with the accession number A0A1I0F3P2.
  • SEQ ID NO: 76 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus firmus with the accession number A0A0J5YTU9.
  • SEQ ID NO: 77 shows the amino acid sequence of the xanthine amide hydrolase expressed by Anaerovirgula multivorans with the accession number A0A239FV01.
  • SEQ ID NO: 78 shows the amino acid sequence of the xanthine amide hydrolase with the accession number Q3AEA1 expressed by Carbydothermus hydrogenoformans (strain ATCC BAA-161/DSM 6008/Z-2901).
  • SEQ ID NO: 79 shows the amino acid sequence of xanthine amide hydrolase expressed by Sporanaerobacter acetigenes DSM 13106 with the accession number A0A1M5YST8.
  • SEQ ID NO: 80 shows the amino acid sequence of the xanthine amide hydrolase expressed by Proteiniborus sp. DW1 with the accession number A0A1M4MA63.
  • SEQ ID NO: 81 shows the amino acid sequence of the xanthine amide hydrolase expressed by Bacillus 7894-2 (Bacillus sp.7894-2) with the accession number A0A268IWM5.
  • SEQ ID NO: 82 shows the amino acid sequence of the xanthine amide hydrolase with the accession number A0A1M4PLJ1 expressed by [Clostridium]ultunense Esp.
  • SEQ ID NO: 83 shows the amino acid sequence of the xanthine amide hydrolase expressed by Virgibacillus indicus with the accession number A0A265N7L4.
  • SEQ ID NO: 84 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus NFR01 (Paenibacillus sp. NFR01) with the accession number A0A1I0KAI3.
  • SEQ ID NO: 85 shows the amino acid sequence of the xanthine amide hydrolase expressed by Andreesenia angus with the accession number A0A1S1V3Q5.
  • SEQ ID NO: 86 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus sp. DMB5 (Paenibacillus sp. DMB5) with the accession number A0A117T0P2.
  • SEQ ID NO: 87 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paludifilum halophilum with the accession number A0A235B9X8.
  • SEQ ID NO: 88 shows the amino acid sequence of the xanthine amide hydrolase expressed by Proteiniborus ethanoligenes with the accession number A0A1H3NJW1.
  • SEQ ID NO: 89 shows the amino acid sequence of the xanthine amide hydrolase expressed by Alkaliphilus metalliredigens (strain QYMF) with the accession number A6TWT7.
  • SEQ ID NO: 90 shows the amino acid sequence of the xanthine amide hydrolase expressed by Fictibacillus solisalsi with the accession number A0A1G9U6Z5.
  • SEQ ID NO: 91 shows the amino acid sequence of the xanthine amide hydrolase expressed by Sporosarcina globispora with the accession number A0A0M0GGH4.
  • SEQ ID NO: 92 shows the amino acid sequence of the xanthine amide hydrolase with the accession number A0A366K523 expressed by Bacillus firmus.
  • SEQ ID NO: 93 shows the amino acid sequence of the xanthine amide hydrolase expressed by Paenibacillus FSL R5-0490 (Paenibacillus sp.FSL R5-0490) with the accession number A0A1R1FFH4.
  • SEQ ID NO: 94 shows the amino acid sequence of the xanthine amide hydrolase expressed by Alkaliphilus sp. with the accession number A0A2G2MLE4.
  • SEQ ID NO: 95 shows the amino acid sequence of the xanthine amide hydrolase with the accession number A8MLA7 expressed by anaerobic arsenic-reducing bacteria (Alkaliphilus oremlandii, strain OhILAs).
  • SEQ ID NO: 96 shows the amino acid sequence of the xanthine amide hydrolase expressed by Caloramator mitchellensis with the accession number A0A0R3JVG6.
  • SEQ ID NO: 97 shows the amino acid sequence of the xanthine amide hydrolase expressed by Clostridium cylindrosporum DSM 605 (Clostridium cylindrosporum DSM 605) with the accession number A0A0J8G334.
  • SEQ ID NO: 98 shows the amino acid sequence of the xanthine amide hydrolase expressed by the ammonia bacterium CFH 90114 (Ammoniphilus sp. CFH 90114) with the accession number A0A4Q1SWC2.
  • SEQ ID NO: 99 shows the amino acid sequence of the xanthine amide hydrolase expressed by Carbydothermus pertinax with the accession number A0A1L8CSM0.
  • SEQ ID NO: 100 shows the amino acid sequence of the xanthine amide hydrolase expressed by Soehngenia sp. 1933P with the accession number A0A4Z0D783.
  • SEQ ID NO: 101 shows the amino acid sequence of the xanthine amide hydrolase expressed by Soehngenia saccharolytica with the accession number A0A4T9ZOT2.
  • SEQ ID NO: 102 shows the amino acid sequence of the xanthine amide hydrolase expressed by Natribacillus halophilus with the accession number A0A1G8N2H8.
  • SEQ ID NO: 103 shows the amino acid sequence of the xanthine amide hydrolase expressed by the compost metagenome (compost metagenome) with the accession number A0A403WBU6.
  • SEQ ID NO: 104 shows the amino acid sequence of the xanthine amide hydrolase with the accession number A0A3R4A6V9 expressed by the compost metagenome (compost metagenome).
  • Figure 1 shows the metabolic pathways of various purines in the human body.
  • the final product of various purine metabolism is urate, and the deposition of urate in joints and kidneys is the cause of gout.
  • Figure 2 shows the process by which the aerobic protein xanthine oxidase catalyzes the degradation of xanthine to produce uric acid.
  • the gout drug allopurinol inhibits the production of uric acid by inhibiting xanthine oxidase, thereby treating gout ( Figure 3).
  • GDVOGELS et al. Degradation of Purines and Pyrimidines by Microorganisms, Bacteriological Reviews, June 1976, Vol. 40, No. 2, p.
  • nucleic acids are written from left to right in the 5'to 3'direction, respectively; amino acid sequences are written from left to right in the amino to carboxy direction, respectively.
  • the number range includes the number that defines the range.
  • Amino acids can be represented herein by their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, the commonly accepted one-letter codes can be used to represent nucleotides. Refer to the specification as a whole to more fully define the terms defined above.
  • Polypeptide and “protein” in this application are used interchangeably herein and refer to polymers of amino acid residues and their variants and synthetic and naturally-occurring analogs. Therefore, these terms apply to naturally-occurring amino acid polymers and their naturally-occurring chemical derivatives, and to non-naturally-occurring amino acids in which one or more amino acid residues are synthetic (such as chemical analogs of the corresponding naturally-occurring amino acids ) Amino acid polymer.
  • Such derivatives include, for example, post-translational modifications and degradation products, including phosphorylated, glycosylated, oxidized, isomerized, carboxylated, and deaminated variants of polypeptide fragments.
  • enzyme active center refers to the part of the enzyme molecule that can directly bind to the substrate molecule and catalyze the chemical reaction of the substrate, and this part becomes the active center of the enzyme. It is generally believed that the active center is mainly composed of two functional sites: the first is the catalytic site, where the bond of the substrate is broken or a new bond is formed to cause certain chemical changes; the second is the binding site, which is the substrate of the enzyme. The substance binds to the enzyme molecule by this site.
  • the functional part is composed of a few amino acid residues or some groups on these residues that are relatively close in the three-dimensional structure of the enzyme molecule. They may be far apart in the primary structure, or even located on different peptide chains.
  • coenzyme molecules such as metal ions Zn 2+ and/or Mn 2+
  • a certain part of the structure of the coenzyme molecule is also a function The part of the site.
  • amino acid refers to a compound in which a hydrogen atom on a carbon atom of a carboxylic acid is replaced by an amino group, and the amino acid molecule contains two functional groups: an amino group and a carboxyl group. It includes naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally-occurring amino acids include 20 kinds of (L)-amino acids used in protein biosynthesis and other amino acids, such as 4-hydroxyproline, hydroxylysine, carboxylated lysine, catenin, isocatenin, high half Cystine, citrulline and ornithine.
  • Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethylthioine, etc., which are known to those skilled in the art.
  • Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution of chemical groups and moieties on amino acids, or derivatization of amino acids.
  • Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge space properties of amino acids.
  • an organic structure that mimics arginine has a positively charged moiety that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of a naturally occurring Arg amino acid.
  • Mimics also include constrained structures to maintain optimal space and charge interactions of amino acids or amino acid functional groups. Those skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
  • isoenzyme refers to enzymes that catalyze the same reaction in an organism but have different molecular structures.
  • nucleic acid refers to mRNA, RNA, cRNA, cDNA or DNA, including single-stranded and double-stranded forms of DNA.
  • the term generally refers to a polymeric form of nucleotides of at least 10 bases in length, the nucleotides being ribonucleotides or deoxynucleotides or a modified form of any type of nucleotide.
  • nucleic acid encoding when used in the context of a specific nucleic acid means that the nucleic acid contains the necessary information to direct the translation of the nucleotide sequence into a specific protein.
  • codons represents information about the encoded protein.
  • a nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) located within the translation region of the nucleic acid or may lack such intervening untranslated sequences (e.g., as in cDNA).
  • a "full-length sequence" related to a specific polynucleotide or the protein encoded by it refers to the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence.
  • the full-length polynucleotide encodes the full-length, catalytically active form of the specific protein.
  • isolated refers to a polypeptide or nucleic acid or a biologically active portion thereof, which is substantially or essentially free of those normally accompanied or reacted with the protein or nucleic acid as found in its naturally occurring environment Components. Therefore, when recombinant technology is used to produce isolated polypeptides or nucleic acids, the isolated polypeptides or nucleic acids are substantially free of other cellular materials or culture media, or when the isolated polypeptides or nucleic acids are chemically synthesized, they are substantially free of chemical precursors or other chemicals. .
  • expression vector as used herein is a recombinantly or synthetically produced nucleic acid construct that has a series of specific nucleic acid elements that allow specific nucleic acid to be transcribed in a host cell.
  • the term "host cell” as used herein refers to a cell that receives a foreign gene in transformation and transduction (infection).
  • the host cell may be a eukaryotic cell such as a yeast cell or a prokaryotic cell such as E. coli.
  • the present application provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has xanthine amide hydrolase activity.
  • the first aspect it has a catalytic site defined as follows in spatial conformation:
  • the catalytic site comprises amino acid residues H59, H61, K151, H186, H242, and D316 that are close to each other in spatial conformation and refer to SEQ ID NO:1.
  • the catalytic site further comprises a divalent metal ion.
  • the catalytic site further comprises two divalent metal ions, such as Zn 2+ and/or Mn 2+ .
  • polypeptide further comprises a binding site having the following definition in a spatial conformation:
  • the binding site comprises amino acid residues I288, A289, P338 and G339 of SEQ ID NO:1 that are close to each other in spatial conformation.
  • the distance between the catalytic site and the binding site is no more than 5 angstroms
  • the functional variant is a natural isozyme of the amino acid sequence shown in SEQ ID NO:1.
  • the natural isozyme is derived from: Bacillus firmus, compost metagenome, Clostridium purinilyticum, Thermoflavimicrobium sp., salt-tolerant marine Filamentous bacteria (Marininema halotolerans), Clostridiaceae bacterium, Bacillus sp., Paenibacillus sp., Thermoflavimicrobium dichotomicum, Fictibacillus enclensis, mesophilic ocean Filamentous bacteria (Marininema mesophilum), Paenibacillus typhae, Tissierella praeacuta, Bradyrhizobium japonicum, Acidaminobacter hydrogenoformans, and warm iron reducing bacteria (Caloranaerobacter sp.), tissue bacteria (Tissierella sp.), Paenibacillus donghaensis, Gottschalkia acidurici
  • the natural isozyme comprises the amino acid sequence shown in any one of SEQ ID NO: 4-104.
