Xanthine oxidase

Abstract

This invention discloses a xanthine oxidase. It also discloses a mutant xanthine oxidase with good thermostability and high activity. Furthermore, this invention discloses the DNA sequence encoding xanthine oxidase, an expression vector containing the DNA sequence, a recombinant bacterial strain, and a method for preparing xanthine oxidase. This invention also discloses the application of xanthine oxidase in the preparation of clinical detection kits for 5'-nucleotidase and adenosine deaminase, demonstrating good reproducibility and high precision.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology. More specifically, the present invention relates to a xanthine oxidase, a mutant xanthine oxidase, a DNA sequence encoding the xanthine oxidase, an expression vector containing the DNA sequence, a recombinant strain, a method for preparing the xanthine oxidase, and the use of the xanthine oxidase in preparing a kit for clinical detection of 5'-nucleotidase and adenosine deaminase. Background Art

[0002] Xanthine oxidase (XOD, EC 1.17.3.2) is a multifunctional molybdenum-flavoprotein and one of the forms of xanthine oxidoreductase (XOR). The characteristic of XOD is that each enzyme unit binds two flavin molecules (FAD), two molybdenum atoms and eight iron atoms. XOD is widely distributed in various tissues and cells of eukaryotic and prokaryotes. Eukaryotic XODs are homodimers with a molecular weight of 300 KDa. Most prokaryotes can grow with purine as nitrogen, carbon or energy source. Different subunits (Mw) from prokaryotic sources reveal several different quaternary structures, such as (αβγ)2 Rhodobacter capsulatus, α3 Pseudomonas putida (PpXOD), (αβ)2 Rhodobacter capsulatus (RcXOD) and Arthrobacter spp., αβγ from Escherichia coli (EcXOD), and αβ from Arthrobacter S-2. The differences in their molecular weight and subunit composition reflect the diversity and evolution of prokaryotic XOD enzymes compared with eukaryotic XOD enzymes. The diversity of the prokaryotic XOD family may reveal different properties and specific functions of these enzymes. However, prokaryotic XODs are less studied.

[0003] XOD belongs to the class of metalloflavoprotein complexes. It is produced by oxidation of thiol residues or by proteolysis by xanthine dehydrogenase (XDH). XOD and XDH play a vital role in the last two steps of urate formation. Elevated levels of XOD can be observed in the serum of patients with chronic liver disease. Therefore, XOD can be used as a biomarker for the detection of liver disease. Xanthine oxidase is a key enzyme in the purine metabolic pathway, catalyzing the hydroxylation of hypoxanthine to xanthine and further hydroxylation of xanthine to uric acid while reducing NAD or molecular oxygen. This enzyme is also involved in caffeine metabolism. XOD also plays a vital role in the catabolism of xenobiotics, including various drugs such as thiopurine drugs, containing 6-mercaptopurine, allopurinol and uric acid. Excessive XOD activity leads to hyperuricemia (high uric acid levels in the blood), which further leads to gout.

[0004] The xanthine oxidase currently used at home and abroad is mainly obtained from milk, with an activity of about 1U / mg. Due to its relatively single source, the production cost is high, and the content and quality of xanthine oxidase are affected by batches, which greatly limits its application. The corresponding products of domestic and foreign companies have poor thermal stability and cannot meet the high temperature problems generated during reagent transportation. Therefore, a new xanthine oxidase gene and Pseudomonas expression system are needed. Summary of the invention

[0005] The object of the present invention is to provide a xanthine oxidase, the amino acid sequence of its B subunit is SEQ ID NO: 1, and the amino acid sequence of its A subunit is SEQ ID NO: 2.

[0006] Another object of the present invention is to provide a mutant xanthine oxidase, whose B subunit amino acid sequence is SEQ ID NO:3; and whose A subunit amino acid sequence is SEQ ID NO:4.

[0007] Another object of the present invention is to provide a DNA sequence encoding xanthine oxidase.

[0008] Another object of the present invention is to provide an expression vector containing the DNA sequence.

[0009] Another object of the present invention is to provide a recombinant strain containing the DNA sequence.

[0010] Another object of the present invention is to provide a method for preparing xanthine oxidase.

[0011] Another object of the present invention is to provide the use of xanthine oxidase in preparing a kit for clinical detection of 5'-nucleotidase and adenosine deaminase.

[0012] In order to achieve these purposes and other advantages according to the present invention, a xanthine oxidase is provided, whose B subunit amino acid sequence is shown in SEQ ID NO:1, and whose A subunit amino acid sequence is shown in SEQ ID NO:2.

[0013] The mutant xanthine oxidase, its B subunit amino acid sequence is shown in SEQ ID NO:3, and its A subunit amino acid sequence is shown in SEQ ID NO:4.

[0014] The DNA sequence encoding the xanthine oxidase is shown in SEQ ID NO:5.

[0015] The DNA sequence encoding the xanthine oxidase is shown in SEQ ID NO:6.

[0016] An expression vector comprising the DNA sequence is a prokaryotic expression vector, an insect cell expression vector, a yeast or fungal expression vector.

[0017] The recombinant strain containing the DNA sequence is obtained by transforming an expression vector containing a xanthine oxidase expression cassette into an expression host, wherein the xanthine oxidase expression cassette consists of a promoter and a xanthine oxidase gene connected to the downstream of the promoter, and the expression host is Pseudomonas mendocina, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas putida, or Pseudomonas fluorescens.

[0018] The preparation method of xanthine oxidase comprises constructing an expression vector of the xanthine oxidase, transforming an expression host to obtain a recombinant strain, inducing the expression of the xanthine oxidase, and separating and purifying the xanthine oxidase.

[0019] The xanthine oxidase is used in preparing a kit for clinical detection of 5'-nucleotidase and adenosine deaminase.

[0020] The present invention has at least the following beneficial effects:

[0021] First, the present invention randomly mutates the xanthine oxidase gene using error-prone PCR, and pre-expresses and screens it in Pseudomonas mendocina NK-01 through an expression vector to obtain a xanthine oxidase mutant with good thermal stability and high activity. The enzymatic properties of the xanthine oxidase mutant are the same as those of the wild-type xanthine oxidase, and its thermal stability is significantly higher than that of the wild-type xanthine oxidase. The activity is 91.1 times that of milk-derived xanthine oxidase, and is suitable for large-scale industrial production of xanthine oxidase.

[0022] Second, the present invention can obtain a large number of xanthine oxidase mutants through fermentation of recombinant strains, providing a large number of raw materials for the development of clinical diagnostic reagents and reducing production costs. The kit prepared by the present invention is used to detect 5'-nucleotidase and adenosine deaminase, with good repeatability and high precision.

[0023] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The pBBR1MCS-5_XOD map and the pBBR1MCS-5_XOD-mut map of the present invention;

[0025] Figure 2 is the PCR identification result of XOD of the present invention;

[0026] Figure 3 The activity curves of XOD and XOD-mut of the present invention at different pH values;

[0027] Figure 4Activity curves of XOD and XOD-mut of the present invention after being kept at different pH values ​​for 15 hours;

[0028] Figure 5 The activity curves of XOD and XOD-mut of the present invention at different temperatures;

[0029] Figure 6 These are the activity curves of XOD, XOD-mut and milk-derived cXOD of the present invention after being kept at different temperatures for 30 minutes. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0031] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0032] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0033] The xanthine oxidase protected by the present invention has a B subunit amino acid sequence of SEQ ID NO: 1 and an A subunit amino acid sequence of SEQ ID NO: 2.

