阅读说明：本技术 一种鲍氏不动杆菌黄嘌呤脱氢酶突变体及其应用 (Acinetobacter baumannii xanthine dehydrogenase mutant and application thereof ) 是由 王成华 朱春燕 张婷 张冉 于 2019-11-12 设计创作，主要内容包括：一种鲍氏不动杆菌黄嘌呤脱氢酶突变体及其应用,该突变体是将鲍氏不动杆菌黄嘌呤脱氢酶进行如下任一突变后获得的：(1)将鲍氏不动杆菌黄嘌呤脱氢酶的ɑ亚基进行F296L突变；(2)将鲍氏不动杆菌黄嘌呤脱氢酶的ɑ亚基进行Y351A突变；(3)将鲍氏不动杆菌黄嘌呤脱氢酶的ɑ亚基进行S392A突变；(4)在鲍氏不动杆菌黄嘌呤脱氢酶的ɑ亚基的E388与D389位之间插入一段长度由GHFCGLLQLSKRFEQ组成的15个氨基酸多肽片段。本发明的鲍氏不动杆菌黄嘌呤脱氢酶突变体可利用空气中氧气作为电子受体,具有更好的底物亲和力和催化效率,可降低生产成本,更加适用于工业化生产应用。(An acinetobacter baumannii xanthine dehydrogenase mutant and application thereof, wherein the mutant is obtained by performing any one of the following mutations on acinetobacter baumannii xanthine dehydrogenase: (1) subjecting the alpha subunit of acinetobacter baumannii xanthine dehydrogenase to F296L mutation; (2) carrying out Y351A mutation on the alpha subunit of the acinetobacter baumannii xanthine dehydrogenase; (3) subjecting the alpha subunit of acinetobacter baumannii xanthine dehydrogenase to S392A mutation; (4) a15 amino acid polypeptide fragment consisting of GHFCGLLQLSKRFEQ in length is inserted between positions E388 and D389 of the alpha subunit of acinetobacter baumannii xanthine dehydrogenase. The acinetobacter baumannii xanthine dehydrogenase mutant can utilize oxygen in the air as an electron acceptor, has better substrate affinity and catalytic efficiency, can reduce production cost, and is more suitable for industrial production and application.)

1. The acinetobacter baumannii xanthine dehydrogenase mutant is composed of an alpha subunit and an β subunit, and is characterized in that the amino acid sequence of the alpha subunit is shown as SEQ ID NO. 1, and the amino acid sequence of the β subunit is shown as SEQ ID NO. 2.

2. The A.baumannii xanthine dehydrogenase mutant according to claim 1, wherein the mutant enzyme comprises an alpha subunit and an β subunit, wherein the alpha subunit is any one of (a1) - (a4), and the β subunit is the following (b);

(a1) the amino acid sequence of the alpha subunit is an amino acid sequence obtained by replacing phenylalanine at position 296 of a sequence 1 in a sequence table with leucine;

(a2) the amino acid sequence of the alpha subunit is an amino acid sequence obtained by replacing the 351-th tyrosine amino acid of the sequence 1 in the sequence table with alanine;

(a3) the amino acid sequence of the alpha subunit is an amino acid sequence obtained by replacing serine at the 392 th site of the sequence 1 in a sequence table with alanine;

(a4) the amino acid sequence of the alpha subunit is obtained by inserting 15 amino acid polypeptide fragments consisting of GHFCGLLQLSKRFEQ between the 388 th alanine and the 389 th aspartic acid of the sequence 1 in the sequence table;

(b) the amino acid sequence of the β subunit is a sequence shown as a sequence 2 in a sequence table.

3. A DNA molecule encoding the protein of claim 1.

4. The DNA molecule of claim 3, wherein said DNA molecule comprises a gene encoding said A subunit and a gene encoding said β subunit.