  • the functional variant is the insertion, substitution and/or substitution of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO:1 or its natural isozymes. Or missing.
  • the insertion, substitution and/or deletion does not occur at the catalytic site.
  • the insertion, substitution and/or deletion does not occur at the binding site.
  • the insertion, substitution and/or deletion does not occur at the catalytic site and the binding site.
  • the number of amino acid insertions, substitutions and/or deletions is 1-30, preferably 1-20, more preferably 1-10, wherein the obtained functional variant is basically The activity of xanthine amide hydrolase remains unchanged.
  • the functional variant differs from the amino acid sequence shown in SEQ ID NO:1 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids Insertions, substitutions and/or deletions.
  • the functional variant differs from the amino acid sequence shown in SEQ ID NO: 1 by about 1, 2, 3, 4 or 5 amino acid insertions, substitutions and/or deletions.
  • the polypeptide is an isolated polypeptide.
  • the present application provides a nucleic acid molecule that encodes the polypeptide described in the first aspect.
  • the nucleic acid molecule of the present application comprises a nucleic acid that hybridizes to a nucleotide sequence encoding the polypeptide shown in any one of SEQ ID NO: 1 and SEQ ID NO: 4-104 under stringent conditions.
  • a nucleotide sequence, or a nucleotide sequence that specifically hybridizes with any one of the polypeptides of SEQ ID NO: 1 and SEQ ID NO: 4-104 and is encoded with SEQ ID NO: 1 and SEQ ID NO: 4-104 The nucleic acid sequence composition of a polypeptide that is functionally equivalent to the polypeptide shown in any one of the items.
  • Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is in an environment below its melting temperature. The higher the degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Therefore, a higher relative temperature tends to make the reaction conditions more stringent, while at a lower temperature, the stringency is lower.
  • stringent conditions of the hybridization reaction please refer to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • stringent conditions used for DNA hybridization include: 1) Low ionic strength and high temperature are used for washing, for example, 0.015M sodium chloride/0.0015M sodium citrate/0.1% at 50°C. Sodium alkyl sulfate; 2) Use formamide and other denaturants during hybridization, such as 50% (v/v) formamide plus 0.1% bovine serum albumin/0.1% Ficoll/0.1% polydiene pyrrolidone/pH 6.5 at 42°C 50mM sodium phosphate buffer and 750mM sodium chloride, 75mM sodium citrate; or (3) hybridize overnight at 42°C, the hybridization solution contains 50% formamide, 5 ⁇ SSC (0.75M sodium chloride, 0.075M citric acid Sodium), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt's solution, sonicated salmon sperm DNA (50mg/mL), 0.1% SDS and 10% dextran sulfate
  • moderately stringent conditions can be determined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989.
  • Moderately stringent conditions include the use of washing solutions and hybridization conditions (such as temperature, ionic strength, and SDS percentage) that are less stringent than those described above.
  • moderately stringent conditions include hybridization with at least about 16% v/v to at least about 30% v/v formamide and at least about 0.5 M to at least about 0.9 M salt at 42°C, and at least about 0.1 M to at least About 0.2M salt is washed at 55°C.
  • Moderately stringent conditions can also include hybridization with 1% bovine serum albumin (BSA), 1mM EDTA, 0.5M NaHPO 4 (pH 7.2), 7% SDS at 65°C, and (i) 2 ⁇ SSC, 0.1% SDS; Or (ii) 0.5% BSA, 1mM EDTA, 40mM NaHPO 4 (pH 7.2), 5% SDS wash at 60-65°C. Professionals will adjust the temperature and ionic strength according to the length of the probe and other factors. The stringency of hybridizing nucleic acids depends on the length and degree of complementarity of the nucleic acid molecules, as well as other variables well known in the art.
  • the minimum length of a hybridizable nucleic acid is at least about 12 nucleotides, preferably at least about 16, more preferably at least about 24, and most preferably at least about 36 nucleotides.
  • nucleic acid molecules of the present application can be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction sites, multiple cloning sites, other coding segments, etc., so that their total The length can vary significantly. It is therefore considered that polynucleotide fragments of almost any length can be utilized; the total length is preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid molecules encoding the polypeptides of the present application or functional variants thereof can be used in recombinant DNA molecules to direct the expression of the polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can also be used in this application, and these sequences can be used to clone and express a given polypeptide.
  • nucleic acid molecules of the present application can be modified, including but not limited to the cloning, processing, expression and/or activity modification of gene products.
  • the nucleic acid molecule is produced by artificial synthesis, such as direct chemical synthesis or enzymatic synthesis.
  • the nucleic acid molecule is produced by recombinant technology.
  • the nucleic acid molecule is an isolated nucleic acid molecule.
  • the present application provides an expression cassette, which contains the nucleic acid molecule described in the second aspect.
  • the expression cassette may additionally include a 5'leader sequence, which can play a role in enhancing translation.
  • various DNA fragments can be manipulated to provide the DNA sequence in the proper orientation and in the proper reading frame when appropriate.
  • adaptors or linkers can be used to connect DNA fragments, or other operations can be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, and so on.
  • it may involve in vitro mutagenesis, primer repair, restriction, annealing, and replacement, such as transition and transversion.
  • the present application provides an expression vector, which comprises the nucleic acid molecule described in the second aspect or the expression cassette described in the third aspect.
  • any suitable expression vector can be used in this application.
  • the expression vector can be any of pET28, pET14, HT-1N-TAG-2691, pRS416 and other vectors.
  • the nucleic acid molecule encoding the polypeptide shown in any one of SEQ ID NO: 1 and SEQ ID NO: 4-104 is cloned into a vector to form a nucleic acid molecule containing the nucleic acid molecule described in the present application. Recombinant vector.
  • the expression vector used to clone the polynucleotide is a plasmid vector.
  • the above-mentioned expression vector further comprises a control sequence for regulating the expression of the nucleic acid molecule, wherein the nucleic acid molecule is operably linked to the control sequence.
  • regulatory sequence refers to a polynucleotide sequence required to achieve expression of a coding sequence linked to it.
  • the nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and terminator; in eukaryotes, such regulatory sequences generally include promoters, terminators, and in some cases enhancers. Therefore, the term “regulatory sequence” includes all sequences whose existence is the minimum necessary for the expression of the target gene, and may also include other sequences whose existence is advantageous for the expression of the target gene, such as leader sequences.
  • operably linked refers to a situation where the involved sequences are in a relationship that allows them to function in a desired manner.
  • a regulatory sequence "operably linked" to a coding sequence allows the expression of the coding sequence to be achieved under conditions compatible with the regulatory sequence.
  • nucleotide sequence that encodes the polypeptide shown in any one of SEQ ID NO: 1 and SEQ ID NO: 4-104 and suitable Expression vector for transcription/translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the nucleotide sequence is operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.
  • promoters include: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, Retrovirus LTRs and some other known promoters that can control gene expression in prokaryotic cells or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the replication initiation point, polyoma enhancers and adenovirus enhancers on the late side of the replication initiation point, and the like.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as those encoding kanamycin sulfate resistance, ampicillin resistance, and the like.
  • the present application provides a host cell, which comprises the nucleic acid molecule described in the second aspect or the expression cassette described in the third aspect or the expression vector described in the fourth aspect.
  • the host cell can express and produce: a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has xanthine amide hydrolysis Enzyme activity.
  • polypeptide has a catalytic site defined as follows in spatial conformation:
  • the catalytic site comprises amino acid residues H59, H61, K151, H186, H242, and D316 that are close to each other in spatial conformation and refer to SEQ ID NO:1.
  • the catalytic site further comprises a divalent metal ion.
  • the catalytic site further comprises two divalent metal ions, such as Zn 2+ and/or Mn 2+ .
  • polypeptide has a catalytic site and a binding site defined as follows in a spatial conformation:
  • the catalytic site comprises amino acid residues H59, H61, K151, H186, H242 and D316 that are close to each other in spatial conformation and with reference to SEQ ID NO:1 and 2 divalent metal ions (e.g. Zn 2+ and/or Mn 2 + ); and
  • the binding site comprises amino acid residues I288, A289, P338 and G339 of SEQ ID NO:1 that are close to each other in spatial conformation.
  • the usable host cell is a cell containing the above-mentioned expression vector, which may be a eukaryotic cell, for example, a yeast cell culture system may be used for the expression of the polypeptide of the present application.
  • the host cell may also be a prokaryotic cell containing the above-mentioned expression vector, for example, may be selected from the genus Escherichia (for example, Escherichia coli), Lactobacillus, Bifidobacterium, Bacteroides, Firmicutes and the like.
  • the host cell is a yeast cell or E. coli.
  • the nucleic acid molecule encoding one or more enzymes of the present application may exist in the host cell in the form of an episomal vector, or may also be integrated into the genome of the host cell.
  • the isolated nucleic acid is operably linked to a regulatory sequence, which can be recognized by a host cell transformed with the expression vector.
  • the expression vector can be introduced into the host cell using any technique known in the art, including transformation, transduction, transfection, viral infection, gene gun or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, ( 1986)). As an example, when the host is a prokaryotic organism such as Escherichia coli, competent cells can be harvested after the exponential growth phase and transformed by the CaCl 2 method well known in the art.
  • the present application provides a pharmaceutical composition or health food, which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • a pharmaceutical composition or health food which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • the carrier or the host cell described in the fifth aspect and a pharmaceutically acceptable carrier or excipient which comprises the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, and the expression described in the fourth aspect
  • the carrier or the host cell described in the fifth aspect and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition or health food is used for the prevention, intervention and/or treatment of gout.
  • the pharmaceutical composition or health food may further comprise one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; Emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate and mineral oil
  • wetting agents such as talc, magnesium stearate and mineral oil
  • Emulsifiers such as talc, magnesium stearate and mineral oil
  • suspending agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents etc.
  • the pharmaceutical composition or health food in the present application can be formulated into tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules, etc. form.
  • any method that can be administered to the intestinal tract can be used to deliver the pharmaceutical composition or health food of the present application, preferably oral administration.
  • the host cells of the fifth invention are prepared into enteric-coated capsules for oral administration.
  • the drug for therapeutic use can be formulated in the form of a lyophilized preparation or an aqueous solution by mixing a reagent with the required purity with a pharmaceutically acceptable carrier, excipient, etc. as appropriate.
  • the composition is used for storage.
  • the present application provides the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, the expression vector described in the fourth aspect, and the host described in the fifth aspect Use of the cell or the pharmaceutical composition or health food of the sixth aspect in degrading purines.
  • the degradation of purines occurs in vitro.
  • the degradation of purines is performed in an anaerobic environment, such as in the intestine.
  • this application provides the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette described in the third aspect, the expression vector described in the fourth aspect, and the host described in the fifth aspect Use of the cell or the pharmaceutical composition or health food of the sixth aspect in the preparation of a medicament for the prevention, intervention and/or treatment of gout.
  • the present application provides a method for preventing, intervening and/or treating gout, which comprises administering the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, and the method of the third aspect to an individual in need
  • the xanthine amide hydrolase shown in SEQ ID NO: 1 degrades xanthine into 4-ureido-5-carboxyimidazole, thereby reducing the acquisition of purine by human cells And it is converted into uric acid, which can degrade xanthine in the intestinal tract to prevent, intervene and/or treat gout.
