CN106318918B

CN106318918B - Alkaline xanthine dehydrogenase and application thereof in detection kit

Info

Publication number: CN106318918B
Application number: CN201510406718.5A
Authority: CN
Inventors: 邢新会; 王成华; 张翀; 苏楠
Original assignee: WUXI TMAXTREE BIOTECHNOLOGY Co Ltd; Wuxi Research Institute of Applied Technologies of Tsinghua University
Current assignee: WUXI TMAXTREE BIOTECHNOLOGY Co Ltd; Wuxi Research Institute of Applied Technologies of Tsinghua University
Priority date: 2015-07-10
Filing date: 2015-07-10
Publication date: 2020-02-21
Anticipated expiration: 2035-07-10
Also published as: CN106318918A

Abstract

The invention discloses alkaline xanthine dehydrogenase and application thereof in a detection kit. The invention discloses proteins, including xanthine dehydrogenase large subunits and xanthine dehydrogenase small subunits. The alkaline xanthine dehydrogenase provided by the invention is an experimental-confirmed xanthine dehydrogenase of the Acinetobacter baumannii which is reported for the first time. The alkaline xanthine dehydrogenase provided by the invention can adapt to samples under alkaline determination conditions, has higher substrate affinity and highest catalytic efficiency than the prior reports, can more sensitively degrade and detect hypoxanthine/xanthine, PNP enzyme and 5' -nucleotidase, is used with PNP enzyme to detect inorganic phosphate and adenosine deaminase, is beneficial to improving the enzymatic efficiency and reducing the cost, and is more beneficial to industrial application processes. The invention also provides a method for detecting the content of xanthine/hypoxanthine in a sample including serum and urine by using the xanthine dehydrogenase. The invention further provides a detection kit for detecting the content of xanthine/hypoxanthine in a sample. The detection method and the detection kit provided by the invention are not influenced by dissolved oxygen in a sample, have wider detection range and higher sensitivity than the prior art, and are convenient to popularize and apply.

Description

Alkaline xanthine dehydrogenase and application thereof in detection kit

Technical Field

The invention relates to the technical field of biology, in particular to xanthine dehydrogenase and application thereof in detection of samples containing hypoxanthine and xanthine, and particularly relates to acinetobacter baumannii alkaline xanthine dehydrogenase and application thereof in degradation of samples containing hypoxanthine and xanthine and detection of content of xanthine/hypoxanthine in samples such as blood, urine and the like.

Background

Xanthine Oxidoreductase (XOR) is a flavoprotein oxidoreductase containing an iron-sulfur cluster, molybdopterin 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. XOR is capable of catalytically oxidizing sp2 hybridized carbon atoms of various heterocyclic molecules including purines, pteridines and aldehydes (lilisk, chenhua, shore-wave, liuyuri, xuping, the structure, function and action of xanthine oxidoreductase) not only using naturally occurring substances such as oxygen, NAD, nitrate as electron acceptors, but also using artificially synthesized dyes such as PMS and methylene blue as electron acceptors. Therefore, XOR has important commercial application value, and is used for producing XOR antibody and detecting Xanthine/hypoxanthine and detecting inorganic phosphorus by enzyme coupling method, and nucleoside drugs such as enzyme biosynthesis ribavirin and degrading organic pollutants (agar, A. Banerjee, and U.C. Banerjee, xanthene oxide uptake: a journey free purine metabolism to cardiac novasular amplification-transformation coupled. crit Rev Biotechnology, 2011.31(3):264-80 Mengjiang, Chenmei, and using Xanthine oxidase to improve ribavirin transformation rate. university of Hanwu (Nature science edition): 1999.45(6):838 840).