[0034] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0035] MAININGAPAEATPRPGGQCLRTFLREQGNLGVRKGCDGGDCGACTVHVDGTDGVPVCIYPAVRAEV

[0036] TTIETTIEGLAEASGLHPGLHPVQQCGFCTRQGFQCGFCTAATFSDMTAATFDDEQGNLCLPRNLKGSI

[0037] ADSCTGYRAIADAVADSAGHPAPQGQGARPDSGSQPDPEPGQLGDDVPAPASGAARYTLARYTLDIP

[0038] QLLHLKLHLKVLRSPHAHARVVSIDSDALKIPRIPGVVAVSPTQLFSAPEQLFSTAQHELFTDDNVVRFI

[0039] GQLDDVVRFIGQRVAAVGTRAVAAAEAGVRALAAEYEELPAVFTPGDKDSRPGAPLVHGGPANNVAA

[0040] ARISRPDRNVVAGFRAGSVADGFSAADFIHEALETHSQRVQHVALETHAAISQVPFLVKEGRLQVRTSS

[0041] QVPDRVRVTLCRVFGLEEDRVQEVLVAGRVGGGFGGKQEVQVEVALAALKLKRPVQLHPFTIQFTAT

[0042] TTRHPFTIRLKAGASDTGAHLTALELDVLGVMFHYGNHGPGVMFHGVMFHAVYKCDNKKVDAHA

[0043] MYTNTVPSGIDERGYGLSQMIFAVLNMMDELAAGIGMDPYPEPEEDVMVREGDHMLSTHPEPEEDR

[0044] GRGSYGLDQCLDLVRPEWVTGRERYRAAGLDQLGPRGHYGAALSMIDTVPPRGHFAHSRIAEFGGT

[0045] YQADVGTAEFGNGTTALGTSAQLAATALSTEAAHDTGAQSDTDLVEHDTGAFGSAEELSTGKATLAA

[0046] AEELAVRIDCHLNAGIRQTQASACVLAELYVVVDRTPVALSELAQAALYEEALAAEGRWGGTNTGTG

[0047] FNVHGFRVAVNRVNPRQRILQSVQAADAGVVVNPRQCRGQIEGGIAQAIGAALTDILRVDSTGKVTTD

[0048] IDVPRSETFADVPRSAKFADTNDKLGPPALANMSESPFNPVIRFSAIRNATGVRFASLLALRDRIYLGAS

[0049] AMVPVGEGVTR

[0050] The amino acid sequence of SEQ ID NO:2 is specifically as follows:

[0051] MDMNTIEAVRPTADPAQWRDGDAWLAGETVLFSYGSYAFGPRQVAGWEPGAGGETPVTVTDDGICT

[0052] VCRTCTIAELPGGAGVPDAAGLSWVYRPCLVRPCCDSFVAWNLSTNMVTVGGNLCTSSLCADISLCA

[0053] GLDGVATHGARGLTSRSVPTGDGTGDAKECRSIRLPLLRSVHLPASALSISLSNLRRLSLSNLGRSGVLI

[0054] GRRGFTALVLTVSTKRPVRQLRFDSLPDAVLAHSLDDAIPDGLYHDDHGLPAWRNDRRDMLAEEIRA

[0055] LCADPTGTGAPGPVSGPKHRSAPASGSTALPKGA

[0056] The mutant xanthine oxidase protected by the present invention has a B subunit amino acid sequence of SEQ ID NO: 3 and an A subunit amino acid sequence of SEQ ID NO: 4.

[0057] The amino acid sequence of SEQ ID NO:3 is as follows:

[0058] MAININGAPAEATPRPGGQCLRTFLREQGNLGVRKGCDGGDCGACTVHVDGTDGVPVCIYPAVRAEV

[0059] TTIETTIEGLAEASGLHPGLHPVQQCGFCTRQGFQCGFCTAATFSDMTAATFDDEQGNLCLPRNLKGSI

[0060] ADSCTGYRAIADAVADSAGHPAPQGQGARPDSGSQPDPEPGQLGDDVPAPASGAARYTLARYTLDIK

[0061] QLLHLKLHLKVLRSPHAHARVVSIDSDALKIPRIPGVVAVSPTQLFSAPEQLFSTAQHELFTDDNVVRFI

[0062] GQLDDVVRFIGQRVAAVGTRAVAAAEAGVRALAAEYEELPAVFTPGDKDSRPGAPLVHGGPANNVAA

[0063] ARISRPDRNVVAGFRAGSVADGFSAADFIHEALETHSQRVQHVALETHAAISQVPFLVKEGRLQVRTSS

[0064] QVPDRVRVTLCRVFGLEEDRVQEVLVAGRVGGGFGGKQEVQVEVALAALKLKRPVQLHPFTIQFTAT

[0065] TTRHPFTIRLKAGASDTGAHLTALELDVLGVMFHYGNHGPGVMFHGVMFHAVYKCDNKKVDAHA

[0066] MYTNTVPSGIDERGYGLSQMIFAVLNMEDELAAGIGMDPYPEPEEDVMVREGDHMLSTHPEPEEDR

[0067] GRGSYGLDQCLDLVRPEWVTGRERYRAAGLDQLGPRGHYGAALSMIDTVPPRGHFAHSRIAEFGGT

[0068] YQADVGTAEFGNGTTALGTSAQLAATALSTEAAHDTGAQSDTDLVEHDTGAFGSAEELSTGKATLAA

[0069] AEELAVRIDCHLNAGIRQTQASACVLAELYVVVDRTPVALSELAQAALYEEALAAEGRWGGTNTGTG

[0070] FNVHGFRVAVNRTNPRQRILQSVQAADAGVVVNPRQCRGQIEGGIAQAIGAALTDILRVDSTGKVTTD

[0071] IDVPRSETFADVPRSAKFADTNDKLGPPALANMSESPFNPVIRFSAIRNATGVRFASLLALRDRIYLGAS

[0072] AMVPVGEGVTR

[0073] The amino acid sequence of SEQ ID NO:4 is as follows:

[0074] MDMNTIEAVRPTADPAQWRDGDAWLAGETVLFSYGSYAFGPRQVAGWEPGAGGETPVTVTDDGICT

[0075] VCRTTCTIAELPGGARVPDAAGLSWVYRPCLVRPCCDSFVAWNLSTNMVTVGGNLCTSSLCADISLCA

[0076] GLDGVATHGARGLTSRSVPTGDGTGDAKECRSIRLPLLRSVHLPASALSISLSNLRRLSLSNLGRSGVLI

[0077] GRRGFTALVLTVSTKRPVRQLRFDSLPDAVLAHSLDDAIPDGLYHDDHGLPAWRNDRRDMLAEEIRA

[0078] LCADPTGTGAPGPVSGPKHRSAPASGSTALPKGA

[0079] Specifically, the xanthine oxidase nucleic acid sequence was subjected to error-prone PCR random mutation, and the 203rd amino acid of the xanthine oxidase B subunit was mutated from P to K, the 568th amino acid was mutated from M to E, and the 819th amino acid was mutated from V to T; the 81st amino acid of the xanthine oxidase A subunit was mutated from G to R, thereby obtaining a xanthine oxidase with good thermal stability and high activity.