  • the expression vector containing gene 1 encoding xanthine amide hydrolase constructed in Example 1 was transformed into Escherichia coli BL21 cells, and spread on LB agar plates containing 50 ⁇ g/mL kanamycin sulfate 37 Incubate overnight at °C. Pick a single colony and culture it overnight in 5mL LB liquid medium containing kanamycin sulfate at 37°C and 220rpm, and transfer it to 800mL LB medium for expansion the next day. When the OD 600 value reaches about 0.8, change the temperature The temperature was reduced to 18°C, and IPTG was added at a final concentration of 0.3 mM to induce protein expression.
  • the cells were collected by centrifugation at 4000 ⁇ g for 10 min at 4°C. Resuspend the bacterial pellet in 35mL lysis buffer [50mM Tris/HCl, pH 8.0, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.2mg/mL lysozyme, 0.03% Triton X-100 and 1 ⁇ L DNase I], And frozen and preserved at -80°C.
  • lysis buffer 50mM Tris/HCl, pH 8.0, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.2mg/mL lysozyme, 0.03% Triton X-100 and 1 ⁇ L DNase I
  • the cells were taken out from -80°C and incubated in a water bath at 25°C for 40 minutes to lyse the cells. Add 6 mL of 6% streptomycin sulfate aqueous solution, mix gently, and centrifuge at 20,000 ⁇ g for 5 minutes at 4°C. The supernatant was filtered with a 0.22 ⁇ m filter membrane and bound to a 5mL TALON cobalt column equilibrated with buffer A (20mM Tris-HCl 7.5, 0.2M KCl), and the column was washed with 10 times the column volume of buffer A.
  • buffer A (20mM Tris-HCl 7.5, 0.2M KCl
  • the target protein was eluted with 5 column volumes of buffer B containing 150 mM imidazole (20 mM Tris-HCl 7.5, 0.2 M KCl, 150 mM imidazole, 5 mM ⁇ -mercaptoethanol).
  • buffer B containing 150 mM imidazole (20 mM Tris-HCl 7.5, 0.2 M KCl, 150 mM imidazole, 5 mM ⁇ -mercaptoethanol.
  • ammonium sulfate to the eluted protein solution to 70% saturated precipitated protein, centrifuge at 10,000 ⁇ g for 10 minutes, discard the supernatant, and use 5mL buffer C (20mM Tris-HCl 7.5, 0.2M KCl, 10 % (V/v) glycerol) was resuspended, and a G25 column was used to remove the salt.
  • the target protein After removing the salt, the target protein is divided into aliquots, quick-frozen with liquid nitrogen, and stored at -80°C for freezer storage.
  • the final concentration of xanthine amide hydrolase is detected by a spectrophotometer. According to the protein sequence, the extinction coefficient of xanthine amide hydrolase is 55,810M -1 cm -1 . On average, about 35mg of protein can be purified per liter.
  • Figure 5 shows the process of expression and purification of xanthine amide hydrolase, and its migration rate on gel electrophoresis conforms to the actual molecular weight (55.7kDa).
  • the PHYRE2 server was used to simulate the structure of the enzyme (Figure 7), showing that it contains 4 histidines, one A catalytic site composed of lysine, an aspartic acid, and two divalent metal ions (Zn 2+ and/or Mn 2+ ). Furthermore, computer software was used to show the anchoring of the substrate xanthine to the simulated xanthine amide hydrolase.
  • the active center also includes an isoleucine, an alanine, a proline and a glycine in the spatial conformation of each other. The binding site close to the composition interacts with the substrate xanthine ( Figure 7).
  • the inventor of the present application further found the sequence of this xanthine amide hydrolase isoenzyme UniRef50 from the Uniprot database (covering 50% sequence identity, and at the same time more than 80% of the xanthine amide hydrolase length of the protein sequence), the above sequences are all in The UniRef50_Q3AEA1 cluster includes a total of 101 protein sequences, and the phylogenetic tree analysis of these 101 proteins from different species ( Figure 8).
  • a representative protein is selected from each evolutionary branch, and there are five proteins in total, which are derived from Bacillus firmus, Clostridium cylindrosporum DSM 605 (Clostridium cylindrosporum DSM 605), Clostridium purinilyticum, and hydrogen-producing carboxyl thermophilic Bacteria (Carbydothermus hydrogenoformans, strain ATCC BAA-161/DSM 6008/Z-2901) and Paenibacillus donghaensis, the accession numbers encoded by them are A0A366K523, A0A0J8G334, A0A0L0W692, Q3AEA1, and A0A2Z2KEH1. Comparison.
  • Escherichia coli capable of expressing xanthine amide hydrolase (SEQ ID NO: 1) was constructed.
  • the key operation of this example is to integrate the gene encoding xanthine amide hydrolase into the E. coli genome, for example, one copy of the multi-copy 16sRNA, so that it can be stably expressed, and at the same time, the control of guanine transporter and guanine deaminase
  • the promoter is replaced with the gapA promoter, allowing the gene encoding xanthine amide hydrolase to continuously and stably express.
  • the promoters encoding guanine transporter and guanine deaminase with the constitutive and continuous and stable expression promoter gapA.
  • the original promoter usually lacks nitrogen sources in the environment and needs to use guanine nitrogen. When the nitrogen source is sufficient, the gene controlled by it is not expressed.
  • the control coded guanine transporter and guanine deamination In order to make the guanine in the food degraded by the xanthine amide hydrolase of the present invention, it is necessary to replace the control coded guanine transporter and guanine deamination.
  • the promoter of the enzyme gene allows it to be expressed at any time, thereby effectively transporting guanine into the above-mentioned engineered E.
  • Example 7 Escherichia coli expressing xanthine amide hydrolase is used for the treatment of gout
  • Example 6 Using oxonic acid-induced hyperuric acid rats or uricase knockout transgenic mice as gout animal models, various Escherichia coli prepared in Example 6 were made into enteric-coated capsules. Under the same feeding conditions, test whether various E. coli enteric-coated capsules can reduce the blood uric acid content of gout rats and mouse models.
  • Example 8 Escherichia coli expressing xanthine amide hydrolase is used for the treatment of gout
  • Animal pre-adaptation ⁇ 7 days of pre-adaptation process, feeding with ordinary feed and ordinary drinking water.
  • Antibiotic pretreatment feed with ordinary feed, add 2mg/mL streptomycin + 1mg/mL ampicillin to the drinking water after feeding rats for 3 days, stop drinking water for more than 6 hours, and then give the subsequent gavage treatment.
  • mice were fed with 1% (w/w) adenine feed every day, and 200 ⁇ L containing 2 ⁇ 10 ⁇ 10 E. coli suspension prepared in Example 6 was used for gavage treatment for two weeks .
  • Hyperuricemia group The rats were fed a diet containing 1% (w/w) adenine every day for two weeks.
  • Control group rats were fed with ordinary diet daily for two weeks.
  • mice choose Balb/c mice (8 weeks old), 5 mice in each group, and divide them into experimental group, hyperuricemia group and control group under the same feeding conditions.
  • the specific experimental process is as follows:
  • Animal pre-adaptation ⁇ 7 days of pre-adaptation process, feeding with ordinary feed and ordinary drinking water.
  • Antibiotic pretreatment feed with ordinary feed, add 2mg/mL streptomycin + 1mg/mL ampicillin to the drinking water. After feeding the mice for 3 days, stop drinking water for more than 6 hours, then give the follow-up gavage treatment.
  • mice were fed with 0.1% (w/w) adenine feed and 200 mg/kg potassium oxycyanate was administered by gavage every day, and 100 ⁇ L contained 1 ⁇ 10 ⁇ 10 large intestine prepared in Example 6 The bacillus suspension was treated by gavage for two weeks.
  • Hyperuricemia group mice were fed 0.1% (w/w) adenine feed and 200 mg/kg potassium oxycyanate by gavage every day for two weeks.
  • Control group normal feed was fed every day for two weeks.