XOR is widely existed in cow's milk, mouse, birds and other animals, insects, plants and bacteria. Among them, XDH is a direct product of transcription and translation of an XOR gene in vivo, and is a main existing state of XOR in vivo. XOD is converted from a precursor protein XDH by post-translational modification, and XOD utilizes the molecule O2 as an electron acceptor, but loses the XDH's ability to utilize NAD as an electron acceptor (Nishino Tomoko, OkamotoKen, Kawaguchi Yuko, horiHiroyuki, Matsumura Tomohiro, Eger Bryan T., Pai, Emil F., Nishino, Takeshi. mechanism of the conversion of conversion to conversion of conversion, 2005, 280-2494). Currently, commercial XOR is mainly made by extraction methods, such as cow milk XOD extracted from cow milk cream by Sigma, and bacterial XOD extracted from wild arthrobacter luteus by TOYOBO of japan. In particular, cow's milk XOD is used in combination with other enzymes for sample detection, including detection of xanthine and hypoxanthine, detection of inorganic phosphate in combination with Purine Nucleoside Phosphorylase (PNP), detection of adenosine deaminase and 5' -nucleotidase in combination with PNP and Peroxidase (POD), and the like. However, in the clinical diagnosis application of XOD, the content of dissolved oxygen in the sample solution is affected by temperature, pressure, salt content, organic matter content and other factors, which often causes inaccuracy of the detection result. Similarly, in the processes of catalyzing by using an XOD enzyme method or generating nucleoside drugs by using whole-cell biotransformation containing the XOD enzyme in a large scale and degrading hybrid organic pollutants, the catalytic conversion process is often influenced by the content of dissolved oxygen. In contrast, XDH utilizes NAD as an electron acceptor, not affected by dissolved oxygen, and can better meet these specific application needs.

The study shows that XDH mainly comprises two major types of eukaryotic XDH represented by milk XDH and bacterial XDH represented by Rhodobacter capsulatus XDH, the milk XDH is spontaneously converted into XOD through partial enzymolysis or oxidation by protease such as trypsin, while the Rhodobacter capsulatus XDH is pure XDH, the catalytic activity of Rhodobacter capsulatus XDH is at least 5 times higher than that of milk XOD/XDH (Leimk ü hlerSikle, HodsonRachael, George Graham N, Rajagopalank.V.rebinand recombinant Rhodobacter calcoaceticus xanthium dehydrogenase, a useful model system for the exchange of protein varians leading to bovine Iin hunmans. journal of Biological Chemistry (2003, 278, 20823) and is more suitable for the development of eukaryotic XDH, the commercial XDH has a higher price and is more suitable for the development of bovine XDH with a higher industrial activity than that of bovine XDH, and the commercial XDH has a higher price, thus providing a more commercially available alternative to the eukaryotic XDH for the bovine XDH with a low industrial XDH.

The inventor obtains a new XDH (application No. 201410764840.5, inventor's New Schchen, Wanghua, Zhang 32704, a xanthine dehydrogenase and encoding genes and applications thereof) from rhodobacter capsulatus, and obtains a variant XDH with a higher catalytic activity in a form of a truncation body by a genetic engineering means (application No. 201510048275.7, inventor's New Schchen, Wanghua, Zhang 32704, a xanthine dehydrogenase truncation body and applications thereof). With the expansion of the field of application, there is a need for further development of novel XDH with stronger substrate affinity, higher pH tolerance range and better catalytic efficiency.

Disclosure of Invention

An object of the present invention is to provide a xanthine dehydrogenase.

The invention provides a protein which is alkaline xanthine dehydrogenase and consists of a subunit A and a subunit B:

the amino acid sequence of the subunit A is a sequence 1 in a sequence table or a sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table;

the amino acid sequence of the subunit B is a sequence 2 in a sequence table or a sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table;

DNA molecules encoding the above proteins are also within the scope of the present invention.

The DNA molecule comprises a DNA molecule for coding the subunit A and a DNA molecule for coding the subunit B.

The nucleotide sequence of the coding DNA molecule of the subunit A is a sequence from 1 st to 1500 th in a sequence 3 in a sequence table or a sequence obtained by substituting and/or deleting and/or adding one or more bases of a base sequence shown from 1 st to 1500 th in the sequence 3 in the sequence table;

the nucleotide sequence of the coding DNA molecule of the subunit B is a sequence obtained by substituting and/or deleting and/or adding one or more bases of the base sequence shown from 1502 th to 3877 th in the sequence 3 in the sequence table or the base sequence shown from 1502 th to 3877 th in the sequence 3 in the sequence table.