5. The DNA molecule of claims 3-4, wherein: the gene is a DNA molecule shown in any one of the following 1) to 8):

1) replacing ttt at the position of 886-888 in a sequence 4 in a sequence table with ctg to obtain a DNA molecule shown in the sequence;

2) replacing ttt at the position of 888-886-th-;

3) replacing tat at the 1051-1053 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the sequence;

4) replacing tat at the 1051-1053 th site of the sequence 4 in the sequence table with a DNA molecule shown at the 1 st-1500 th site of the obtained sequence after gca;

5) replacing tct at the 1174-1176 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the sequence;

6) replacing tct of the 1174 th-1176 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the 1 st-1500 th site of the sequence;

7) a DNA molecule which hybridizes under stringent conditions with a DNA molecule as defined in any one of 1) to 6) and which encodes a protein as claimed in claim 1;

8) a DNA molecule having 90% or more homology with the DNA sequence defined in any one of 1) to 7) and encoding the protein of claim 1 or 2.

6. A recombinant vector, expression cassette, host cell or recombinant bacterium comprising a DNA molecule according to any one of claims 3 to 5.

7. Use of the protein of claim 1 as xanthine oxidase.

8. Use according to claim 6, characterized by the use for degrading hypoxanthine and/or xanthine containing and for degrading hypoxanthine and/or xanthine containing materials.

9. Use of a DNA molecule according to any one of claims 2 to 5 or a recombinant vector, expression cassette, host cell or recombinant bacterium according to claim 6 or 7 in any one of:

(a) preparing a product having xanthine oxidase activity;

(b) preparing the product with xanthine oxidase activity and xanthine dehydrogenase activity.

10. A method for producing the protein of claim 1, comprising introducing the nucleic acid molecule of any one of claims 3 to 5 into Escherichia coli, and inducing expression to obtain the protein.

Technical Field

The invention relates to acinetobacter baumannii xanthine dehydrogenase and application thereof, and belongs to the technical field of biology.

Background

Xanthine Oxidoreductase (XOR) is an oxidoreductase comprising an iron-sulfur cluster, molybdopterin and a flavin prosthetic group, and has two different forms of Xanthine oxidase (XOD, EC1.17.3.2) and Xanthine dehydrogenase (XDH, EC 1.17.1.4) present. It can utilize O₂、NAD⁺And methylene blue, etc. as electron acceptors, oxidatively catalyze the oxidation of a variety of substrates including purines, pterins, heterocyclic molecules, etc. (Lilisu, Chenghua, Shaoybo, etc.. xanthine oxidoreductase structure, function and action [ J]Journal of cell biology, 2004,26(4): 381-384). X0R has important commercial application value, wherein the application of xanthine oxidase mainly relates to five aspects, namely drug metabolism, nucleoside drug synthesis, in vitro detection, bioremediation, health food (Wanchenhua, New Prochen society, research progress and development prospect of xanthine oxidase [ J]Guangxi science, 2017,24(1): 15-24.). Can be clinically used for preparing immune antibody, tumor inhibitor and detecting diseases such as cell ischemia reperfusion injury (fermentation, purification, characteristics and gene research of the xanthine oxidase of the plum loyalty qin arthrobacter [ D)]Jiangsu, Jiangnan university, 2007).

XOD is prepared from precursor protein XDH through reversible conversion by sulfydryl oxidation or irreversible conversion by enzymolysis and cleavage, and utilizes molecule O₂As an electron acceptor, but lost the utilization NAD possessed by XDH⁺Ability to act as an electron acceptor. They are products of transcriptional expression of the same gene, with XDH being the direct product of the gene after transcription, and being the predominant state in vivo (Woolfolk, C.A. and J.s.Downard, Distribution of the enzyme oxidation and enzyme catalysis type bacteria. J.Bacteriol, 1977.130(3): 1175-91). Utilization of expensive NAD relative to XDH⁺As the electron acceptor, 0 in air can be directly used₂XOD as an electron acceptor has become commercially usefulThe enzyme is the most preferred.

Currently, commercial X0D is limited to extraction processes. For example, milk XOD extracted from animal-derived materials such as milk cream, and bacteria XOD extracted from wild microorganisms such as Arthrobacter luteus. The milk xanthine oxidase is the most important XOD on the market at present and is the enzyme which is patented and launched to the market at the earliest. However, the complicated conversion process from XDH to X0D has the problems of mixing of active X0D with precursor protein and low content of active XOD, which not only affects the activity and yield of XOD, but also increases the use cost of enzyme, which causes high price of XOD and limits the application development (Enroth, C., et al., crystals structures of bone milk protein dehydrogenase and bone oxidase: structure-based mechanism of conversion. Proc Natl Acad Sci USA, 2000.97(20): 10723-8).