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Abstract

黄嘌呤酰胺水解酶及其用途，特别是在治疗痛风中的用途。

Description

黄嘌呤酰胺水解酶及其用途
相关申请的交叉引用
本申请要求于2020年1月14日递交的中国专利申请第202010036500.6号和第202010036519.0号的优先权，其全部内容通过引用整体并入本文。
技术领域
本申请大体涉及生物医药技术领域；具体而言，本申请提供了新的嘌呤降解通路、参与该通路的酶及它们的用途，特别是在治疗痛风中的应用。
背景技术
嘌呤的吸收主要发生在肠道，肠道为无氧环境。痛风是嘌呤代谢异常导致的一种关节炎，其特征是反复发作的红色、触痛、灼热和肿胀的关节。疼痛通常很快发生，在不到12小时内达到最大强度。造成痛风的原因是体内尿酸水平的升高，造成了尿酸盐在关节和肾脏部位的沉积，而尿酸是嘌呤代谢的产物。
目前已有的治疗痛风的药物有秋水仙碱、别嘌呤醇、非布司他以及其它非甾体类抗炎药和糖皮质激素等。秋水仙碱通过抑制中性白细胞的趋化、黏附和吞噬作用等，从而达到控制关节局部的疼痛、肿胀及炎症反应。别嘌呤醇和非布司他为选择性黄嘌呤氧化酶抑制剂，可通过降低血尿酸盐浓度而发挥治疗痛风的作用，但以上药物均有较大的副作用。
新的用于预防、干预和/或治疗痛风的药物的开发和应用是本领域所需要的。
发明概述
第一方面，本申请提供了多肽，其包含SEQ ID NO:1所示的氨基酸序列或其功能变体，其中所述功能变体具有黄嘌呤酰胺水解酶活性。
在第一方面的一些实施方案中，其中所述多肽在空间构象上具有如下定义的催化部位：
所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基。
在第一方面的一些实施方案中，所述催化部位还包含二价金属离子。
在第一方面的一些实施方案中，所述多肽还包含在空间构象上具有如下定义的结合部位：
所述结合部位包含在空间构象上相互靠近的、参照SEQ ID NO:1的I288、A289、P338和G339氨基酸残基。
在第一方面的一些实施方案中，其中所述催化部位与所述结合部位相距不大于5埃
Figure PCTCN2021071501-appb-000001
在第一方面的一些实施方案中，所述功能变体为SEQ ID NO:1所示的氨基酸序列的天然同工酶。
在第一方面的一些实施方案中，其中所述功能变体为在SEQ ID NO:1所示的氨基酸序列或其天然同工酶的基础上发生一个或多个氨基酸的插入、取代和/或缺失而产生的。
在第一方面的一些实施方案中，其中所述插入、取代和/或缺失不发生在催化部位和/或结合部位。
第二方面，本申请提供了核酸分子，其编码第一方面所述的多肽。
第三方面，本申请提供了表达盒，其包含第二方面所述的核酸分子。
第四方面，本申请提供了表达载体，其包含第二方面所述的核酸分子或第三方面所述的表达盒。
第五方面，本申请提供了宿主细胞，其包含第二方面所述的核酸分子、第三方面所述的表达盒或第四方面所述的表达载体。
在第五方面的一些实施方案中，所述宿主细胞能够表达并产生：多肽，其包含SEQ ID NO:1所示的氨基酸序列或其功能变体，其中所述功能变体具有黄嘌呤酰胺水解酶活性。
在第五方面的一些实施方案中，其中所述多肽在空间构象上具有如下定义的催化部位：
所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基。
在第五方面的一些实施方案中，所述催化部位还包含二价金属离子。
在第五方面的一些实施方案中，所述宿主细胞为真核细胞或原核细胞。
在第五方面的一些实施方案中，所述真核细胞为酵母细胞。
在第五方面的一些实施方案中，所述原核细胞选自埃希氏菌属、乳酸杆菌属、双歧杆菌属、拟杆菌门和厚壁菌门
第六方面，本申请提供了药物组合物或保健食品，其包含第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体或第五方面所述的宿主细胞和药学上可接受的载体或赋形剂。
在第六方面的一些实施方案中，所述药物组合物或保健食品用于预防和/或干预和/或治疗痛风。
第七方面，本申请提供了第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品在降解嘌呤中的用途。
在第七方面的一些实施方案中，所述降解嘌呤在体外发生。
第八方面，本申请提供了第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品在制备用于预防、干预和/或治疗痛风的药物中的用途。
第九方面，本申请提供了预防、干预和/或治疗痛风的方法，其 包括向有需要的个体给予第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品。
附图说明
图1为人体降解各种嘌呤的代谢通路及催化每一步反应的酶的EC编号。
图2为黄嘌呤氧化酶降解黄嘌呤产生尿酸的代谢反应及其涉及复杂含Mo辅基的催化机理。
图3为别嘌呤醇通过抑制黄嘌呤氧化酶阻止尿酸生成的治疗痛风的机理。
图4为黄嘌呤酰胺水解酶在无氧条件下降解黄嘌呤的反应式(1)及整个代谢通路。
图5为纯化后的黄嘌呤酰胺水解酶的电泳结果，其中泳道M代表分子量标识，泳道T为表达黄嘌呤酰胺水解酶的细菌裂解液，泳道U代表为经Co亲和柱后的细菌裂解液，泳道E 1、E 2、E 3分别代表经钴亲和柱纯化洗脱的1、2和4μg黄嘌呤酰胺水解酶。
图6为黄嘌呤酰胺水解酶催化黄嘌呤水解开环的酶活性LC-MS(液质联用)检测结果，其中A为不加酶的阴性对照的LC-MS检测结果，B为实验组的LC-MS检测结果。
图7为经PHYRE2服务器(PHYRE2Protein Fold Recognition Server)进行计算机模拟得到的黄嘌呤酰胺水解酶结构的活性中心的图，其中催化部位包含空间构象上相互靠近的4个组氨酸、一个羧基化的赖氨酸、一个天冬氨酸以及两个二价金属离子(例如Zn 2+和/或Mn 2+)；结合部位包含空间构象上相互靠近的与黄嘌呤相互作用的一个异亮氨酸、一个丙氨酸、一个脯氨酸和一个甘氨酸残基。
图8为UniRef50_Q3AEA1所有黄嘌呤酰胺水解酶的进化系统树图。灰色标记为在各树状分支中选取菌株代表坚强芽孢杆菌(Bacillus firmus)、柱胞梭菌DSM 605(Clostridium cylindrosporum DSM 605)、Clostridium purinilyticum、产氢羧基嗜热菌(Carbydothermus  hydrogenoformans，菌株ATCC BAA-161/DSM 6008/Z-2901)以及Paenibacillus donghaensis，将它们编码的登录号分别为A0A366K523、A0A0J8G334、A0A0L0W692、Q3AEA1和A0A2Z2KEH1的黄嘌呤酰胺水解酶的氨基酸序列在图9中进行序列对比。
图9为五个不同物种坚强芽孢杆菌(Bacillus firmus)、柱胞梭菌DSM 605(Clostridium cylindrosporum DSM 605)、Clostridium purinilyticum、产氢羧基嗜热菌(Carbydothermus hydrogenoformans，菌株ATCC BAA-161/DSM 6008/Z-2901)以及Paenibacillus donghaensis，编码的登录号分别为A0A366K523、A0A0J8G334、A0A0L0W692、Q3AEA1和A0A2Z2KEH1的黄嘌呤酰胺水解酶的序列对比结果，其中催化部位包含两个二价金属(例如Zn 2+或Mn 2+等)及与其配位的背景为灰色的四个组氨酸(H)、一个赖氨酸(K)和一个天冬氨酸(D)残基(实线方框)；结合部位包含与黄嘌呤结合的背景为灰色的一个异亮氨酸(I)、一个丙氨酸(A)、一个脯氨酸(P)和一个甘氨酸残基(G)。
序列说明
SEQ ID NO：1显示了坚强芽孢杆菌(Bacillus firmus，CGMCC1.2010)表达的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：2显示了用于PCR扩增编码黄嘌呤酰胺水解酶的核酸分子的上游引物的核苷酸序列。
SEQ ID NO：3显示了用于PCR扩增编码黄嘌呤酰胺水解酶的核酸分子的下游引物的核苷酸序列。
SEQ ID NO：4显示了Clostridium purinilytica表达的登录号为A0A0L0W692的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：5显示了高温黄色微球菌FBKL4.011(Thermoflavimicrobium sp.FBKL4.011)表达的登录号为A0A364K317的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：6显示了耐盐海洋丝状菌(Marininema halotolerans)表达的登录号为A0A1I6SUY8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：7显示了梭菌科细菌(Clostridiaceae bacterium)表达的登录号为A0A3D2NSM2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：8显示了芽孢杆菌2_A_57_CT2(Bacillus sp.2_A_57_CT2)表达的登录号为E5WNF6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：9显示了类芽孢杆菌FSL H8-0259(Paenibacillus sp.FSL H8-0259)表达的登录号为A0A1R1CK18的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：10显示了双岐高温黄色微球菌(Thermoflavimicrobium dichotomicum)表达的登录号为A0A1I3U261的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：11显示了Fictibacillus enclensis表达的登录号为A0A0V8JCN2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：12显示了中温海洋丝状菌(Marininema mesophilum)表达的登录号为A0A1H2QVP9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：13显示了类芽孢杆菌FSL R5-0912(Paenibacillus sp.FSL R5-0912)表达的登录号为A0A089K5P4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：14显示了香蒲类芽孢杆菌(Paenibacillus typhae)表达的登录号为A0A1G9DGW2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：15显示了极尖组织菌(Tissierella praeacuta)DSM18095表达的登录号为A0A1M4UHZ6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：16显示了芽孢杆菌cl95(Bacillus sp.cl95)表达的登录号为A0A1I6C7J4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：17显示了慢生型大豆根瘤菌(Bradyrhizobium japonicum)表达的登录号为A0A0A3XEE6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：18显示了坚强芽孢杆菌(Bacillus firmus)表达的登录号为A0A380XTT6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：19显示了产氢氨基酸杆菌(Acidaminobacter hydrogenoformans)DSM 2784表达的登录号为A0A1G5S5P6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：20显示了温铁还原细菌TR13(Caloranaerobacter sp.TR13)表达的登录号为A0A0P8Z9T7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：21显示了组织菌P1(Tissierella sp.P1)表达的登录号为A0A265Q2B2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：22显示了芽孢杆菌3-2-2(Bacillus sp.3-2-2)表达的登录号为A0A429Y566的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：23显示了Paenibacillus donghaensis表达的登录号为A0A2Z2KEH1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：24显示了Gottschalkia acidurici(菌株ATCC7906/DSM 604/BCRC 14475/CIP 104303/NCIMB 10678/9a)表达的登录号为K0AWA5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：25显示了强壮芽孢杆菌(Bacillus fortis)表达的登录号为A0A443IKX3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：26显示了海泥芽孢杆菌(Bacillus oceanisediminis)表达的登录号为A0A2V2ZNB0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：27显示了Virgibacillus profundi表达的登录号为A0A2A2IEE5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：28显示了类芽孢杆菌FSL R7-0331(Paenibacillus sp.FSL R7-0331)表达的登录号为A0A089MDW3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：29显示了芽孢杆菌FJAT-21945(Bacillus sp.FJAT-21945)表达的登录号为A0A0M0X8C9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：30显示了极尖组织菌(Tissierella praeacuta)表达的登录号为A0A3F3S6I2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：31显示了海泥芽孢杆菌(Bacillus