The DNA molecule is at least one of the following 1) to 4):

1) the coding region is a DNA molecule shown by nucleotides from 1 st to 3877 th in a sequence 3 in a sequence table;

2) the coding region is a DNA molecule shown by nucleotide of a sequence 3 in a sequence table;

3) a DNA molecule which hybridizes under stringent conditions with a DNA molecule defined in 1) or 2) and which encodes a protein according to claim 1;

4) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) or 2) and encoding the protein of

claim

1 or 2.

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

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above DNA molecules are also within the scope of the present invention.

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

The recombinant vector is obtained by replacing a DNA molecule shown as a sequence 3 in a sequence table with a DNA fragment between pTrc99A plasmid NcoI and HindIII enzyme recognition sites, is named as pTRA, namely the recombinant plasmid of the acinetobacter baumannii xanthine dehydrogenase is obtained, and the xanthine dehydrogenase is expressed.

The application of the protein as xanthine dehydrogenase is also within the protection scope of the invention.

The application of the DNA molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in the preparation of xanthine dehydrogenase is also within the protection scope of the invention.

The application of the protein or the DNA molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in preparing products with the xanthine dehydrogenase activity is also within the protection scope of the invention;

or the protein or the DNA molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium of claim 5 in degrading xanthine or hypoxanthine, also belongs to the protection scope of the invention.

In the above application, the xanthine dehydrogenase has at least one of the following characteristics 1) to 3):

1) the subunit configuration of the xanthine dehydrogenase is type (αβ) 2;

2) the optimal reaction temperature of the xanthine dehydrogenase is 40-45 ℃;

3) the xanthine dehydrogenase has an optimum pH of 8.5 to 9.0 and can withstand a wide range of alkaline environmental conditions of pH7.0 to 11.0.

Another objective of the invention is to provide a method for preparing the alkaline xanthine dehydrogenase.

The method provided by the invention comprises the following steps: inducing and expressing the recombinant bacterium to obtain the protein of claim 1.

The xanthine dehydrogenase provided by the invention consists of a small subunit with the size of 56kDa and the sequence shown as the sequence 1 and a large subunit with the size of 87kDa and the sequence shown as the sequence 2, wherein the optimum pH of the xanthine dehydrogenase is 8.5-9.0, the optimum pH is stable within the range of 6.0-12, the optimum temperature is 40-45 ℃, the optimum temperature is stable within the range of 30-50 ℃, and the semi-inactivation temperature is 59 ℃; when xanthine is used as the substrate, K_mThe value was 23.66. + -. 1.56. mu.M, and the conversion number was 76.36. + -. 1.07s^-1The maximum specific activity can reach 32.12 +/-0.45U/mg protein, and the catalytic efficiency is 3.23s^-1.μM^-1(ii) a When hypoxanthine is used as a substrate, K_mThe value was 22.48. + -. 1.53. mu.M, and the conversion number was 98.85. + -. 1.31s^-1The maximum specific activity can reach 41.58 +/-0.55U/mg protein, and the catalytic efficiency is 4.40s^-1.μM^-1。

Experiments prove that compared with the reported xanthine dehydrogenase, the xanthine dehydrogenase provided by the invention can tolerate a more alkaline pH range of at least pH11.0, and has higher catalytic efficiency and stronger substrate affinity. For example, in the database of enzymatic information (BREDA, http:// www.brenda-enzymes. org/enzyme. php. Typical examples include the optimum pH of rhodobacter capsulatus XDH of 8.0 to 8.5, the pH tolerance range of pH 5.5 to 10, the optimum pH of Arthrobacter luteus XOD from TOYOBO of 7.5 to 8.0, the pH tolerance range of pH6.0 to 9.0, and the optimum pH of commercial bovine XOD from Sigma of 8.0 to 8.4, the pH tolerance range of pH7.0 to 9.0 (Lizhong, Schuipine, Wuwaru. butter xanthine oxidase, research on enzymatic properties, food science, 2008 (09): 420-.