The inventor researches in earlier period to obtain a new XDH (application No. 201410764840.5, inventor's New Congress, Wanghua, Zhang \32704), a xanthine dehydrogenase and a coding gene and application thereof) from Acinetobacter baumannii, a new XDH (application No. 201510406718.5, inventor's New Congress, Wanghua, Zhang \32704m, Sunan, an alkaline xanthine dehydrogenase and application thereof in a detection kit). And a variant XDH (application No. 201510048275.7, inventor's New Schchen, Wanghua, Zhang 32704c, a xanthine dehydrogenase truncation and application thereof) with higher catalytic activity and a xanthine dehydrogenase mutant (application No. 201710152663.9, inventor's New Schchen, Wanghua, Zhang 32704c, xanthine dehydrogenase mutant and application thereof) are obtained by genetic engineering means. With the expansion of the field, there is a need to further develop novel XODs with higher pH tolerance range, stronger substrate affinity and better catalytic efficiency. It would be of great significance if an A.baumannii-derived XDH protein could be engineered to be directly transcriptable and translatable XOD.

Disclosure of Invention

The provided acinetobacter baumannii xanthine dehydrogenase mutant has enhanced xanthine oxidase activity, and is obtained by performing site-directed mutagenesis on an acinetobacter baumannii xanthine dehydrogenase sequence which is composed of an alpha subunit of an amino acid sequence shown in sequence 1 and an β subunit of the amino acid sequence shown in sequence 2, wherein the site-directed mutagenesis is to perform mutagenesis only on the alpha subunit (β subunit is not mutated).

The invention provides an acinetobacter baumannii xanthine dehydrogenase mutant, which consists of an alpha subunit and an β subunit, wherein the amino acid sequence of the alpha subunit is shown as SEQ IN NO 1 IN a sequence table, and the amino acid sequence of the β subunit is shown as SEQ IN NO 2 IN the sequence table.

The acinetobacter baumannii xanthine dehydrogenase mutant provided by the invention is composed of the following alpha subunit and β subunit.

The alpha subunit is any one of the following (a1) - (a 5):

(a1) the amino acid sequence is a subunit shown as a sequence 3 in a sequence table;

(a2) a subunit shown in an amino acid sequence obtained by replacing phenylalanine (F) at the 296 th site of the sequence 1 in the sequence table with leucine (L);

(a3) a subunit shown as an amino acid sequence obtained by replacing the 351-position tyrosine amino acid (Y) of the sequence 1 in the sequence table with alanine (A);

(a4) the subunit of the amino acid sequence is obtained by replacing the 392 th serine (S) of the sequence 1 in the sequence table with alanine (A);

(a5) and (b) any one of the amino acid sequences (a1) - (a4) is subjected to substitution and/or deletion and/or addition of one or more amino acid residues to obtain the subunit with the same function.

The β subunit is as follows (b):

(b) the amino acid sequence is a subunit shown as a sequence 2 in a sequence table;

for the above (a1), the site-directed mutation is specifically to insert an amino acid polypeptide sequence with the length of 15 amino acids, specifically the sequence of GHFCGLLQLSKRFEQ, between the amino acids of the a subunit, E388 and D389, of the xanthine dehydrogenase of acinetobacter baumannii. This mutation was noted as E388 Insert.

For the above (a2), the site-directed mutation is specifically to replace phenylalanine (F) at position 296 of the a subunit of a xanthine dehydrogenase of acinetobacter baumannii (sequence 1) with leucine (L). This mutation was designated as F296L.

In the case of (a3) above, the site-directed mutation is specifically to replace the alanine (A) with the tyrosine (Y) at position 351 of the a subunit of the xanthine dehydrogenase of A.baumannii (SEQ ID NO: 1). This mutation was designated as Y351A.

In the case of (a4) above, the site-directed mutation is specifically to replace serine (S) at position 392 of the A subunit (SEQ ID NO: 1) of the A dehydrogenase of A.baumannii with alanine (A). This mutation was designated as S392A.

Nucleic acid molecules encoding said acinetobacter baumannii xanthine dehydrogenase mutants are also within the scope of the present invention.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA;

the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.