oceanisediminis)表 达的登录号为A0A1S1YDG4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：32显示了芽孢杆菌OV194(Bacillus sp.OV194)表达的登录号为A0A1I1X526的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：33显示了Anaeromicrobium sediminis表达的登录号为A0A267MJF0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：34显示了解脂嗜热互营杆菌(Thermosyntropha lipolytica)DSM 11003表达的登录号为A0A1M5RDL0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：35显示了Alkaliphilus peptidifermentans DSM 18978表达的登录号为A0A1G5KXH0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：36显示了米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)表达的登录号为A0A1G8Q7H9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：37显示了类芽孢杆菌IHB B 3415(Paenibacillus sp.IHB B 3415)表达的登录号为A0A0B2F3Z2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：38显示了Marinisporobacter balticus表达的登录号为A0A4R2KME8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：39显示了土地芽孢杆菌(Bacillus terrae)表达的登录号为A0A429X0W8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：40显示了加利福尼亚丁达尔氏菌(Tindallia californiensis)表达的登录号为A0A1H3Q6T8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：41显示了Clostridium acidurici(菌株ATCC7906/DSM 604/BCRC 14475/CIP 104303/NCIMB 10678/9a)表达的登录号为K0B360的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：42显示了Romboutsia lituseburensis DSM 797表达的登录号为A0A1G9M635的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：43显示了Paraclostridium bifermentans ATCC 19299表达的登录号为T4V5T0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：44显示了Bacillus praedii表达的登录号为A0A4R1ATL6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：45显示了Carbydothermus islandicus表达的登录号为A0A1L8D5S0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：46显示了Caloramator australicus RC3表达的登录号为I7KTT3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：47显示了Paraclostridium bifermentans ATCC 638表达的登录号为T4VRB8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：48显示了Tepidimicrobium xylanilyticum表达的登录号为A0A1H2RLU1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：49显示了米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)表达的登录号为A0A0K2WJ73的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：50显示了芽孢杆菌属(Bacillus bacterium)表达的登录号为A0A3D0EBN7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：51显示了Bacillus notoginsengisoli表达的登录号为A0A417YW08的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：52显示了海泥芽孢杆菌 2691(Bacillus oceanisediminis 2691)表达的登录号为A0A160MBB4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：53显示了类芽孢杆菌FSL R7-0273(Paenibacillus sp.FSL R7-0273)表达的登录号为A0A089LXU3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：54显示了[Clostridium]ultunense Esp表达的登录号为M1ZGS5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：55显示了Bacillus freudenreichii表达的登录号为A0A448FF64的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：56显示了Caloramator fervidus表达的登录号为A0A1H5RUL9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：57显示了海泥芽孢杆菌(Bacillus oceanisediminis)表 达的登录号为A0A4R8GVS7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：58显示了非解糖卟啉单胞菌(Soehngenia saccharolytica)表达的登录号为A0A4T9ZWH0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：59显示了坚强芽孢杆菌DS1(Bacillus firmus DS1)表达的登录号为W7LB72的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：60显示了Thermotalea metallivorans表达的登录号为A0A140L3I5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：61显示了Ornithinibacillus halophilus表达的登录号为A0A1M5FNK3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：62显示了解脂嗜热互营杆菌(Thermosyntropha lipolytica)DSM 11003表达的登录号为A0A1M5LQE8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：63显示了虚构芽孢杆菌S7(Fictibacillus sp.S7)表达的登录号为A0A4Q2HRQ1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：64显示了Bacillus mesonae表达的登录号为A0A3Q9QZ31的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：65显示了Clostridium purinilytica表达的登录号为A0A0L0W688的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：66显示了Tindallia magadiensis表达的登录号为A0A1I3ETA9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：67显示了北拟杆菌(Paenibacillus borealis)表达的登录号为A0A089LHF8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：68显示了Paraclostridium benzoelyticum表达的登录号为A0A0M3DN30的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：69显示了Bacillus solani表达的登录号为A0A0Q3QMR7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：70显示了Thermohalobacter berrensis表达的登录号为A0A419T2I0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：71显示了不动杆菌RIT592(Acinetobacter sp.RIT592) 表达的登录号为A0A369PBX6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：72显示了Maledivibacter halophilus表达的登录号为A0A1T5JUM9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：73显示了Tissierella creatinini表达的登录号为A0A4T9WHZ3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：74显示了堆肥宏基因组(compost metagenome)表达的登录号为A0A3R1HSK2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：75显示了Natronincola peptidivorans表达的登录号为A0A1I0F3P2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：76显示了坚强芽孢杆菌(Bacillus firmus)表达的登录号为A0A0J5YTU9的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：77显示了Anaerovirgula multivorans表达的登录号为A0A239FV01的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：78显示了产氢羧基嗜热菌(Carbydothermus hydrogenoformans，菌株ATCC BAA-161/DSM 6008/Z-2901)表达的登录号为Q3AEA1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：79显示了产乙酸互营单包菌DSM13106(Sporanaerobacter acetigenes)DSM 13106表达的登录号为A0A1M5YST8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：80显示了Proteiniborus sp.DW1表达的登录号为A0A1M4MA63的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：81显示了芽孢杆菌7894-2(Bacillus sp.7894-2)表达的登录号为A0A268IWM5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：82显示了[Clostridium]ultunense Esp表达的登录号为A0A1M4PLJ1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：83显示了Virgibacillus indicus表达的登录号为A0A265N7L4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：84显示了类芽孢杆菌NFR01(Paenibacillus sp.NFR01)表达的登录号为A0A1I0KAI3的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：85显示了Andreesenia angusta表达的登录号为A0A1S1V3Q5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：86显示了类芽孢杆菌DMB5(Paenibacillus sp.DMB5)表达的登录号为A0A117T0P2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：87显示了Paludifilum halophilum表达的登录号为A0A235B9X8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：88显示了Proteiniborus ethanoligenes表达的登录号为A0A1H3NJW1的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：89显示了Alkaliphilus metalliredigens(菌株QYMF)表达的登录号为A6TWT7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：90显示了盐渍土假芽胞杆菌(Fictibacillus solisalsi)表达的登录号为A0A1G9U6Z5的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：91显示了Sporosarcina globispora表达的登录号为A0A0M0GGH4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：92显示了坚强芽孢杆菌(Bacillus firmus)表达的登录号为A0A366K523的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：93显示了类芽孢杆菌FSL R5-0490(Paenibacillus sp.FSL R5-0490)表达的登录号为A0A1R1FFH4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：94显示了嗜碱菌(Alkaliphilus sp.)