It should be noted that although some acinetobacter baumannii genome sequence information, some of which are annotated as xanthine dehydrogenase activities (xanthhinedehydrogenase) and have high homology with the sequence 1, the sequence 2 and the sequence 3 provided by the present invention, has been included in the National Center for Biotechnology Information (NCBI) database, all of the existing sequence information has not been verified by experiments and is a theoretical prediction result. To further illustrate the innovativeness of the present invention, the present invention performed gene synthesis of Acinetobacter baumannii AB307-0294XdhB (GI:446999566) and XdhA (GI:446816421) having the highest homology in NCBI, which had sequence homologies of 94.7% and 95.5% with the corresponding region of the present invention sequence 3, and amino acid sequences having homologies of 98.7% and 97.4% with sequence 1 and sequence 2, respectively, with 15 and 20 different amino acids. The characterization result by adopting the same method as the invention shows that compared with Acinetobacter baumannii AB307-0294XDH, the XDH provided by the invention has the advantages that the catalytic activity is higher than 25 percent, and the pH tolerance range is wider.

The acinetobacter baumannii alkaline xanthine dehydrogenase provided by the invention is an experimental-confirmed acinetobacter baumannii xanthine dehydrogenase reported for the first time. The alkaline xanthine dehydrogenase provided by the invention can adapt to samples under alkaline determination conditions, and solves the problem of the pH effective action range of the existing xanthine dehydrogenase/oxidase. The alkaline xanthine dehydrogenase has higher substrate affinity and highest catalytic efficiency, so that hypoxanthine/xanthine can be degraded and detected more sensitively.

Another object of the present invention is to provide a method using xanthine dehydrogenase.

In order to achieve the above object, the present invention adopts a technical scheme that a method for detecting the hypoxanthine/xanthine content in a sample comprises the following steps:

1) mixing a sample with a reagent mainly consisting of alkaline xanthine dehydrogenase, oxidized coenzyme and a buffer reagent, and reacting the mixture as follows:

2) and (3) detecting the increase of the product uric acid at 295nm of the main wavelength or the increase of NADH at 340nm, calculating the hypoxanthine/xanthine content in the sample, and preferably detecting the change of NADH at 340 nm.

The sample in the step 1) may be serum, urine, muscle tissue and the like extracted according to a conventional physical examination, or muscle tissue and the like of aquatic products such as fish, or plant tissue such as fruits and vegetables;

the XDH may be a basic xanthine dehydrogenase, which consists of: subunit A coded by SEQ ID NO. 1 and subunit B coded by SEQ ID NO. 2;

the pH value of the detection system is in an alkaline range, particularly between pH7.0 and 12, preferably between pH 7.5 and 10.5; the reaction conditions are such that the temperature is between 15 and 50 ℃, preferably between 25 and 40 ℃; the reaction time is 1-60 min;

the ratio of sample to reagent may be between 1/2 and 1/200.

And the step 2) is that the final reactant is placed on an ultraviolet/visible spectrophotometry analyzer or a semi-automatic biochemical analyzer, and the enzyme activity is quantified through the product change, wherein the change of uric acid can be detected through the absorbance change at 295nm, or the change of NADH can be detected at 340nm, and the change of NADH at 340nm is preferred.

The method for quantifying the hypoxanthine/xanthine in the sample adopts a standard curve method, draws a standard curve by measuring the change of the absorbance of standard xanthine with different concentrations in the same reaction system, and then calculates the content of the hypoxanthine/xanthine according to the change of the absorbance corresponding to the sample.

Another object of the present invention is to provide a detection kit comprising a xanthine dehydrogenase. The kit comprises: XDH enzyme, oxidized coenzyme, xanthine standard, buffer system and stabilizer; the components are as follows:

basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

oxidized coenzyme 0.1-20 mmol/L;

0.001-20mmol/L of xanthine standard;

0.1-80% (total volume) of stabilizer.

The kit is characterized in that:

wherein the XDH enzyme is alkaline xanthine dehydrogenase consisting of: subunit A coded by SEQ ID NO. 1 and subunit B coded by SEQ ID NO. 2.

The pH range of the buffer is pH 7.0-12.0, preferably pH 7.4-9.0, and the buffer reagent is one of tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, tris (hydroxymethyl) aminomethane-diaminoethane tetraacetic acid buffer solution, phosphate buffer solution, triethanolamine buffer solution, 2-amino-2-methyl-1-propanol buffer solution, imidazole buffer solution, glycylglycine buffer solution or glycine-hydrochloric acid buffer solution.

The oxidized coenzyme is NAD⁺、NADP、thio-NAD⁺Oxidized nicotinamide coenzyme or one of its derivatives.