[0080] The amino acid sequences SEQ ID NO: 1-4 in which one or more amino acids are replaced, deleted or added but whose xanthine oxidase function remains unchanged also fall within the protection scope of the present application.

[0081] If the amino acid residues of the same group are replaced in the amino acid sequence, such as R replaced by K or I replaced by L, the role of the residues in the protein domain is not changed, so the three-dimensional structure of the protein will not be affected, so the function of the protein can still be achieved. For example, D and E, S and T, A and G, Q and N, P and G, F and W, A and V, C and M, etc., are replaced with each other in pairs, which will not affect the three-dimensional structure and functional activity of the protein. The amino acid residues belonging to the same category can be replaced at any amino acid residue position on xanthine oxidase. On the contrary, the amino acid residues of different categories are replaced, or the amino acid replacement does not conform to the above-listed replacement rules, which is likely to change the structure of the protein and cause differences in function.

[0082] The xanthine oxidase provided by the present invention can also be modified or mutated to obtain a derived protein. The "derived protein" described in the present invention refers to a protein that has a difference in amino acid sequence from the xanthine oxidase having the above amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants can be obtained by various techniques, such as random mutations produced by radiation or mutagens, or by techniques such as site-directed mutagenesis or other known molecular biology techniques.

[0083] Modifications include: chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as mammalian glycosylation acid or deglycosylation enzyme). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0084] In a further preferred embodiment, the amino acid sequence of the xanthine oxidase has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1, preferably has at least 95% identity, and more preferably has at least 99% identity.

[0085] The DNA sequence encoding xanthine oxidase XOD is shown in SEQ ID NO:5.

[0086] The nucleotide sequence of SEQ ID NO:5 is as follows:

[0087] ATGGACATGAACACCATCGAGGCCGTGCGTCCGACCGCCGATCCAGCGCAATGGCGTGACGGT

[0088] GATGCCTGGCTGGCCGGTGAAACCGTGCTGTTCAGCTACGGCAGCTACGCCTTCGGTCCGCGT

[0089] CAGGTGGCCGGTTGGGAACCAGGTGCCGGTGGCGAAACCCCAGTGACCGTGACCGACGACGG

[0090] CATCTGCACCGTGTGCCGTACCTGCACCATCGCCGAGCTGCCAGGTGGTGCGGGTGTGCCGGA

[0091] TGCCGCCGGTCTGAGCTGGGTGTATCGTCCGTGCCTGGTGCGTCCGTGCTGCGACAGCTTCGTG

[0092] GCCTGGAACCTGAGCACCAACATGGTGACCGTCGGTGGCAACCTGTGCACCAGCAGCCTGTGC

[0093] GCCGACATCTCGCTGTGCGCCGGTCTGGATGGCGTGGCGACCCATGGTGCCCGTGGTCTGACC

[0094] AGCCGTTCGGTGCCAACCGGTGACGGCACCGGTGATGCGAAAGAGTGCCGCAGCATCCGCCT

[0095] GCCGCTGCTGCGTAGCGTGCATCTGCCAGCGAGCGCCCTGAGCATCAGCCTGAGCAACCTGCG

[0096] TCGCCTGAGCCTGTCGAACCTGGGTCGCAGCGGTGTGCTGATCGGTCGCCGTGGCTTCACCGC

[0097] GCTGGTGCTGACCGTGTCGACCAAGCGTCCGGTGCGCCAGCTGCGCTTCGACAGCCTGCCGGA

[0098] CGCCGTGCTGGCCCATAGCCTGGATGACGCGATCCCGGACGGCCTGTACCACGACGACCATGG

[0099] CCTGCCAGCCTGGCGCAACGACCGTCGCGATATGCTGGCCGAAGAGATCCGTGCGCTGTGCGC

[0100] GGATCCAACCGGCACCGGTGCGCCAGGTCCGGTGAGCGGTCCGAAACATCGTAGCGCCCCAG

[0101] CCAGCGGTTCGACCGCGCTGCCAAAGGGTGCCTGAGCATGGCCATCAACATCAACGGTGCCCC

[0102] AGCCGAGGCGACCCCACGTCCAGGCGGTCAGTGCCTGCGTACCTTCCTGCGCGAGCAGGGCA

[0103] ACCTGGGCGTGCGCAAAGGTTGCGACGGTGGCGATTGCGGTGCCTGCACCGTCCATGTGGATG

[0104] GCACCGATGGCGTGCCGGTCTGCATCTACCCAGCCGTGCGTGCGGAGGTGACCACCATCGAAA

[0105] CCACCATCGAGGGCCTGGCCGAGGCCAGCGGTCTGCACCCAGGCCTGCATCCGGTGCAGCAGT

[0106] GCGGCTTCTGCACCCGTCAGGGCTTCCAATGCGGTTTCTGCACCGCCGCCACCTTCAGCGACAT

[0107] GACCGCGGCGACCTTCGACGACGAACAGGGTAACCTGTGCCTGCCACGCAACCTGAAGGGCT

[0108] CGATCGCCGACTCGTGCACCGGCTACCGTGCGATCGCGGATGCCGTGGCCGATAGCGCCGGTCA

[0109] TCCAGCGCCACAAGGTCAGGGTGCCCGTCCGGATAGCGGCAGCCAGCCGGATCCAGAACCGG

[0110] GTCAGCTGGGCGACGATGTGCCAGCGCCAGCCTCGGGTGCCGCGCGTTATACCCTGGCGCGTT

[0111] ACACCCTGGACATCCCACAGCTGCTGCATCTGAAGCTGCACCTGAAGGTCCTGCGCAGCCCAC

[0112] ACGCGCATGCCCGTGTCGTGAGCATCGACTCGGACGCCCTGAAGATCCCACGCATCCCAGGCG

[0113] TGGTGGCCGTGTCGCCGACCCAACTGTTCTCGGCCCCAGAGCAGCTGTTCTCCACCGCGCAGC

[0114] ACGAGCTGTTCACCGACGACAACGTGGTGCGCTTCATCGGCCAGCTGGACGACGTGGTCCGTT

[0115] TCATCGGTCAACGCGTGGCCGCCGTGGGCACCCGTGCCGTCGCCGCCGCCGAAGCCGGTGTGC

[0116] GTGCCCTGGCCGCCGAGTACGAAGAACTGCCAGCCGTGTTCACCCCAGGCGACAAGGACAGC

[0117] CGTCCAGGTGCGCCACTGGTGCATGGCGGTCCAGCCAACAACGTGGCCGCGGCGCGTATCTCG

[0118] CGTCCGGACCGCAACGTGGTCGCCGGTTTCCGTGCCGGTTCGGTGGCCGATGGTTTCAGCGCC

[0119] GCCGACTTCATCCACGAGGCCCTGGAAACCCACTCGCAGCGCGTGCAGCACGTCGCGCTGGA

[0120] AACCCATGCCGCGATCAGCCAGGTGCCATTCCTGGTGAAAGAGGGTCGCCTGCAGGTCCGCAC

[0121] CAGCTCGCAAGTGCCGGATCGCGTGCGTGTGACCCTGTGCCGTGTGTTCGGCCTGGAAGAGGA

[0122] CCGCGTGCAAGAGGTGCTGGTCGCGGGTCGTGTCGGTGGCGGTTTCGGTGGTAAGCAAGAGG

[0123] TCCAGGTGGAAGTGGCCCTGGCGGCCCTGAAGCTGAAGCGTCCAGTGCAACTGCATCCGTTCA

[0124] CCATCCAGTTCACCGCCACCACCACCCGTCATCCATTCACCATCCGCCTGAAAGCCGGTGCCAG

[0125] CGATACCGGTGCGCATCTGACCGCGCTGGAACTGGACGTGCTGGGCGTGATGTTCCACTACGG

[0126] CAACCACGGTCCGGGTGTCATGTTCCACGGCGTCATGTTCCATGCCGTGTACAAGTGCGACAA

[0127] CAAGAAGGTGGACGCCCACGCCATGTACACCAACACCGTGCCGAGCGGCATCGACGAGCGTG

[0128] GCTATGGCCTGAGCCAGATGATCTTCGCCGTCCTGAACATGATGGACGAGCTGGCGGCCGGTAT

[0129] CGGCATGGACCCGTATCCGGAACCGGAAGAGGATGTGATGGTGCGCGAGGGCGACCACATGCT

[0130] GAGCACCCATCCAGAGCCGGAAGAGGACCGTGGCCGTGGCAGCTACGGCCTGGACCAGTGCC

[0131] TGGACCTGGTCCGTCCGGAATGGGTGACCGGTCGCGAGCGTTATCGTGCGGCCGGTCTGGACC

[0132] AACTGGGTCCGCGTGGCCATTACGGTGCGGCCCTGTCCATGATCGATACCGTGCCACCGCGTGG

[0133] TCACTTCGCCCACAGCCGTATCGCGGAATTCGGTGGCACCTACCAGGCCGATGTGGGCACCGC

[0134] CGAGTTCGGCAACGGCACCACCGCGCTGGGCACCTCGGCGCAACTGGCCGCCACCGCGCTGT

[0135] CGACCGAAGCCGCGCATGACACCGGTGCGCAGAGCGACACCGACCTGGTCGAGCATGATACC

[0136] GGTGCCTTCGGCAGCGCCGAGGAACTGTCCACCGGCAAAGCGACCCTGGCGGCGGCCGAAGA

[0137] ACTGGCCGTGCGCATCGACTGCCACCTGAACGCCGGTATCCGTCAGACCCAGGCCAGCGCCTG

[0138] CGTCCTGGCCGAACTGTACGTGGTGGTCGATCGCACCCCAGTCGCGCTGAGCGAACTGGCCCA

[0139] AGCGGCGCTGTACGAGGAAGCGCTGGCCGCGGAAGGCCGTTGGGGTGGCACCAACACCGGCA

[0140] CCGGCTTCAACGTGCACGGCTTCCGCGTCGCCGTGAACCGCGTGAACCCACGCCAGCGTATCC

[0141] TGCAGAGCGTGCAAGCGGCCGATGCGGGTGTCGTGGTCAACCCACGTCAGTGCCGTGGTCAGA

[0142] TCGAAGGCGGTATCGCCCAGGCGATCGGTGCCGCGCTGACCGATATCCTGCGCGTGGACAGCA

[0143] CCGGCAAGGTCACCACCGATATCGACGTGCCACGCAGCGAAACCTTCGCGGACGTCCCACGCT

[0144] CGGCCAAGTTCGCGGACACCAACGACAAGCTGGGTCCGCCAGCGCTGGCCAACATGAGCGAG

[0145] AGCCCGTTCAACCCGGTGATCCGCTTCAGCGCCATCCGTAACGCCACCGGTGTGCGCTTCGCCT

[0146] CGCTGCTGGCCCTGCGCGACCGCATCTACCTGGGTGCGAGCGCCATGGTGCCGGTCGGTGAAG

[0147] GCGTGACCCGCTGA

[0148] The DNA sequence encoding the mutant xanthine oxidase XOD-mut is shown in SEQ ID NO:6.

[0149] The nucleotide sequence of SEQ ID NO:6 is specifically as follows:

[0150] ATGGACATGAACACCATCGAGGCCGTGCGTCCGACCGCCGATCCAGCGCAATGGCGTGACGGTGA

[0151] TGCCTGGCTGGCCGGTGAAACCGTGCTGTTCAGCTACGGCAGCTACGCCTTCGGTCCGCGTCAGG

[0152] TGGCCGGTTGGGAACCAGGTGCCGGTGGCGAAACCCCAGTGACCGTGACCGACGACGGCATCTG

[0153] CACCGTGTGCCGTACCTGCACCATCGCCGAGCTGCCAGGTGGTGCGCGTGTGCCGGATGCCGCCG

[0154] GTCTGAGCTGGGTGTATCGTCCGTGCCTGGTGCGTCCGTGCTGCGACAGCTTCGTGGCCTGGAAC

[0155] CTGAGCACCAACATGGTGACCGTCGGTGGCAACCTGTGCACCAGCAGCCTGTGCGCCGACATCTC

[0156] GCTGTGCGCCGGTCTGGATGGCGTGGCGACCCATGGTGCCCGTGGTCTGACCAGCCGTTCGGTGC

[0157] CAACCGGTGACGGCACCGGTGATGCGAAAGAGTGCCGCAGCATCCGCCTGCCGCTGCTGCGTAG

[0158] CGTGCATCTGCCAGCGAGCGCCCTGAGCATCAGCCTGAGCAACCTGCGTCGCCTGAGCCTGTCGA

[0159] ACCTGGGTCGCAGCGGTGTGCTGATCGGTCGCCGTGGCTTCACCGCGCTGGTGCTGACCGTGTCG

[0160] ACCAAGCGTCCGGTGCGCCAGCTGCGCTTCGACAGCCTGCCGGACGCCGTGCTGGCCCATAGCCT

[0161] GGATGACGCGATCCCGGACGGCCTGTACCACGACGACCATGGCCTGCCAGCCTGGCGCAACGAC

[0162] CGTCGCGATATGCTGGCCGAAGAGATCCGTGCGCTGTGCGCGGATCCAACCGGCACCGGTGCGCC

[0163] AGGTCCGGTGAGCGGTCCGAAACATCGTAGCGCCCCAGCCAGCGGTTCGACCGCGCTGCCAAAG

[0164] GGTGCCTGAGCATGGCCATCAACATCAACGGTGCCCCAGCCGAGGCGACCCCACGTCCAGGCGG

[0165] TCAGTGCCTGCGTACCTTCCTGCGCGAGCAGGGCAACCTGGGCGTGCGCAAAGGTTGCGACGGT

[0166] GGCGATTGCGGTGCCTGCACCGTCCATGTGGATGGCACCGATGGCGTGCCGGTCTGCATCTACCC

[0167] AGCCGTGCGTGCGGAGGTGACCACCATCGAAACCACCATCGAGGGCCTGGCCGAGGCCAGCGGT

[0168] CTGCACCCAGGCCTGCATCCGGTGCAGCAGTGCGGCTTCTGCACCCGTCAGGGCTTCCAATGCGG

[0169] TTTCTGCACCGCCGCCACCTTCAGCGACATGACCGCGGCGACCTTCGACGACGAACAGGGTAACC

[0170] TGTGCCTGCCACGCAACCTGAAGGGCTCGATCGCCGACTCGTGCACCGGCTACCGTGCGATCGCG

[0171] GATGCCGTGGCCGATAGCGCCGGTCATCCAGCGCCACAAGGTCAGGGTGCCCGTCCGGATAGCGG

[0172] CAGCCAGCCGGATCCAGAACCGGGTCAGCTGGGCGACGATGTGCCAGCGCCAGCCTCGGGTGCC

[0173] GCGCGTTATACCCTGGCGCGTTACACCCTGGACATCAAACAGCTGCTGCATCTGAAGCTGCACCT

[0174] GAAGGTCCTGCGCAGCCCACACGCGCATGCCCGTGTCGTGAGCATCGACTCGGACGCCCTGAAG

[0175] ATCCCACGCATCCCAGGCGTGGTGGCCGTGTCGCCGACCCAACTGTTCTCGGCCCCAGAGCAGCT

[0176] GTTCTCCACCGCGCAGCACGAGCTGTTCACCGACGACAACGTGGTGCGCTTCATCGGCCAGCTGG

[0177] ACGACGTGGTCCGTTTCATCGGTCAACGCGTGGCCGCCGTGGGCACCCGTGCCGTCGCCGCCGCC

[0178] GAAGCCGGTGTGCGTGCCCTGGCCGCCGAGTACGAAGAACTGCCAGCCGTGTTCACCCCAGGCG

[0179] ACAAGGACAGCCGTCCAGGTGCGCCACTGGTGCATGGCGGTCCAGCCAACAACGTGGCCGCGGC

[0180] GCGTATCTCGCGTCCGGACCGCAACGTGGTCGCCGGTTTCCGTGCCGGTTCGGTGGCCGATGGTT

[0181] TCAGCGCCGCCGACTTCATCCACGAGGCCCTGGAAACCCACTCGCAGCGCGTGCAGCACGTCGC

[0182] GCTGGAAACCCATGCCGCGATCAGCCAGGTGCCATTCCTGGTGAAAGAGGGTCGCCTGCAGGTC

[0183] CGCACCAGCTCGCAAGTGCCGGATCGCGTGCGTGTGACCCTGTGCCGTGTGTTCGGCCTGGAAG

[0184] AGGACCGCGTGCAAGAGGTGCTGGTCGCGGGTCGTGTCGGTGGCGGTTTCGGTGGTAAGCAAGA

[0185] GGTCCAGGTGGAAGTGGCCCTGGCGGCCCTGAAGCTGAAGCGTCCAGTGCAACTGCATCCGTTC

[0186] ACCATCCAGTTCACCGCCACCACCACCCGTCATCCATTCACCATCCGCCTGAAAGCCGGTGCCAG

[0187] CGATACCGGTGCGCATCTGACCGCGCTGGAACTGGACGTGCTGGGCGTGATGTTCCACTACGGCA

[0188] ACCACGGTCCGGGTGTCATGTTCCACGGCGTCATGTTCCATGCCGTGTACAAGTGCGACAACAAG

[0189] AAGGTGGACGCCCACGCCATGTACACCAACACCGTGCCGAGCGGCATCGACGAGCGTGGCTATG

[0190] GCCTGAGCCAGATGATCTTCGCCGTCCTGAACATGGAGGACGAGCTGGCGGCCGGTATCGGCATG

[0191] GACCCGTATCCGGAACCGGAAGAGGATGTGATGGTGCGCGAGGGCGACCACATGCTGAGCACCC

[0192] ATCCAGAGCCGGAAGAGGACCGTGGCCGTGGCAGCTACGGCCTGGACCAGTGCCTGGACCTGGT

[0193] CCGTCCGGAATGGGTGACCGGTCGCGAGCGTTATCGTGCGGCCGGTCTGGACCAACTGGGTCCGC

[0194] GTGGCCATTACGGTGCGGCCCTGTCCATGATCGATACCGTGCCACCGCGTGGTCACTTCGCCCACA

[0195] GCCGTATCGCGGAATTCGGTGGCACCTACCAGGCCGATGTGGGCACCGCCGAGTTCGGCAACGGC

[0196] ACCACCGCGCTGGGCACCTCGGCGCAACTGGCCGCCACCGCGCTGTCGACCGAAGCCGCGCATG

[0197] ACACCGGTGCGCAGAGCGACACCGACCTGGTCGAGCATGATACCGGTGCCTTCGGCAGCGCCGA

[0198] GGAACTGTCCACCGGCAAAGCGACCCTGGCGGCGGCCGAAGAACTGGCCGTGCGCATCGACTGC

[0199] CACCTGAACGCCGGTATCCGTCAGACCCAGGCCAGCGCCTGCGTCCTGGCCGAACTGTACGTGGT

[0200] GGTCGATCGCACCCCAGTCGCGCTGAGCGAACTGGCCCAAGCGGCGCTGTACGAGGAAGCGCTG

[0201] GCCGCGGAAGGCCGTTGGGGTGGCACCAACACCGGCACCGGCTTCAACGTGCACGGCTTCCGCG

[0202] TCGCCGTGAACCGCACGAACCCACGCCAGCGTATCCTGCAGAGCGTGCAAGCGGCCGATGCGGG

[0203] TGTCGTGGTCAACCCACGTCAGTGCCGTGGTCAGATCGAAGGCGGTATCGCCCAGGCGATCGGTG

[0204] CCGCGCTGACCGATATCCTGCGCGTGGACAGCACCGGCAAGGTCACCACCGATATCGACGTGCCA

[0205] CGCAGCGAAACCTTCGCGGACGTCCCACGCTCGGCCAAGTTCGCGGACACCAACGACAAGCTGG

[0206] GTCCGCCAGCGCTGGCCAACATGAGCGAGAGCCCGTTCAACCCGGTGATCCGCTTCAGCGCCATC

[0207] CGTAACGCCACCGGTGTGCGCTTCCGCCTCGCTGCTGGCCCTGCGCGACCGCATCTACCTGGGTGC

[0208] GAGCGCCATGGTGCCGGTCGGTGAAGGCGTGACCCGCTGA

[0209] Using the gene of xanthine oxidase as a template, the gene was randomly mutated by error-prone PCR, and pre-expressed and screened in Pseudomonas mendocinae NK-01 through the constructed expression vector. The mutant genetic engineering bacteria obtained expressed xanthine oxidase with good thermal stability and could withstand high temperatures of 70°C. After testing, the enzymatic properties were as follows: the molecular weight of the B subunit was about 101KDa, and the molecular weight of the A subunit was about 24.4KDa; thermal stability: 70°C, 30min. The K of XOD at 37°C and pH 7.5 was m The value is 2.5×10 -5 M (xanthine as substrate), 3.6×10 -5 M (hypoxanthine as substrate); K of XOD-mut m The value is 1.8×10 -5 M (xanthine as substrate), 3.2×10 -5M (using hypoxanthine as a substrate). Moreover, in the application of fermentative production of xanthine oxidase, it can improve the production capacity of producing xanthine oxidase by microbial fermentation without changing the original production process and technological steps, is suitable for large-scale industrial production of xanthine oxidase, and has high application value and industrial practicability.

[0210] <Construction of XOD Vector and Flask Fermentation>

[0211] 1. Experimental Materials

[0212] The pBBR1MCS-5 vector plasmid and Pseudomonas mendocina NK-01 strain are preserved by our company; DNA marker 15000 is purchased from Beijing Tiangen Biotech Co., Ltd.; peptone and yeast extract are purchased from OXOID; Ni column and SP column are purchased from GE Company; EcoR I and Sal I enzymes are purchased from NEB (Beijing) Co., Ltd.