表达的登录号为A0A2G2MLE4的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：95显示了厌氧砷还原菌(Alkaliphilus oremlandii，菌株OhILAs)表达的登录号为A8MLA7的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：96显示了Caloramator mitchellensis表达的登录号为A0A0R3JVG6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：97显示了柱胞梭菌DSM 605(Clostridium cylindrosporum DSM 605)表达的登录号为A0A0J8G334的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：98显示了嗜氨菌CFH 90114(Ammoniphilus sp.CFH 90114)表达的登录号为A0A4Q1SWC2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：99显示了Carbydothermus pertinax表达的登录号为A0A1L8CSM0的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：100显示了Soehngenia sp.1933P表达的登录号为A0A4Z0D783的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：101显示了非解糖卟啉单胞菌(Soehngenia saccharolytica)表达的登录号为A0A4T9ZOT2的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：102显示了Natribacillus halophilus表达的登录号为A0A1G8N2H8的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：103显示了堆肥宏基因组(compost metagenome)表达的登录号为A0A403WBU6的黄嘌呤酰胺水解酶的氨基酸序列。
SEQ ID NO：104显示了堆肥宏基因组(compost metagenome)表达的登录号为A0A3R4A6V9的黄嘌呤酰胺水解酶的氨基酸序列。
发明详细描述
图1显示了人体中各种嘌呤的代谢通路，其中各种嘌呤代谢的终产物为尿酸盐，而尿酸盐在关节和肾脏等部位的沉积是导致痛风的原因。图2显示了需氧型蛋白黄嘌呤氧化酶催化黄嘌呤降解生成尿酸的过程。治疗痛风的药物别嘌呤醇通过抑制黄嘌呤氧化酶来抑制尿酸的产生，从而治疗痛风(图3)。G.D.VOGELS等人(Degradation of Purines and Pyrimidines by Microorganisms,Bacteriological Reviews,June 1976,Vol.40,No.2,p.403-468)报道了柱胞梭菌(Clostridium cylindrosporum)在无氧条件下可以将黄嘌呤降解为亚胺甲基甘氨酸，图4显示了黄嘌呤酰胺水解酶在无氧条件下降解黄嘌呤的反应式。本申请的发明人意识到无氧条件下的嘌呤降解具有重要意义，因为人体的肠道为无氧环境，如果能先于肠道对嘌呤的吸收，在肠道内降解嘌呤，这对于预防、干预、缓解和/或治疗痛风是一种新思路。本申请的发明人利用生物信息学 的方法在进行了大量工作后，成功地鉴定出无氧条件下降解黄嘌呤的黄嘌呤酰胺水解酶。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
除非另有所指，分别地，核酸以5'至3'方向从左向右书写；氨基酸序列以氨基至羧基方向从左向右书写。数字范围包括限定该范围的数字。氨基酸在本文可以用其通常所知的三字母符号或IUPAC-IUB生物化学命名委员会推荐的一字母符号来表示。同样地，可以用通常接受的单字母码表示核苷酸。参考说明书整体更为充分地定义以上定义的术语。
本申请的“多肽”和“蛋白”在本文可互换使用，指氨基酸残基的聚合物及其变体和合成的和天然存在的类似物。因此，这些术语适用于天然存在的氨基酸聚合物及其天然存在的化学衍生物，以及其中一个或多个氨基酸残基是合成的非天然存在的氨基酸(诸如相应的天然存在的氨基酸的化学类似物)的氨基酸聚合物。此类衍生物包括例如翻译后修饰和降解产物，包括多肽片段的磷酸化的、糖基化的、氧化的、异构化、羧基化的和脱氨基化的变体。
如本文所用的术语“酶活性中心”是指酶分子中能够直接与底物分子结合，并催化底物化学反应的部位，这一部位就成为酶的活性中心。一般认为活性中心主要由两个功能部位组成：第一个是催化部位，底物的键在此被打断或形成新的键从而发生一定的化学变化；第二个是结合部位，酶的底物靠此部位结合到酶分子上。组成功能部位的是酶分子中在三维结构上比较靠近的少数几个氨基酸残基或是这些残基上的某些基团，它们在一级结构上可能相距甚远，甚至位于不同肽链上，而是通过肽链的盘绕、折叠在空间构象上相互靠近；对于需要辅酶的酶来说，辅酶分子(如金属离子Zn 2+和/或Mn 2+)或辅酶分子的某一部分结构也是功能部位的组成部分。
如本文所用的术语“氨基酸”是指羧酸碳原子上的氢原子被氨基取代后的化合物，氨基酸分子中含有氨基和羧基两种官能团。其包括天然存在的和非天然存在的氨基酸以及氨基酸类似物和模拟物。天然存在的氨基酸包括蛋白生物合成中使用的20种(L)-氨基酸以及其他氨基酸，例如4-羟脯氨酸、羟赖氨酸、羧化赖氨酸、锁链素、异锁链素、高半胱氨酸、瓜氨酸和鸟氨酸。非天然存在的氨基酸包括例如(D)-氨基酸、正亮氨酸、正缬氨酸、p-氟苯丙氨酸、乙基硫氨酸等，这些是本领域技术人员已知的。氨基酸类似物包括天然和非天然存在的氨基酸的修饰形式。这种修饰可以包括例如取代氨基酸上的化学基团和部分，或者氨基酸的衍生化。氨基酸模拟物包括例如表现出功能上类似性质的有机结构，所述性质例如氨基酸的电荷和电荷空间特性。例如，模拟精氨酸(Arg或R)的有机结构具有位于类似分子空间并且具有与天然存在的Arg氨基酸的侧链的e-氨基相同程度的移动性的正电荷部分。模拟物还包括约束结构以维持氨基酸或氨基酸官能团的最佳空间和电荷相互作用。本领域技术人员可以确定什么结构构成功能上等效的氨基酸类似物和氨基酸模拟物。
如本文所用的术语“同工酶”是指生物体内催化相同反应而分子结构不同的酶。
如本文所用的术语“核酸”是指指mRNA、RNA、cRNA、cDNA或DNA，包括单链和双链形式的DNA。该术语通常指至少10个碱基长度的核苷酸多聚形式，所述核苷酸是核糖核苷酸或脱氧核苷酸或任一类型的核苷酸的修饰形式。
如本文所用的术语“编码”用于特定核酸的上下文时，指该核酸包含指导该核苷酸序列翻译成特定蛋白的必需信息。使用密码子表示编码蛋白的信息。编码蛋白的核酸可以包含位于该核酸翻译区内的非翻译序列(例如，内含子)或者可以缺少这样的居间非翻译序列(例如，如同在cDNA中)。
如本文所用的，涉及特定多核苷酸或其所编码的蛋白的“全长序列”指具有天然(非合成)内源序列的整个核酸序列或整个氨基酸 序列。全长多核苷酸编码该特定蛋白的全长、催化活性形式。
如本文所用的术语“分离的”是指多肽或核酸或其生物学活性部分，其基本上或本质上不含如在其天然存在的环境中所发现的通常伴随或反应于该蛋白质或核酸的组分。因而，用重组技术产生分离的多肽或核酸时，分离的多肽或核酸基本上不含其它细胞物质或培养基，或者化学合成分离的多肽或核酸时，基本上不含化学前体或其它化学品。
如本文所用的术语“表达载体”是重组或合成产生的核酸构建体，其具有一系列允许特定的核酸在宿主细胞中转录的特异性核酸元件。
如本文所用的术语“宿主细胞”是指在转化和转导(感染)中接受外源基因的细胞。宿主细胞可以是诸如酵母细胞的真核细胞或诸如大肠杆菌的原核细胞。
具体实施方案
第一方面，本申请提供了多肽，其包含SEQ ID NO:1所示的氨基酸序列或其功能变体，其中所述功能变体具有黄嘌呤酰胺水解酶活性。
在第一方面的一些实施方案中，其在空间构象上具有如下定义的催化部位：
所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基。
在第一方面的一些实施方案中，所述催化部位还包含二价金属离子。
在第一方面的一些具体实施方案中，所述催化部位还包含2个二价金属离子，例如Zn 2+和/或Mn 2+。
在第一方面的一些实施方案中，其中所述多肽还包含在空间构象上具有如下定义的结合部位：
所述结合部位包含在空间构象上相互靠近的、参照SEQ ID NO:1的I288、A289、P338和G339氨基酸残基。
在第一方面的一些实施方案中，所述催化部位与所述结合部 位相距不大于5埃
Figure PCTCN2021071501-appb-000002
在第一方面的一些实施方案中，其中所述功能变体为SEQ ID NO:1所示的氨基酸序列的天然同工酶。
在第一方面的一些实施方案中，所述天然同工酶来自：坚强芽孢杆菌(Bacillus firmus)、堆肥宏基因组(compost metagenome)、Clostridium purinilyticum、高温黄色微球菌(Thermoflavimicrobium sp.)、耐盐海洋丝状菌(Marininema halotolerans)、梭菌科细菌(Clostridiaceae bacterium)、芽孢杆菌(Bacillus sp.)、类芽孢杆菌(Paenibacillus sp.)、双岐高温黄色微球菌(Thermoflavimicrobium dichotomicum)、Fictibacillus enclensis、中温海洋丝状菌(Marininema mesophilum)、香蒲类芽孢杆菌(Paenibacillus typhae)、极尖组织菌(Tissierella praeacuta)、慢生型大豆根瘤菌(Bradyrhizobium japonicum)、产氢氨基酸杆菌(Acidaminobacter hydrogenoformans)、温铁还原细菌(Caloranaerobacter sp.)、组织菌(Tissierella sp.)、Paenibacillus donghaensis、Gottschalkia acidurici、Clostridium acidurici、强壮芽孢杆菌(Bacillus fortis)、海泥芽孢杆菌(Bacillus oceanisediminis)、Virgibacillus profundi、Anaeromicrobium sediminis、解脂嗜热互营杆菌(Thermosyntropha lipolytica)、Alkaliphilus peptidifermentans、米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)、芽孢杆菌细菌属(Bacillus bacterium)、Marinisporobacter balticus、土地芽孢杆菌(Bacillus terrae)、加利福尼亚丁达尔氏菌(Tindallia californiensis)、Romboutsia lituseburensis、Paraclostridium bifermentans、Bacillus praedii、Carbydothermus islandicus、Caloramator australicus、Paraclostridium bifermentans、Tepidimicrobium xylanilyticum、Bacillus notoginsengisoli、[Clostridium]ultunense Esp、Bacillus freudenreichii、Caloramator fervidus、非解糖卟啉单胞菌(Soehngenia saccharolytica)、Thermotalea metallivorans、Ornithinibacillus halophilus、虚构芽孢杆菌(Fictibacillus sp.)、Bacillus mesonae、Tindallia magadiensis、北拟杆菌(Paenibacillus borealis)、Paraclostridium benzoelyticum、 Bacillus solani、Thermohalobacter berrensis、不动杆菌(Acinetobacter sp.)、Maledivibacter halophilus、Tissierella creatinini、Natronincola peptidivorans、Anaerovirgula multivorans、产氢羧基嗜热菌(Carbydothermus hydrogenoformans)、产乙酸互营单包菌(Sporanaerobacter acetigenes)、Proteiniborus sp.、Virgibacillus indicus、Andreesenia angusta、Paludifilum halophilum、Proteiniborus ethanoligenes、Alkaliphilus metalliredigens、盐渍土假芽胞杆菌(Fictibacillus solisalsi)、Sporosarcina globispora、嗜碱菌(Alkaliphilus sp.)、厌氧砷还原菌(Alkaliphilus oremlandii)、Caloramator mitchellensis、柱胞梭菌(Clostridium cylindrosporum)、嗜氨菌(Ammoniphilus sp.)、Carbydothermus pertinax、Soehngenia sp.和Natribacillus halophilus。
在第一方面的一些具体实施方案中，所述天然同工酶包含SEQ ID NO:4-104中任一项所示的氨基酸序列。
在第一方面的一些具体实施方案中，其中所述功能变体为在SEQ ID NO:1所示的氨基酸序列或其天然同工酶的基础上发生一个或多个氨基酸的插入、取代和/或缺失而产生的。
在第一方面的一些实施方案中，所述插入、取代和/或缺失不发生在催化部位。
在第一方面的一些实施方案中，所述插入、取代和/或缺失不发生在结合部位。
在第一方面的一些实施方案中，所述插入、取代和/或缺失不发生在催化部位和结合部位。
在第一方面的一些实施方案中，所述氨基酸插入、取代和/或缺失的数目为1-30个，优选为1-20个，更优选为1-10个，其中获得的功能变体基本上保持未改变的黄嘌呤酰胺水解酶活性。