The stabilizer is at least one of ethylene glycol, propylene glycol, glycerol, trehalose, glycan, polyalcohol, ammonium sulfate, Bovine Serum Albumin (BSA), oteracil potassium or salt.

The hypoxanthine/xanthine diagnostic kit used to carry out the method of the invention may be a dual or triple dose, wherein a dual dose comprises:

reagent I

Basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

oxidized coenzyme 0.1-20 mmol/L;

0.1-80% (total volume) of stabilizer.

Reagent II

The xanthine standard is 0.001-20 mmol/L.

The reagent can also be prepared into three agents, which is more favorable for eliminating the pollution of hypoxanthine and xanthine of internal and external sources and simultaneously is favorable for stabilizing the reagent:

reagent I

Basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

0.1-80% (total volume) of stabilizer.

Reagent II

Oxidized coenzyme 0.1-20 mmol/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

0.1-80% (total volume) of stabilizer.

Reagent III

The xanthine standard is 0.001-20 mmol/L.

The formulation of the three agents is not limited to the above formulation, wherein the alkaline xanthine dehydrogenase in the agent I and the oxidized coenzyme in the agent II are put together, and the stabilizing reagent and the buffer solution are put together, thus forming various formulations, which will not be described in detail.

To reduce cross-talk between reagents and to keep the reagents stable and convenient to store, the kit may be presented in a single component form.

In addition, in order to reduce the cross influence among the components of each reagent, and to maintain the stability of the reagent for long-term storage, a stabilizer is usually added to the above single-dose, double-dose, reagent I, reagent II, or triple-dose reagent I, reagent II, or reagent III at a concentration of 0.1 to 80% or10 to 100mmol/L, and the stabilizer may be at least one of ethylene glycol, propylene glycol, glycerol, trehalose, polysaccharide, polyol, ammonium sulfate, Bovine Serum Albumin (BSA), or salt.

It should be understood that the detection kit provided by the present invention can be conveniently used with other enzymes to expand the detection range, such as Purine Nucleoside Phosphorylase (PNP) for detecting inorganic phosphate, PNP and Peroxidase (POD) for detecting adenosine deaminase and 5' -nucleotidase, etc. All of which are intended to fall within the scope of the present invention.

The acinetobacter baumannii alkaline xanthine dehydrogenase provided by the invention has the characteristics of independence on molecular oxygen (xanthine dehydrogenase utilizes NAD as an electron acceptor and can react regardless of the existence of oxygen), wide pH action range and tolerance and wide temperature action range, is favorable for being combined with other enzymes to further develop new application fields, is particularly suitable for sample detection, comprises the detection of xanthine and hypoxanthine, the detection of PNP enzyme and the detection of 5' -nucleotidase, and is combined with PNP enzyme to detect inorganic phosphate and adenosine deaminase, and is suitable for industrial application, including commercial application of degrading byproducts such as hypoxanthine and the like generated in the production process of nucleoside drugs such as ribavirin and the like, degrading various heterocyclic molecular organic pollutants containing sp2 hybridized carbon atoms such as purine, pteridine and aldehyde and the like. The application field of the xanthine dehydrogenase provided by the invention can be expanded to other catalytic substrates, such as the oxidation reaction of other purine, pteridine, heterocyclic molecules and aldehydes and various electron acceptors such as methylene blue, benzoquinone, ferricyanide and nitrate, and the xanthine dehydrogenase is further applied to the field of biosensors.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: 1) the affinity of the substrate is high, which is beneficial to combining and detecting low-concentration substrates; 2) the catalytic efficiency is high, the use amount of enzyme is reduced, the cost is lowered, the acting time is shortened, and the working efficiency is improved; 3) the optimum pH is alkaline, so that the pH value can be tolerated, and the application field can be expanded; 4) the method is independent of oxygen, has little dependence on the conditions of a sample treatment and detection system, is not influenced by the dissolved oxygen state of the sample, and has simple and convenient operation and accurate result; 5) the components of the detection system are few, so that the detection steps are reduced, and the test cost is reduced; 6) no special additional equipment is required for the detection application process. Therefore, the xanthine dehydrogenase and the application thereof in the detection kit provided by the invention are more convenient to popularize and apply.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of purified recombinant A.baumannii xanthine dehydrogenase.