In the embodiment of the invention, the nucleic acid molecule is specifically a gene encoding the acinetobacter baumannii xanthine dehydrogenase mutant, and the gene is specifically a DNA molecule shown in any one of the following 1) to 8):

1) DNA molecule shown in sequence 5 in the sequence table;

2) DNA molecules shown in 1 st to 1545 th sites of a sequence 5 in a sequence table;

3) replacing ttt at the position of 886-888 in a sequence 4 in a sequence table with ctg to obtain a DNA molecule shown in the sequence;

4) replacing ttt at the position of 888-886-th-;

5) replacing tat at the 1051-1053 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the sequence;

6) replacing tat at the 1051-1053 th site of the sequence 4 in the sequence table with a DNA molecule shown at the 1 st-1500 th site of the obtained sequence after gca;

7) replacing tct at the 1174-1176 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the sequence;

8) replacing tct of the 1174 th-1176 th site of the sequence 4 in the sequence table with gca to obtain a DNA molecule shown in the 1 st-1500 th site of the sequence;

9) a DNA molecule which hybridizes under stringent conditions with a DNA molecule as defined in any one of 1) to 8) and which encodes said protein;

10) a DNA molecule having 90% or more homology with the DNA sequence defined in any one of 1) to 9) and encoding the protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The genes shown in the 1) and the 2) are genes for coding an acinetobacter baumannii xanthine dehydrogenase mutant E388 Insert; 3) and 4) above are genes encoding acinetobacter baumannii xanthine dehydrogenase mutant F296L; 5) and 6) above are genes encoding acinetobacter baumannii xanthine dehydrogenase mutant Y351A; 7) and 8) above are genes encoding the A.baumannii xanthine dehydrogenase mutant S392A.

The parent gene used for producing the xanthine dehydrogenase mutant is an xanthine dehydrogenase gene derived from acinetobacter baumannii, and the nucleotide sequence thereof is represented by seq id No. 15, wherein a DNA molecule represented by seq id No. 1 to 1500 nucleotides from the 5' -end of seq id No. 15 encodes an a subunit, and a DNA molecule represented by seq id No. 1502 to 3877 nucleotides encodes β subunit.

Recombinant vectors, expression cassettes, host cells or recombinant bacteria comprising the above-described DNA molecules are also within the scope of the present invention.

The transgenic cell lines do not include animal and plant propagation material.

In one embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a DNA molecule shown as sequence 5 in a sequence table with a DNA fragment between recognition sites of enzymes NcoI and HindIII of a pTrc99A plasmid, and is named as pTRX 0388. In one embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing DNA molecule shown in the sequence obtained by replacing ttt at position 886-888 of sequence 4 in the sequence table with ctg, and replacing DNA fragment between NcoI and HindIII enzyme recognition sites of pTrc99A plasmid, and is named as pTRX 0296. In another embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing DNA molecule shown in the sequence "after replacing tat at 1051-1053 position of sequence 4 in the sequence table with gca" with DNA fragment between NcoI and HindIII enzyme recognition sites of pTrc99A plasmid, and is named as pTRX 0351. In still another embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a DNA molecule represented by the sequence indicated by "replacing the tct at 1174-1176 of the sequence 4 in the sequence table with gca" with a DNA fragment between the NcoI and HindIII enzyme recognition sites of the pTrc99A plasmid, and is named as pTRX 0392. The four recombinant vectors are recombinant plasmids of the acinetobacter baumannii xanthine dehydrogenase mutant, and the four acinetobacter baumannii xanthine dehydrogenase mutants are expressed respectively.

In the invention, the microorganism is specifically a recombinant escherichia coli obtained by transforming escherichia coli with the recombinant vector (one of the four recombinant vectors).

The transformation may be any method used for introducing the recombinant vector into a host cell. For example, methods for introducing the expression vector into a host cell include, but are not limited to, calcium chloride and thermal shock transformation, ion gun impact, electroporation, sonication, precipitation by PEG.

The application of the acinetobacter baumannii xanthine dehydrogenase mutant as xanthine oxidase also belongs to the protection scope of the invention.

The application of the acinetobacter baumannii xanthine dehydrogenase mutant as xanthine oxidase in any one of the following applications also belongs to the protection scope of the invention:

(a) degrading hypoxanthine and/or xanthine;

(b) degrading hypoxanthine and/or xanthine containing materials.