在第一方面的一些实施方案中，所述功能变体与SEQ ID NO:1所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9、或10个氨基酸的插入、取代和/或缺失。
在第一方面的一些实施方案中，所述功能变体与SEQ ID NO: 1所示的氨基酸序列相差约1、2、3、4或5个氨基酸的插入、取代和/或缺失。
在第一方面的一些实施方案中，所述多肽为分离的多肽。
第二方面，本申请提供了核酸分子，其编码第一方面所述的多肽。
在第二方面的一些实施方案中，本申请的核酸分子包含与编码SEQ ID NO:1和SEQ ID NO:4-104中任一项所示多肽的核苷酸序列在严格条件下杂交的核苷酸序列，或者由与编码SEQ ID NO:1和SEQ ID NO:4-104中任一项多肽的核苷酸序列特异性杂交且编码与SEQ ID NO:1和SEQ ID NO:4-104中任一项所示多肽在功能上等同的多肽的核酸序列组成。
本领域技术人员可以常规选择DNA杂交的严格条件。通常较长的探针需要较高的温度，以便进行适当的退火，而较短的探针需要较低的温度。杂交通常取决于当互补链处于低于其解链温度的环境时变性DNA的重退火能力。探针与可杂交序列之间同源性程度越高，所能采用的相对温度越高。于是，较高的相对温度往往使反应条件更严格，而在较低的温度下，则严格度较低。关于杂交反应严格条件的详细描述，可参阅Ausubel等人，Current Protocols in Molecular Biology,Wiley Interscience Publishers,(1995)。
在第二方面的一些实施方案中，DNA杂交采用的严格条件包括：1)洗涤时采用低离子强度和高温，例如50℃下的0.015M氯化钠/0.0015M柠檬酸钠/0.1％十二烷基硫酸钠；2)杂交时采用甲酰胺等变性剂，例如42℃下50％(v/v)甲酰胺加0.1％牛血清白蛋白/0.1％Ficoll/0.1％聚二烯吡咯烷酮/pH 6.5的50mM磷酸钠缓冲液以及750mM氯化钠、75mM柠檬酸钠；或(3)在42℃下过夜杂交，杂交溶液含50％甲酰胺，5×SSC(0.75M氯化钠，0.075M柠檬酸钠)、50mM磷酸钠(pH 6.8)、0.1％焦磷酸钠、5×Denhardt’s溶液、超声波处理的鲑鱼精子DNA(50mg/mL)、0.1％SDS和10％硫酸葡聚糖，然后在0.2×SSC(氯化钠/柠檬酸钠)中于42℃洗涤10分钟，再以含有EDTA的0.1×SSC于55℃进行高严格度洗涤。中等严格条件可按Sambrook 等人，Molecular Cloning：A Laboratory Manual,NewYork：Cold Spring Harbor Press,1989中的描述加以确定。中等严格条件包括采用严格度低于以上描述的洗涤溶液和杂交条件(如温度、离子强度和SDS百分比)。例如，中等严格条件包括用至少约16％v/v到至少约30％v/v的甲酰胺和至少约0.5M到至少约0.9M的盐在42℃杂交，以及用至少约0.1M到至少约0.2M盐在55℃洗涤。中等严格条件还可以包括用1％牛血清白蛋白(BSA)、1mM EDTA、0.5M NaHPO 4(pH 7.2)、7％SDS在65℃杂交，以及用(i)2×SSC、0.1％SDS；或(ii)0.5％BSA、1mM EDTA、40mM NaHPO 4(pH 7.2)、5％SDS在60-65℃洗涤。专业人员将会根据探针长度等因素调节温度、离子强度等。杂交核酸时的严格度取决于核酸分子长度和互补程度，以及其它本领域内众所周知的变量。两个核苷酸序列之间的相似性或同源性越大，则含有这些序列的核酸杂合体的Tm越大。核酸杂交的相对稳定性(对应于较高的Tm)以下列顺序递减：RNA:RNA、DNA:RNA,DNA:DNA。优选地，可杂交核酸的最低长度至少约为12个核苷酸，优选至少约为16个、更优选至少约为24个、最优选至少约为36个核苷酸。
本申请的核酸分子可以与其他DNA序列组合，所述其他DNA序列例如启动子、聚腺苷化信号、其他限制性酶切位点、多克隆位点、其他编码节段等，使得它们的总长度可以显著不同。因此考虑可以利用几乎任意长度的多核苷酸片段；总长度优选地受预期重组DNA方案中制备和使用的便利性的限制。
可以利用本领域内已知的和可获得的多种成熟技术中的任何一种来制备、操控和/或表达多核苷酸及其融合物。例如，编码本申请的多肽或其功能变体的核酸分子可以用于重组DNA分子中以指导多肽在适当的宿主细胞中表达。由于遗传密码子固有的简并性，编码基本上相同或在功能上等同的氨基酸序列的其他DNA序列也可以用于本申请中，并且这些序列可以用于克隆和表达给定的多肽。
此外，可以使用本领域内公知的方法改造本申请的核酸分子，包括但不限于基因产物的克隆、加工、表达和/或活性的改变。
在第二方面的一些实施方案中，所述核酸分子通过人工合成产生，例如直接的化学合成或酶合成。
在第二方面的一些实施方案中，所述核酸分子通过重组技术产生。
在第二方面的一些实施方案中，所述核酸分子为分离的核酸分子。
第三方面，本申请提供了表达盒，其包含第二方面所述的核酸分子。
在第三方面的一些具体实施方案中，表达盒可以另外包含5'前导序列，所述前导序列能发挥增强翻译的作用。
在制备表达盒时，可以操作各种DNA片段以提供合适方向的和适当时合适读框的DNA序列。为达到这一目的，可以采用接头或连接子来连接DNA片段，或者可以涉及其它操作来提供方便的限制性位点、移除多余DNA、移除限制性位点等等。为了这一目的，可以涉及体外诱变，引物修复，限制性，退火，再取代，例如转换(transition)和颠换(transversion)。
第四方面，本申请提供了表达载体，其包含第二方面所述的核酸分子或第三方面所述的表达盒。
在第四方面的一些实施方案中，任何合适的表达载体都可以用于本申请。例如，所述表达载体可以为pET28、pET14、HT-1N-TAG-2691、pRS416等载体中的任一种。
在第四方面的一些实施方案中，编码SEQ ID NO:1和SEQ ID NO:4-104中任一项所示多肽的核酸分子被克隆到载体中，以构成含有本申请所述核酸分子的重组载体。
在第四方面的一些实施方案中，用于克隆多核苷酸的表达载体为质粒载体。
在第四方面的一些的实施方案中，上述表达载体还包含调节核酸分子表达的调控序列，其中所述核酸分子与所述调控序列可操作地连接。
如本文所用的术语“调控序列”是指实现与其连接的编码序列表达所需的多核苷酸序列。这类调控序列的性质随宿主生物而改变。在原核生物中，这类调控序列一般包括启动子、核糖体结合位点和终止 子；在真核生物中，这类调控序列一般包括启动子、终止子以及在某些情况下的增强子。因此，术语“调控序列”包括其存在对目的基因的表达是必需的最低限度的所有序列，也可以包括其存在对目的基因表达是有利的其它序列，例如前导序列。
如本文所用的术语“可操作地连接”是指如下情形：所涉及到的序列处于允许它们以希望的方式起作用的关系之中。因此，例如“可操作地连接”到一编码序列的调控序列使得在与所述调控序列相容的条件下实现该编码序列的表达。
在第四方面的一些实施方案中，利用本领域的技术人员熟知的方法构建包含编码SEQ ID NO:1和SEQ ID NO:4-104中任一项所示多肽的核苷酸序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等(Sambroook,et al.Molecular Cloning,a Laboratory Manual,cold Spring Harbor Laboratory.New York,1989)。核苷酸序列可操作地连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性实例包括：大肠杆菌的lac或trp启动子；λ噬菌体的PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是DNA表达的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。实例包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如编码抗硫酸卡那霉素、抗氨苄青霉素等的那些基因。
第五方面，本申请提供了宿主细胞，其包含第二方面所述的核酸分子或第三方面所述的表达盒或第四方面所述的表达载体。
在第五方面的一些实施方案中，所述宿主细胞能够表达并产生：多肽，其包含SEQ ID NO:1所示的氨基酸序列或其功能变体，其中所述功能变体具有黄嘌呤酰胺水解酶活性。
在第五方面的一些实施方案中，其中所述多肽在空间构象上具有如下定义的催化部位：
所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基。
在第五方面的一些实施方案中，所述催化部位还包含二价金属离子。
在第五方面的一些具体实施方案中，所述催化部位还包含2个二价金属离子，例如Zn 2+和/或Mn 2+。
在第五方面的一些具体实施方案中，其中所述多肽在空间构象上具有如下定义的催化部位和结合部位：
所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基以及2个二价金属离子(例如Zn 2+和/或Mn 2+)；和
所述结合部位包含在空间构象上相互靠近的、参照SEQ ID NO:1的I288、A289、P338和G339氨基酸残基。
在第五方面的一些实施方案中，可用的宿主细胞为含有上述表达载体的细胞，可以为真核细胞，例如酵母细胞培养系统可用于本申请的多肽的表达。所述宿主细胞也可以为含有上述表达载体的原核细胞，例如可以选自埃希氏菌属(例如大肠杆菌)、乳酸杆菌属、双歧杆菌属、拟杆菌门和厚壁菌门等。
在第五方面的一些具体实施方案中，所述宿主细胞为酵母细胞或大肠杆菌。
在第五方面的一些具体实施方案中，编码本申请的一种或多种酶的核酸分子可以以游离载体的形式存在于宿主细胞中，或者也可以整合至宿主细胞的基因组中。
在上述任一方面的一些实施方案中，所述分离的核酸可操作地连接到调控序列，调控序列可以被用所述表达载体转化过的宿主细胞 识别。
可以利用本领域已知的任何技术将表达载体导入宿主细胞中，包括转化、转导、转染、病毒感染、基因枪或Ti-介导的基因转移。具体的方法包括磷酸钙转染、DEAE-葡聚糖介导的转染、脂转染或电穿孔等(Davis,L.,Dibner,M.,Battey,I.,Basic Methods in Molecular Biology,(1986))。作为示例，当宿主为原核生物如大肠杆菌时，可在指数生长期后收获感受态细胞，用本领域熟知的CaCl 2法进行转化。
第六方面，本申请提供了药物组合物或保健食品，其包含第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体或第五方面所述的宿主细胞和药学上可接受的载体或赋形剂。
在第六方面的一些实施方案中，所述药物组合物或保健食品，用于预防、干预和/或治疗痛风。
在第六方面的一些实施方案中，所述药物组合物或保健食品还可包含下述中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在第六方面的一些实施方案中，可将本申请中的药物组合物或保健食品配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在第六方面的一些实施方案中，可以利用任何能给药至肠道的方式递送本申请的药物组合物或保健食品，优选口服给药。
在第六方面的一些具体实施方案中，将第五发明所述的宿主细胞制备成肠溶胶囊用于口服给药。
在第六方面的一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第七方面，本申请提供了第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载 体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品在降解嘌呤中的用途。
在第七方面的一些实施方案中，所述降解嘌呤在体外发生。
在第七方面的一些实施方案中，降解嘌呤在无氧的环境下进行，例如肠道中。
第八方面，本申请提供了第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品在制备用于预防、干预和/或治疗痛风的药物中的用途。
第九方面，本申请提供了预防、干预和/或治疗痛风的方法，其包括向有需要的个体给予第一方面所述的多肽、第二方面所述的核酸分子、第三方面所述的表达盒、第四方面所述的表达载体、第五方面所述的宿主细胞或第六方面所述的药物组合物或保健食品。
作为示例性的实施方案，在黄嘌呤的无氧降解中，SEQ ID NO:1所示的黄嘌呤酰胺水解酶将黄嘌呤降解为4-脲基-5-羧基咪唑，从而减少人体细胞获得嘌呤并将其转化为尿酸，可以通过此途径在肠道内降解黄嘌呤从而预防、干预和/或治疗痛风。
实施例
下述实施例仅是说明性的，并不意图限制本申请实施方案的范围或者所附的权利要求的范围。
实施例1.编码黄嘌呤酰胺水解酶的基因克隆的制备
以坚强芽孢杆菌(Bacillus firmus)(CGMCC 1.2010)的基因组作为模板，使用SEQ ID NO：2和3所示的上游引物和下游引物PCR扩增编码黄嘌呤酰胺水解酶(SEQ ID NO:1)的核酸分子，获得编码黄嘌呤酰胺水解酶的基因序列。利用Gibson的方法将编码黄嘌呤酰胺水解酶的基因序列插入HT-1N-TAG-2691载体(其含有His 6标签和TEV蛋白酶切割位点)的SspI限制性位点，获得含有编码黄嘌呤酰胺水解酶的基因的表达载体。
实施例2.黄嘌呤酰胺水解酶的重组表达和纯化
2.1重组表达
将实施例1中构建的含有编码黄嘌呤酰胺水解酶的基因1的表达载体转化到大肠杆菌(Escherichia coli)BL21细胞中，涂布在含有50μg/mL硫酸卡那霉素的LB琼脂平板上37℃过夜培养。挑取单克隆菌落在5mL的含硫酸卡那霉素LB液体培养基中37℃、220rpm过夜培养，次日转移到800mL的LB培养液中扩大培养，当OD 600值达到0.8左右时,将温度降到18℃，并加入终浓度为0.3mM的IPTG诱导蛋白表达。诱导表达16小时后在4℃下4000×g离心10min收集菌体。将菌体沉淀用35mL裂解液重悬[50mM Tris/HCl,pH 8.0，1mM苯甲基磺酰氟(PMSF)，0.2mg/mL溶菌酶，0.03％Triton X-100和1μL的DNase I]，并在-80℃下冷冻保藏。
2.2纯化
将菌体从-80℃中取出，置于25℃水浴中孵育40分钟以裂解细胞。加入6mL的6％硫酸链霉素水溶液，温和混匀，在4℃下以20,000×g转速离心5分钟。将上清用0.22μm的滤膜过滤，结合到预先用缓冲液A(20mM Tris-HCl 7.5，0.2M KCl)平衡过的5mL TALON钴柱上，用10倍柱体积的缓冲液A洗涤柱子，然后用5倍柱体积的含有150mM咪唑的缓冲液B(20mM Tris-HCl 7.5，0.2M KCl，150mM咪唑，5mMβ-巯基乙醇)洗脱目的蛋白。向洗脱下来的蛋白溶液中加入硫酸铵至70％饱和沉淀蛋白，在10,000×g下离心10分钟，弃去上清，沉淀用5mL缓冲液C(20mM Tris-HCl 7.5，0.2M KCl，10％(v/v)丙三醇)重悬，使用G25柱，除去盐。将除去盐后的目的蛋白分装，用液氮速冻，置于-80℃中冷冻保存。通过分光光度计检测最终所得到的黄嘌呤酰胺水解酶的浓度。根据蛋白序列，黄嘌呤酰胺水解酶的消光系数是55,810M -1cm -1。每升菌平均大约能纯化35mg蛋白。图5显示黄嘌呤酰胺水解酶表达、纯化的过程，其在凝胶电泳上的迁移速率符合实际的分子量大小(55.7kDa)。