[0213] 2. Construction of Xanthine Oxidase Protein Expression

[0214] The XOD gene fragment is synthesized by Sangon Biotech, ligated with the pBBR1MCS-5 vector after double digestion with EcoR I and Sal I using homologous recombinase, and the PCR identification results are as Figure 2 shown. M: marker. The C-terminus of the xanthine oxidase DNA sequence contains an 8×His tag, and then it is transformed into DH5α competent cells, spread on LB solid plates (10 μg / mL gentamicin). For each gene, 24 transformants are screened by colony PCR, and the recombinants are identified by double digestion, and sent to a sequencing company for gene sequencing. The successfully constructed vector is named pBBR1MCS-5-XOD, and the map is as Figure 1 shown in Figure A.

[0215] 3. Small-scale Extraction of Xanthine Oxidase Expression Plasmid

[0216] The xanthine oxidase positive clone DH5α strain is inoculated into 10 mL of LB medium containing 10 μg / mL gentamycin, cultured until the mid-logarithmic growth phase (OD 600 = 0.5 - 0.8), then 1 mL of the bacterial solution is taken out and added with sterile glycerol and stored at -80 °C to preserve the strain, and the remaining bacteria continue to be cultured overnight at 37 °C. The plasmid is extracted using a plasmid small-scale extraction kit and stored in aliquots.

[0217] LB medium: Trypton 1%, Yeast Extract 0.5%, NaCl 1%, pH 7.5. Add 2% agar powder when making plates. Autoclave at 121℃ for 20min. Can be stored at room temperature for several months. When used to culture pBBR1MCS-5 transformed host bacteria DH5α, let the medium cool to at least 50℃, then add gentamycin to a final concentration of 10μg / mL, and store at 4℃ for 1-2 weeks.

[0218] 4. Preparation of Pseudomonas mendocinae Electroporation Competent Cells

[0219] (1) Streak Pseudomonas mendocinae stored at -80°C on a solid LB plate;

[0220] (2) Inoculate a single colony from a freshly cultured plate into 5-10 mL of LB liquid medium and culture overnight (16 h) at 200 rpm and 37°C.

[0221] (3) Inoculate 1.5 mL of overnight cultured bacterial solution into 150 mL (in a 500 mL Erlenmeyer flask) of fresh LB medium and culture at 200 rpm and 37°C for 3-4 h until the bacteria reach the logarithmic growth phase and the bacterial count reaches about 1 × 10 8 cells / mL;

[0222] (4) Transfer the bacterial solution to a sterile 50 mL centrifuge tube, place on ice for 15 min, and then centrifuge at 4°C, 4500 rpm, for 5 min to collect the bacteria;

[0223] (5) Discard the supernatant, resuspend the cells in 30 mL of ice-cold 10% glycerol, place on ice for 5 min, and then centrifuge at 4°C, 4500 rpm, for 5 min to collect the cells;

[0224] (6) Discard the supernatant, resuspend the cells in 15 mL of ice-cold 10% glycerol, place on ice for 5 min, and then centrifuge at 4°C, 4500 rpm, for 5 min to collect the cells;

[0225] (7) Resuspend the cells in 2 mL of pre-cooled 10% glycerol to a cell concentration of about 1 × 10 10 cells / mL;

[0226] (8) The competent cells were dispensed into 1.5 mL centrifuge tubes, 50 μL each, and quickly frozen at ultra-low temperature (liquid nitrogen), and then stored at -80°C.

[0227] 5. Electroporation of Pseudomonas mendocinae

[0228] Take 5 μL of plasmid to electrotransform 250 μL of NK-01 electroporation competent cells, mix well and add to the pre-cooled 0.2 cm (electrode gap) electroporation transformation cup (cuvette), the specific parameters are high voltage pulse 2.5 kV / cm, capacitance 25 μF, resistance 200 Ω, electroporation 5 ms; after the electroporation, take out the cup and immediately add 800 μl LB culture medium and transfer to the original centrifuge tube, 37 ° C, 200 rpm, resuscitation for 1 hour, take 500 μL of bacterial solution and spread on the LB plate containing 30 μg / mL gentamycin, and invert and culture at 37 ° C overnight for 24-36 hours.

[0229] 6. Shake flask fermentation of pBBR1MCS-5-XOD strain

[0230] Pick 3 monoclonal colonies, culture at 29-30℃, 150rpm for 4h, take out the bacteria and add glycerol to preserve the strain at -80℃. At the same time, inoculate into TB medium (lactose-free) at 1:1000, culture at 29-30℃, 150rpm for 8h, add ammonium molybdate to a final concentration of 1mM, continue to culture for 20h, take 2mL of bacterial solution, centrifuge at 12000rpm and discard the supernatant, add 2mL Tris-HCl buffer, pH7.5, to the precipitate, ultrasonicate for 1-2min, centrifuge at 12000rpm to obtain the supernatant, and obtain the crude cell enzyme solution.

[0231] 7. Purification

[0232] Xanthine oxidase was purified by nickel affinity chromatography.

[0233] Equilibration buffer: 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl and 20 mM imidazole.

[0234] Elution buffer: ① 50 mM Tris buffer at pH 7.5 containing 0.15 M NaCl and 50 mM imidazole.

[0235] ②50 mM Tris buffer at pH 7.5 containing 0.15 M NaCl and 100 mM imidazole.

[0236] ③ 50 mM Tris buffer at pH 7.5 containing 0.15 M NaCl and 300 mM imidazole.

[0237] (1) Take a purified Ni column, clean the column, add 3-4 mL of nickel agarose gel, wash the column 5 times with a wash bottle, and then equilibrate the column twice with equilibration solution;

[0238] (2) Pass through the column, effluent FT, sample FT;

[0239] (3) Wash away the unadsorbed miscellaneous proteins with 20 column volumes of equilibration buffer at a flow rate of 1 - 2 mL / min until the optical absorption of the effluent measured by the ultraviolet spectrophotometer is stable, and then start eluting the XOD adsorbed on the column;

[0240] (4) Elute the column in stages with imidazole concentrations of 50 mM, 100 mM, and 300 mM, eluting about 5 - 10 column volumes at a flow rate of 1 - 2 mL / min, and collect in 4 mL tubes; (Determination of the starting point for collecting proteins: The reading of the ultraviolet spectrophotometer rises rapidly; Determination of the ending point for collecting proteins: The reading of the ultraviolet spectrophotometer drops steadily)

[0241] (5) Take samples of the XOD proteins eluted at each concentration gradient for activity measurement and run SDS - page electrophoresis;

[0242] (6) According to the activity results and the SDS - page electrophoresis pattern, perform dialysis and buffer exchange on the elution fractions of the active XOD proteins.

[0243] <Determination of XOD enzyme activity>

[0244] One unit of XOD enzyme activity is defined as the amount of enzyme that catalyzes the formation of one micromole of uric acid per minute at 37 °C and pH 7.5.

[0245] Reaction formula:

[0246] Uric acid has light absorption at 293 nm.

[0247] The reagent preparation is shown in Table 1.

[0248] Table 1

[0249]

[0250]

[0251] Steps for enzyme activity detection:

[0252] 1. Prepare the reaction mixture in Table 2 in the reaction tube (cuvette) and equilibrate at 37 °C for 5 min.

[0253] Table 2

[0254]

[0255] 2. Add 0.1 mL of the enzyme solution to be tested (pre - dilute the enzyme solution to 0.1 - 0.2 U / mL with pre - cooled E enzyme diluent), and invert to mix evenly.