The application of the acinetobacter baumannii xanthine dehydrogenase mutant or the nucleic acid molecule or the recombinant vector, the expression cassette, the host cell or the recombinant bacterium in any one of the following is also within the protection scope of the invention:

(A) preparing a product having xanthine oxidase activity;

(B) preparing the product with xanthine dehydrogenase activity and xanthine oxidase activity.

Any of the following biological materials is also within the scope of the present invention:

A) a protein, the amino acid sequence of which is any one of (a1) - (a4) as follows:

(a1) sequence 3 in the sequence table:

(a2) a sequence obtained by replacing phenylalanine (F) at position 296 of a sequence 1 in a sequence table with leucine (L);

(a3) a sequence obtained by replacing the 351 th tyrosine amino acid (Y) of the sequence 1 in the sequence table with alanine (A);

(a4) a sequence obtained by replacing the 392 th serine (S) of the sequence 1 in the sequence table with alanine (A);

B) a gene, the nucleotide sequence of which is any one of the following b1) -b 4):

b1) 1-1545 of the sequence 5 in the sequence table;

b2) replacing ttt of the 886-888 th site of the sequence 4 in the sequence table with ctg to obtain the 1 st-1500 th site of the sequence;

b3) replacing tat at the 1051-1053 th site of the sequence 4 in the sequence table with the 1 st-1500 th site of the sequence obtained after gca;

b4) replacing tct of the 1174 th-1176 th site of the sequence 4 in the sequence table with gca to obtain the 1 st-1500 th site of the sequence;

C) a recombinant vector, an expression cassette, a host cell or a microorganism comprising the gene of B).

The application of the biological material in any one of the following is also within the protection scope of the invention:

(a) preparing a product having xanthine oxidase activity;

(b) preparing the product with xanthine dehydrogenase activity and xanthine oxidase activity.

In addition, the invention also protects a method for preparing the acinetobacter baumannii xanthine dehydrogenase mutant, and specifically, the protein is obtained by introducing nucleic acid molecules for encoding the acinetobacter baumannii xanthine dehydrogenase mutant into escherichia coli for induction expression.

The nucleic acid molecule is introduced into the E.coli through a recombinant expression vector containing the nucleic acid molecule (e.g., the above-mentioned four recombinant vectors).

When the acinetobacter baumannii xanthine dehydrogenase mutant E388Insert utilizes oxygen in air as an electron acceptor, the optimum pH is 7, the optimum temperature is 40 ℃, the Km value of the acinetobacter baumannii xanthine dehydrogenase mutant to xanthine is 0.27 mu M, and the conversion number is 7.48s^-1The specific activity was 0.13U/mg protein. When NAD is used as an electron acceptor, the optimum pH is 7, the optimum temperature is 45 ℃, the Km value to xanthine is 0.82 mu M, and the conversion number is 15.69s^-1The corresponding specific activity was 0.03U/mg egg. The ratio of the specific activity of the two receptors is 4.3, which is 17.2 times higher than that of the wild type (the ratio of the two in the wild type is 0.25).

The acinetobacter baumannii xanthine dehydrogenase mutant F296L has an optimum pH of 7.5, an optimum temperature of 50 ℃, a Km value of 1.32 mu M for xanthine and a conversion number of 34.76s when oxygen in the air is used as an electron acceptor^-1The specific activity was 0.25U/mg protein. When NAD is used as an electron acceptor, the optimum pH is 9, the optimum temperature is 50 ℃, the Km value to xanthine is 1.16 mu M, and the conversion number is 62.43s^-1The corresponding specific activity was 0.16U/mg egg. The ratio of the specific activity of the two receptors is 1.56, which is 6.24 times higher than that of the wild type (the ratio of the two in the wild type is 0.25).

The acinetobacter baumannii xanthine dehydrogenase mutant Y351A has an optimum pH of 9, an optimum temperature of 40 ℃, a Km value of 10.26 mu M for xanthine and a conversion number of 26.64s when oxygen in air is used as an electron acceptor^-1The specific activity was 1.49U/mg protein. When NAD is used as an electron acceptor, the optimum pH is 9, the optimum temperature is 40 ℃, the Km value to xanthine is 28.02 mu M, and the conversion number is 196.02s^-1The corresponding specific activity was 0.77U/mg protein. The ratio of the specific activity of the two receptors is 1.94, which is 7.76 times higher than that of the wild type (the ratio of the two in the wild type is 0.25).