实施例3.黄嘌呤酰胺水解酶的活性检测
将1mL含有1mM黄嘌呤、4μM实施例2中获得的黄嘌呤酰胺水解酶、0.1mM Mn 2+、200mM Tris-HCl 7.5的溶液在30℃水浴中孵育0.5h，使体系充分反应，然后加入等体积的甲醇溶液混匀，并在14,000×g下离心10分钟，通过0.22μm滤膜过滤掉蛋白沉淀，记为实验组。对照组为未添加黄嘌呤酰胺水解酶及二价金属离子的反应，对照组与实验组的其它实验条件相同(表1)。实验在无氧手套箱内进行，所有溶液包括蛋白溶液，反应缓冲液等在进入手套箱前均通过Schlenk(希莱克)线除氧。
利用LC-MS检测黄嘌呤酰胺水解酶催化黄嘌呤水解开环，对照组未检测到产物峰，而实验组检测到了分子量为169Da的产物峰，证明了黄嘌呤酰胺水解酶可以降解黄嘌呤(图6)。
表1
Figure PCTCN2021071501-appb-000003
实施例4.黄嘌呤酰胺水解酶的活性中心鉴定
在体外证实含有SEQ ID NO:1所示的氨基酸序列的多肽具有黄嘌呤酰胺水解酶活性后，利用PHYRE2服务器对该酶进行结构模拟(图7)，显示出了含有4个组氨酸、一个赖氨酸和一个天冬氨酸以及两个二价金属离子(Zn 2+和/或Mn 2+)构成的催化部位。进一步用计算机软件对模拟的黄嘌呤酰胺水解酶进行底物黄嘌呤的锚定显示，活性中心还包括由一个异亮氨酸、一个丙氨酸、一个脯氨酸和一个甘氨酸在空间构象上相互靠近构成的结合部位与底物黄嘌呤相互作用(图7)。
实施例5.黄嘌呤酰胺水解酶的同工酶鉴定
本申请的发明人进一步从Uniprot数据库中找到此黄嘌呤酰胺水解酶同工酶UniRef50的序列(涵盖50％序列相同，同时超过该黄嘌呤酰胺水解酶80％长度的蛋白序列)，上述序列均在UniRef50_Q3AEA1的簇中，共包括101种蛋白序列，并对这101种不同物种来源的蛋白进行进化树分析(图8)。在每一个进化分支中各选取一个代表性蛋白，共五种蛋白，分别来源于坚强芽孢杆菌(Bacillus firmus)、柱胞梭菌DSM 605(Clostridium cylindrosporum DSM 605)、Clostridium purinilyticum、产氢羧基嗜热菌(Carbydothermus hydrogenoformans，菌株ATCC BAA-161/DSM 6008/Z-2901)以及Paenibacillus donghaensis，将它们编码的登录号分别为A0A366K523、A0A0J8G334、A0A0L0W692、Q3AEA1和A0A2Z2KEH1的黄嘌呤酰胺水解酶的氨基酸序列进行序列比对。序列对比结果显示上述催化位点和结合位点的氨基酸残基高度保守，所有五个序列都含有上述氨基酸残基，证明这些酶均为黄嘌呤酰胺水解酶(图9)。表2显示了从Uniprot数据库中找到的101种黄嘌呤酰胺水解酶的登录号、菌株来源以及氨基酸序列号。
表2
Figure PCTCN2021071501-appb-000004
Figure PCTCN2021071501-appb-000005
Figure PCTCN2021071501-appb-000006
Figure PCTCN2021071501-appb-000007
实施例6.表达黄嘌呤酰胺水解酶的大肠杆菌的构建
本实施例构建了能表达黄嘌呤酰胺水解酶(SEQ ID NO:1)的大肠杆菌。
本实施例的关键操作是把编码黄嘌呤酰胺水解酶的基因整合到大肠杆菌基因组，例如多拷贝16sRNA其中一个拷贝处，让其稳定表达，同时把控制鸟嘌呤转运蛋白和鸟嘌呤脱氨酶的启动子置换为gapA启动子，让编码黄嘌呤酰胺水解酶的基因持续稳定表达。
本实施例优选用组成型持续稳定表达的启动子gapA取代编码鸟嘌呤转运蛋白以及鸟嘌呤脱氨酶的启动子，原启动子通常在环境中氮源缺少，需要利用鸟嘌呤的氮的情况下才表达，在氮源充足的情况下，其控制的基因不表达，为了使食物中的鸟嘌呤能通过本发明的黄嘌呤酰胺水解酶降解，需要取代控制编码鸟嘌呤转运蛋白和鸟嘌呤脱氨酶的基因的启动子，使其在任何时候都获得表达，从而有效的把鸟嘌呤转运进入经上述工程改造的大肠杆菌细胞内，并通过鸟嘌呤脱氨酶和黄嘌呤酰胺水解酶顺序作用降解，从而有效阻止人体肠道对鸟嘌呤的吸收并将其转化成尿酸。
实施例7.表达黄嘌呤酰胺水解酶的大肠杆菌用于治疗痛风
将利用氧嗪酸(oxonic acid)诱导的高尿酸大鼠或尿酸酶敲除转基因小鼠作为痛风动物模型，将实施例6制备的各种大肠杆菌制成肠溶胶囊。在同等喂食的条件下，检测各种大肠杆菌肠溶胶囊能否降低痛风大鼠、小鼠模型的血液尿酸含量。
实施例8.表达黄嘌呤酰胺水解酶的大肠杆菌用于治疗痛风
8.1大鼠实验
选用SD雄性大鼠(体重120g-150g)，每组6只，在同等喂食的条件下分为实验组、高尿酸血症组和对照组，具体实验过程如下：
1.动物预适应：≥7天预适应过程，采用普通饲料和普通饮用水喂养。
2.抗生素预处理：采用普通饲料喂养，在饮用水中添加2mg/mL链霉素+1mg/mL氨卞青霉素喂养大鼠3天后，停饮用水＞6h后，给予后续灌胃处理。
3.实验组：每天饲喂大鼠含1％(w/w)腺嘌呤的饲料，并用200μL含有2×10^10个实施例6制备的大肠杆菌混悬液进行灌胃处理，持续两周。
高尿酸血症组：每天饲喂大鼠含1％(w/w)腺嘌呤饲料，持续两周。
对照组：每天饲喂大鼠普通饲料，持续两周。
两周喂养后，眼眶取血，用尿酸试剂盒检测血清中尿酸含量。
8.2小鼠实验
选用小鼠Balb/c(8周龄)，每组5只，在同等喂食的条件下分为实验组、高尿酸血症组和对照组，具体实验过程如下：
1.动物预适应：≥7天预适应过程，采用普通饲料和普通饮用水喂养。
2.抗生素预处理：采用普通饲料喂养，在饮用水中添加2mg/mL链霉素+1mg/mL氨卞青霉素喂养小鼠3天后，停饮用水＞6h后，给予后续灌胃处理。
3.实验组：每天饲喂小鼠0.1％(w/w)腺嘌呤饲料并给予200 mg/kg氧氰酸钾灌胃给药，并用100μL含有1×10^10个实施例6制备的大肠杆菌混悬液进行灌胃处理，持续两周。
高尿酸血症组：每天饲喂小鼠0.1％(w/w)腺嘌呤饲料和给予200mg/kg氧氰酸钾灌胃给药，持续两周。
对照组：每天饲喂普通饲料，持续两周。
两周喂养后，眼眶取血，用尿酸试剂盒检测血清中尿酸含量。
上文对本申请的各项发明的示例性实施方案进行了描述，但是，在不脱离本申请的实质和范围的情况下，本领域技术人员能够对本申请描述的示例性实施方案进行修改或改进，由此得到的变形方案或等同方案也属于本申请的范围。

Claims (18)

  1. 多肽，其包含SEQ ID NO:1所示的氨基酸序列或其功能变体，其中所述功能变体具有黄嘌呤酰胺水解酶活性。
  2. 如权利要求1所述的多肽，其在空间构象上具有如下定义的催化部位：
    所述催化部位包含空间构象上相互靠近的、参照SEQ ID NO:1的H59、H61、K151、H186、H242和D316氨基酸残基。
  3. 如权利要求2所述的多肽，所述催化部位还包含二价金属离子，任选地2个二价金属离子(例如Zn 2+和/或Mn 2+)。
  4. 如权利要求1-3中任一项所述的多肽，其还包含在空间构象上具有如下定义的结合部位：
    所述结合部位包含在空间构象上相互靠近的、参照SEQ ID NO:1的I288、A289、P338和G339氨基酸残基。
  5. 如权利要求4所述的多肽，其中所述催化部位与所述结合部位相距不大于5埃
    Figure PCTCN2021071501-appb-100001
  6. 如权利要求1-5中任一项所述的多肽，其中所述功能变体为SEQ ID NO:1所示的氨基酸序列的天然同工酶；
    优选地，所述天然同工酶来自：坚强芽孢杆菌(Bacillus firmus)、堆肥宏基因组(compost metagenome)、Clostridium purinilyticum、高温黄色微球菌(Thermoflavimicrobium sp.)、耐盐海洋丝状菌(Marininema halotolerans)、梭菌科细菌(Clostridiaceae bacterium)、芽孢杆菌(Bacillus sp.)、类芽孢杆菌(Paenibacillus sp.)、双岐高温黄色微球菌(Thermoflavimicrobium dichotomicum)、Fictibacillus enclensis、中温海洋丝状菌(Marininema mesophilum)、香蒲类芽孢杆菌(Paenibacillus typhae)、极尖组织菌(Tissierella praeacuta)、慢 生型大豆根瘤菌(Bradyrhizobium japonicum)、产氢氨基酸杆菌(Acidaminobacter hydrogenoformans)、温铁还原细菌(Caloranaerobacter sp.)、组织菌(Tissierella sp.)、Paenibacillus donghaensis、Gottschalkia acidurici、Clostridium acidurici、强壮芽孢杆菌(Bacillus fortis)、海泥芽孢杆菌(Bacillus oceanisediminis)、Virgibacillus profundi、Anaeromicrobium sediminis、解脂嗜热互营杆菌(Thermosyntropha lipolytica)、Alkaliphilus peptidifermentans、米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)、芽孢杆菌属(Bacillus bacterium)、Marinisporobacter balticus、土地芽孢杆菌(Bacillus terrae)、加利福尼亚丁达尔氏菌(Tindallia californiensis)、Romboutsia lituseburensis、Paraclostridium bifermentans、Bacillus praedii、Carbydothermus islandicus、Caloramator australicus、Paraclostridium bifermentans、Tepidimicrobium xylanilyticum、Bacillus notoginsengisoli、[Clostridium]ultunense Esp、Bacillus freudenreichii、Caloramator fervidus、非解糖卟啉单胞菌(Soehngenia saccharolytica)、Thermotalea metallivorans、Ornithinibacillus halophilus、虚构芽孢杆菌(Fictibacillus sp.)、Bacillus mesonae、Tindallia magadiensis、北拟杆菌(Paenibacillus borealis)、Paraclostridium benzoelyticum、Bacillus solani、Thermohalobacter berrensis、不动杆菌(Acinetobacter sp.)、Maledivibacter halophilus、Tissierella creatinini、Natronincola peptidivorans、Anaerovirgula multivorans、产氢羧基嗜热菌(Carbydothermus hydrogenoformans)、产乙酸互营单包菌(Sporanaerobacter acetigenes)、Proteiniborus sp.、Virgibacillus indicus、Andreesenia angusta、Paludifilum halophilum、Proteiniborus ethanoligenes、Alkaliphilus metalliredigens、盐渍土假芽胞杆菌(Fictibacillus solisalsi)、Sporosarcina globispora、嗜碱菌(Alkaliphilus sp.)、厌氧砷还原菌(Alkaliphilus oremlandii)、Caloramator mitchellensis、柱胞梭菌(Clostridium cylindrosporum)、嗜氨菌(Ammoniphilus sp.)、Carbydothermus pertinax、Soehngenia sp. 和Natribacillus halophilus；
    更优选地，所述天然同工酶包含SEQ ID NO:4-104中任一项所示的氨基酸序列。
  7. 如权利要求1-6中任一项所述的多肽，其中所述功能变体为在SEQ ID NO:1所示的氨基酸序列或其天然同工酶的基础上发生一个或多个氨基酸的插入、取代和/或缺失而产生的，可选择地，所述插入、取代和/或缺失不发生在催化部位和/或结合部位。
  8. 核酸分子，其编码权利要求1-7中任一项所述的多肽。
  9. 表达盒，其包含权利要求8所述的核酸分子。
  10. 表达载体，其包含权利要求8所述的核酸分子、或权利要求9所述的表达盒。
  11. 宿主细胞，其包含权利要求8所述的核酸分子、权利要求9所述的表达盒、或权利要求10所述的表达载体。
  12. 如权利要求11所述的宿主细胞，所述细胞为真核细胞或原核细胞；
    优选地，所述真核细胞为酵母细胞；
    优选地，所述原核细胞选自埃希氏菌属、乳酸杆菌属、双歧杆菌属、拟杆菌门和厚壁菌门；更优选地所述埃希氏菌属为大肠杆菌。
  13. 药物组合物或保健食品，其包含权利要求1-7中任一项所述的多肽、权利要求8所述的核酸分子、权利要求9所述的表达盒、权利要求10所述的表达载体、或者权利要求11或12所述的宿主细胞和药学上可接受的载体或赋形剂。
  14. 如权利要求13所述的药物组合物或保健食品，其用于预防、干预和/或治疗痛风。
  15. 权利要求1-7中任一项所述的多肽、权利要求8所述的核酸分子、权利要求9所述的表达盒、权利要求10所述的表达载体、权利要求11或12所述的宿主细胞、或者权利要求13或14所述的药物组合物或保健食品在降解嘌呤中的用途。
  16. 如权利要求15所述的用途，其中所述降解嘌呤在体外发生。
  17. 权利要求1-7中任一项所述的多肽、权利要求8所述的核酸分子、权利要求9所述的表达盒、权利要求10所述的表达载体、权利要求11或12所述的宿主细胞、或者权利要求13或14所述的药物组合物或保健食品在制备用于预防、干预和/或治疗痛风的药物中的用途。
  18. 预防、干预和/或治疗痛风的方法，包括向有需要的个体给予权利要求1-7中任一项所述的多肽、权利要求8所述的核酸分子、权利要求9所述的表达盒、权利要求10所述的表达载体、权利要求11或12所述的宿主细胞、或者权利要求13或14所述的药物组合物或保健食品。
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