[0256] 3. Pre - heat the spectrophotometer to 37 °C, zero it with distilled water, and detect the increase in OD value at 293 nm for 3 - 4 min, and calculate ΔOD per minutetest (Calculated from the starting point of the linear curve).

[0257] At the same time, replace the enzyme solution with the E enzyme diluent, and measure ΔOD in the same way blank .

[0258] <Calculation of XOD enzyme activity>

[0259] Calculation method of enzyme activity unit:

[0260] Volume XOD enzyme activity

[0261] Weight XOD enzyme activity (U / mg) = (U / mL) × 1 / C

[0262] Among them, the meanings of each symbol or number are as follows: V t , total volume (2.5 mL); V s , sample volume (0.1 mL); 12.5, millimolar extinction coefficient of uric acid under the measurement condition of pH 7.5 cm 2 / mM); 1.0, light passing path (cm); d f , dilution factor; C, enzyme concentration (c mg / mL).

[0263] Take the crude cell enzyme solution to measure the enzyme activity, screen the high-yield transformants, purify XOD by fermentation and freeze-dry it, and then measure the activity. The activity of the mutant strain XOD-mut is 126% higher than that of XOD, as shown in Table 3. The control group cXOD was purchased from Sigma, catalog number 10110434001.

[0264] Table 3

[0265] name Activity (U / mg-solid) XOtM 1 XOD 40.2 XOD-mut 91.1

[0266] <Random mutagenesis of XOD gene for thermal stability>

[0267] Perform random mutagenesis on the xanthine oxidase sequence expression vector pBBR1MCS-5-XOD by error-prone PCR, cut and recover the mutant DNA fragment, ligate the mutant DNA fragment with the pBBR1MCS-5 plasmid to construct the recombinant vector pBBR1MCS-5-XOD-mut, as Figure 1 shown in B. Extract the plasmid, and then transform it into the electrocompetent cells of Pseudomonas mendocina NK-01 by electroporation, spread the plate to pick clones, and expand the culture to obtain the transformed product culture solution.

[0268] The conditions for random mutagenesis by the error-prone PCR method are as follows:

[0269] 25 μL of 2× error-prone PCR buffer (100 mM KCl, 15 mM MgCl2, 50 mM Tris-HCl pH 8.2, 0.02% gelatin; 5 μM dATP, 5 μM dGTP, 5 μM dTTP, and 5 μM dCTP; 0.1 mM MnCl2, 0.5 U Taq DNA polymerase), 500 nM primer XOD-For, 500 nM primer XOD-Rev, 25 ng of plasmid DNA, and sterile water were used to adjust the reaction volume to 50 μL;

[0270] The amplification program was as follows: The above reaction system was incubated at 95°C for 8 min; 31 cycles of "95°C for 30 s, 56°C for 30 s, 72°C for 4 min"; 72°C for 10 min; The PCR amplified gene products obtained were separated by 1% agarose gel electrophoresis, and the DNA fragments were recovered.

[0271] Furthermore, in the PCR amplification reaction of pBBR1MCS-5-XOD, the following primer pair was used:

[0272] XOD-For: 5’-ATTTCACACAGGAAACAGCTATGGACATGAACACCATCG-3’;

[0273] XOD-Rev: 5’-TAACAAAATATTAACGCTCAGCGGGTCACGCCTTCACCG-3’;

[0274] <Mutant Strain Screening>

[0275] The randomly mutated clones were cultured, and the bacterial liquid was further cultured and induced, followed by ultrasonic cell disruption. Then, the disrupted cell lysate was heated in a water bath at multiple different increasing temperatures, and the enzyme activity of the cell lysate after heating at different temperatures was measured.

[0276] In the random mutagenesis experiment of pBBR1MCS-5-XOD, clones with no loss of xanthine oxidase activity after heating at 70°C were obtained, and this mutation increased the enzyme activity by 126%. This clone is the genetically engineered strain of xanthine oxidase with good thermal stability and high activity, and its amino acid sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, and its vector is pBBR1MCS-5-XOD-mut.

[0277] <Effect of pH on the Enzyme Activity and Stability of XOD and XOD-mut>

[0278] Prepare enzyme detection solution (phosphate buffer) with buffers of different pH (6-9), and determine the enzyme activity according to the above method to determine the optimal reaction pH value of XOD. At 37°C, the activities of XOD and XOD-mut were measured in buffers of pH 6-9, respectively. The results are as follows: Figure 3 As shown in the figure, the activity of XOD and XOD-mut was 40% at pH 6.0, and the activity of XOD and XOD-mut gradually increased with the increase of pH, reaching the maximum at pH 8. Then it began to decrease. Therefore, XOD and XOD-mut have activity between pH 7-9, and the optimal reaction pH of XOD and XOD-mut is 8.0.

[0279] The enzyme solution was diluted with Britton-Robinson buffer of different pH (4-10), and the residual enzyme activity was measured after being kept at 25℃ for 15h. The pH-enzyme activity curve of XOD and XOD-mut at the optimal pH was prepared to investigate the pH stability of XOD and XOD-mut. Figure 4 As shown, the activities of XOD and XOD-mut gradually increased when treated at pH 4-6, had no effect on the activities of XOD and XOD-mut when treated at pH 6-9, and then the enzyme activity decreased with the increase of pH.

[0280] <Effects of temperature on the activity and stability of XOD and XOD-mut enzymes>

[0281] In 50 mM phosphate buffer at pH 7.5, the enzyme activity at different temperatures was measured according to the above method to determine the optimal reaction temperature of XOD. The activity data of XOD and XOD-mut were obtained at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C. The highest activity temperature was 65°C. The activity curve was prepared. The results are shown in Figure 5 As shown, the activities of XOD and XOD-mut increased at 25-65℃, and gradually decreased at 65-80℃.

[0282] The thermal stability of cXOD, XOD and XOD-mut was studied. In 50 mM phosphate buffer at pH 7.5, enzyme solutions with an enzyme activity of 15 U / mL of cXOD, XOD and XOD-mut were kept at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 30 min each, then quickly cooled to room temperature. The residual enzyme activity was measured and the enzyme activity curves of cXOD, XOD and XOD-mut were prepared after keeping at different temperatures for 30 min. The results are shown in the figure. Figure 6As shown, when cXOD was treated at 30 - 50 °C for 30 min, there was a small loss of enzyme activity. After being treated at 60 °C for 30 min, the remaining activity of cXOD was 45%. After being treated at 70 °C for 30 min, the remaining activity of cXOD was 16%. It was completely lost under the treatment condition of 80 °C. When XOD was treated at 30 - 60 °C for 30 min, there was a small loss of enzyme activity. After being treated at 70 °C for 30 min, the remaining activity of XOD was 50%. It was basically lost under the treatment condition of 80 °C. When XOD-mut was treated at 30 - 70 °C for 30 min, the enzyme activity was basically not lost. After being treated at 80 °C for 30 min, the remaining activity of XOD-mut was 40%. The data at 30 °C and 40 °C in the figure overlapped, resulting in an overlapping view.