The acinetobacter baumannii xanthine dehydrogenase mutant S392A has an optimum pH of 7, an optimum temperature of 45 ℃, a Km value of 0.09 mu M for xanthine and a conversion number of 5.88S when oxygen in air is used as an electron acceptor^-1The specific activity was 5.06U/mg protein. When NAD is used as an electron acceptor, the optimum pH is 9, the optimum temperature is 50 ℃, the Km value to xanthine is 0.28 mu M, and the conversion number is 19.68s^-1The corresponding specific activity was 7.2U/mg egg. The ratio of the specific activity of the two receptors is 0.7, which is 2.8 times higher than that of the wild type (the ratio of the two in the wild type is 0.25).

The invention has the outstanding advantages that:

the acinetobacter baumannii xanthine dehydrogenase mutant has enhanced xanthine oxidase activity compared with acinetobacter baumannii xanthine dehydrogenase, can utilize molecular oxygen as an electron acceptor to react, degrade byproducts such as hypoxanthine and the like generated in the production process of nucleoside drugs such as ribavirin and the like, can also be expanded to other catalytic substrates, such as oxidation reactions of other substrates such as purine, pteridine, heterocyclic molecules and aldehydes, and various electron acceptors such as methylene blue, benzoquinone, ferricyanide and nitrate, and further applies the variant xanthine oxidase to the field of biosensors.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of purified recombinant xanthine dehydrogenase mutants. Lane M: standard protein Marker, band sizes of 245kDa, 180kDa, 135kDa, 100kDa, 75kDa, 63kDa and 48kDa, respectively; lane 1: mutant F296L; lane 2: mutant Y351A; lane 3: mutant E388 Insert; lane 4: S392A

FIG. 2 is a graph showing the time-dependent change of absorbance of a purified A.baumannii xanthine dehydrogenase mutant xanthine substrate degradation enzyme activity assay system, which is based on a product uric acid assay. F296L, Y351A, E388Insert and S392A are acinetobacter baumannii xanthine dehydrogenase mutants. The reaction conditions are illustrated by using E388Insert-xanthine as an example in the figure, namely, the degradation reaction of the acinetobacter baumannii xanthine dehydrogenase mutant E388Insert with xanthine (xanthine) as a substrate. .

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents, kits and the like used in the following examples are commercially available unless otherwise specified.

The gene source strain is (Acinetobacter baumannii) which is purchased from China center for culture Collection of Industrial microorganisms (CICC) and has the strain number of CICC 10254.

pTrc99A is product of China plasmid vector Strain cell line Gene Collection Biovector Science Lab, Inc., catalog number Biovector 108321(GenBank accession number M22744.1, Amann, E., Ochs, B. andAbel, K. J. light regulated tac promoter genes used for the expression of unfused and fused proteins in Escherichia coli. Gene,1988,69(2): 301-315.).

The recombinant plasmid pTRAN is a recombinant plasmid for expressing wild type acinetobacter baumannii xanthine dehydrogenase, and is a vector obtained by inserting a 6X histidine purification tag coding sequence into a 5' end of an acinetobacter baumannii xanthine dehydrogenase gene shown as a sequence 4 in a sequence table into a pTrc99A vector NcoI and Hind III double enzyme cutting sites. A patent application from a previous application of the applicant (application No. 201510406718.5, inventor chenchenchenchenchen, wangchua, zhang 32704c, sunan, an alkaline xanthine dehydrogenase and its use in a detection kit) was the recombinant plasmid pTRAN prepared in example 1 of the patent application.

Nickel ion metal chelate affinity chromatography media

The nickel column affinity resin is Qiagen, catalog number 30210.

Xanthine and NAD are products of Sigma company, and the catalog numbers are Sigma X7375-10g and SigmaN1636 respectively.

The acinetobacter baumannii xanthine dehydrogenase mutants in the following examples refer to the acinetobacter baumannii xanthine dehydrogenase mutant prepared in example 1, and the constituent small subunit and the large subunit thereof are abbreviated as alpha subunit (XDHA) and β subunit (XDHB), respectively.