[0283] <Fermentation tank process of XOD>

[0284] Flask culture of XOD fermentation seed liquid:

[0285] The recombinant strain culture was cultured in a 125 mL baffled flask with 25 mL. The flask contained seeds inoculated from a single colony on an LB plate or a frozen glycerol stock solution, which was about 5 - 10% of the initial fermentation broth volume. The flask was placed at 30 °C and shaken at 180 rpm. If necessary, gentamicin (30 mg / L) was used to maintain the plasmid in the medium. The seed medium consisted of 5 g / L glucose, 3 g / L K2HPO4, 2 g / L (NH4)2Cl, 5 g / L yeast extract, 6 g / L Na2HPO4, and pH 7.0. After the growth of the culture, the optical density at 600 nm (OD 600 ) was measured using a Beckman DU 640 spectrophotometer (Beckman Coulter, Brea CA).

[0286] Fed-batch fermentation of XOD:

[0287] 1. Sterilize the fermenter and the fed-batch medium. The fed-batch fermentation was carried out in a 10 L fermenter and 5 L fermentation medium. The fed-batch medium consisted of 40 g / L glucose, 10 g / L (NH4)2SO4, 25 g / L yeast extract, 25 g / L peptone, 10 g / L MgSO4·7H2O, 6 g / L disodium hydrogen phosphate, 3 g / L potassium hydrogen phosphate, 1 g / L L-aspartic acid, 40 μg / mL L-phenylalanine, 40 μg / mL L-tyrosine, 40 μg / mL L-tryptophan, 10 μg / mL 4-hydroxybenzoic acid, and 1 mL / L trace element solution.

[0288] 2. After sterilization and cooling, when the temperature dropped to 30 °C, start stirring and aeration, and adjust the pH of the medium to 7.0 with ammonia water (10%, v / v).

[0289] 3. Inoculate the seed solution in an amount of 5-10% of the initial fermentation volume from the seed solution shake flask into the fermenter. The stirring speed (200 rpm - 700 rpm) is feedback-controlled by dissolved oxygen (DO) to maintain the oxygen level at about 30%. The fermentation temperature is set at 30°C, and sterile air is maintained at a flow rate of 1 vvm. The feeding medium contains: 500 g / L glucose and 2 mL / L trace element solution.

[0290] 4. Sampling is required for each completed fermentation stage and at least twice a day. Take 10 mL of samples at each time point, and take another 1 mL of samples from the 10 mL. The samples are used to analyze cell growth (OD 600 and cell wet weight), pH, microscopic observation, protein concentration or activity. The bacterial cells and supernatant (after centrifugation) are stored at -80°C for subsequent analysis.

[0291] <Application of XOD in 5'-Nucleotidase Detection Kit>

[0292] The 5'-nucleotidase detection kit includes reagent R1 and reagent R2. The components and concentrations contained in reagent R1 are: 150 mM Tris buffer, 0.15 M glutamic acid, 0.5 KU / L purine nucleotide phosphorylase, 1.2 KU / L xanthine oxidase, 3 KU / L peroxidase, 0.03 wt% PC300, 1.2 KU / L ascorbic acid oxidase, 5 μM potassium ferrocyanide, 0.5 wt% Triton X-100, 1 wt% Tween 20, 100 mM magnesium chloride, and the pH of reagent R1 is 7.2; the components and concentrations contained in reagent R2 are: 130 mM phosphate buffer, 80 mM disodium inosinate, 2 mM 4-AAP, 0.03 wt% PC300, 200 mM disodium hydrogen phosphate, and the pH of reagent R2 is 7.2.

[0293] With other components of the kit fixed, only the source of xanthine oxidase is changed. Xanthine oxidase from milk (abbreviated as cXOD, purchased from Sigma, catalog number 10110434001, activity 1 U / mg), the XOD of the present invention, and mutant XOD-mut are used for comparative experiments. The prepared 5'-nucleotidase detection kit is used to perform multiple repeated determinations on two different concentrations of samples to be measured, and the precision SD (standard deviation) and CV (coefficient of variation) of the obtained results are calculated. The results are shown in Table 4.

[0294] Table 4

[0295] Precision 1 XOtM XOD XOD-mut Precision 2 XOtM XOD XOD-mut 1 29.6 28.7 29.4 1 10.9 10.7 10.5 2 28.4 29.1 28.3 2 10.6 10.8 10.2 3 30.2 28.5 28.6 3 10.5 10.4 10.7 4 27.9 28.5 28.1 4 10.6 10.2 10.6 5 28.6 28.0 28.5 5 10.2 10.6 10.4 6 26.5 29.3 28.8 6 10.7 10.4 10.8 7 28.4 28.4 28.9 7 9.5 10.6 10.5 8 27.6 28.0 28.2 8 10.1 10.2 10.9 9 27.1 29.1 28.8 9 10.9 10.6 10.4 10 29.9 28.4 29.0 10 9.4 10.2 10.4 AVE 28.42 28.60 28.66 AVE 10.34 10.47 10.54 SD 1.21 0.45 0.40 SD 0.54 0.22 0.21 CV 4.25％ 1.57％ 1.40％ CV 5.18％ 2.11％ 2.01％

[0296] It can be seen that the precision of the kit prepared by XOD and XOD-mut of the present invention is good, while the cXOD in the comparative example has a certain degree of influence on the precision of the detection result, and the CV value is obviously large, which reduces the precision. Compared with the reagent formula containing cXOD derived from milk, the present invention has the following beneficial effects: good repeatability and high precision.

[0297] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0298] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and the embodiments. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Under the premise of not substantially affecting the activity of xanthine oxidase, the sequence of the present invention is replaced, added and / or deleted by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids to obtain variants of the amino acid sequence of xanthine oxidase, which are all considered to be included in the scope of protection of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the legends shown and described herein.

Claims

1. Xanthine oxidase, characterized in that The amino acid sequence of its B subunit is shown in SEQ ID NO:1, and the amino acid sequence of its A subunit is shown in SEQ ID NO:

2.

2. A mutant xanthine oxidase, characterized in that The amino acid sequence of its B subunit is shown in SEQ ID NO:3, and the amino acid sequence of its A subunit is shown in SEQ ID NO:

4.

3. The DNA sequence encoding the xanthine oxidase according to claim 1, characterized in that As shown in SEQ ID NO:

5.

4. The DNA sequence encoding the mutant xanthine oxidase according to claim 2, characterized in that: As shown in SEQ ID NO:

6.

5. An expression vector comprising the DNA sequence according to claim 3 or 4, characterized in that: The expression vector is a prokaryotic expression vector, an insect cell expression vector or a fungal expression vector.

6. A recombinant strain comprising the DNA sequence according to claim 3 or 4, characterized in that: The method is obtained by transforming an expression vector containing a xanthine oxidase expression cassette into an expression host, wherein the xanthine oxidase expression cassette consists of a promoter and a xanthine oxidase gene connected to the downstream of the promoter, and the expression host is Pseudomonas mendocinae.

7. A method for preparing xanthine oxidase, characterized in that: The method comprises constructing the expression vector of the xanthine oxidase according to claim 1, transforming the expression host to obtain a recombinant strain, inducing the expression of the xanthine oxidase, and separating and purifying the xanthine oxidase.

8. A method for preparing xanthine oxidase, characterized in that: The method comprises constructing an expression vector of the mutant xanthine oxidase according to claim 2, transforming an expression host to obtain a recombinant strain, inducing the expression of the xanthine oxidase, and separating and purifying the xanthine oxidase.

9. Use of the xanthine oxidase according to claim 1 in preparing a kit for clinical detection of 5'-nucleotidase and adenosine deaminase.

10. Use of the mutant xanthine oxidase according to claim 2 in preparing a kit for clinical detection of 5'-nucleotidase and adenosine deaminase.

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