FIG. 2 is a graph showing the activity (A) and stability (B) of purified xanthine dehydrogenase as a function of temperature.

FIG. 3 is a graph showing the change in enzyme activity (A) and stability (B) of purified xanthine dehydrogenase with pH.

FIG. 4 is a graph showing the reaction rate of purified xanthine dehydrogenase as a function of the concentrations of xanthine (A) and hypoxanthine (B).

FIG. 5 is a graph showing the time dependence of purified xanthine dehydrogenase on the degradation of xanthine and hypoxanthine substrates, and A is a uric acid assay; b is NADH detection method.

FIG. 6 is a correlation diagram (B) of the detection value and the real value of the xanthine content in fetal bovine serum detected by the xanthine dehydrogenase method and a standard curve diagram (A) of the xanthine content.

FIG. 7 is a correlation diagram of a detection value and a real value for detecting the content of xanthine in human serum by a kit method, wherein A is a standard curve diagram of the content of xanthine; b is the kit of the invention; c is a Sigma company kit.

FIG. 8 is a graph showing the correlation between the detection value of the content of xanthine in human urine and the true value using the kit method, wherein A is the kit of the present invention; and B is a Sigma company kit.

Detailed Description

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 (Acinetobacter baumannii) which is purchased from China industrial microorganism culture Collection management center (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 Biovector108321(GenBank accession number M22744.1, Amann, E., Ochs, B. andAbel, K. J. light regulated tac promoter genes use effect for the expression of unfused and fused proteins in Escherichia coli. Gene,1988,69(2): 301-315.).

The recombinant plasmid pTRAN is a vector obtained by introducing a 6x histidine purification tag coding sequence into a DNA molecule shown in a sequence 3 at a 5' end and then inserting a pTrc99A vector NcoI and Hind III double enzyme cutting sites.

Engineering Escherichia coli DH5 α containing pTRAN recombinant plasmid, and transferring the recombinant plasmid pTRAN into engineering Escherichia coli DH5 α to obtain recombinant bacteria.

Nickel ion metal chelate affinity chromatography media

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

Xanthine and hypoxanthine are available from Sigma under the catalogues Sigma X7375-10g and Sigma H9377, respectively.

NAD and NADH are both products of Sigma, catalog numbers Sigma N1636 and N1161, respectively.

The xanthine dehydrogenases in the following examples are all the basic xanthine dehydrogenases of Acinetobacter baumannii prepared in example 1, abbreviated as AbXDH, and the constituent large and small subunits thereof are abbreviated as AbXDHB and AbXDHA, respectively.

Example 1 preparation of xanthine dehydrogenase and encoding Gene

Firstly, obtaining xanthine dehydrogenase coding gene and constructing recombinant plasmid pTRAN

1. Extraction of Acinetobacter baumannii genome DNA

Selecting an Acinetobacter baumannii (purchased from China center for culture Collection of Industrial microorganisms and cell culture Collection (CICC) with strain number of CICC 10254) which is environment-friendly and is hidden on an agar slant by using an inoculating loop, streaking the Acinetobacter baumannii on a solid culture medium (the formula is 5.0g of peptone, 3.0g of beef extract, 5.0g of NaCl, 15.0g of agar, 1.0L of distilled water and pH7.0), and then inverting the solid culture medium to perform static culture in an incubator at 37 ℃ for 16 hours; selecting single colony, inoculating to 5ml liquid culture medium (formula: peptone 5.0g, beef extract 3.0g, NaCl 5.0g, distilled water 1.0L, pH7.0), and culturing in shaking table at 37 deg.C and 220rpm overnight; total genomic DNA was extracted using a bacterial genomic DNA extraction kit (OMEGA, product code D3350-01).

2. Design and Synthesis of the following primers

The following primer pair (pTRAN-gS1 and pTRAN-gA2) sequences were designed to amplify xanthine dehydrogenase genes based on the Acinetobacter baumannii genome data published in NCBI,

the upstream primer pTRAN-gS 1:

(the sequence underlined is the Gibsen junction complement region, identical to the pTrc99A vector sequence, the RBS sequence underlined in bold, the start codon in bold, the histidine purification tag coding region in italics, and the sequence complementary to the xanthine dehydrogenase gene in positive)

The downstream primer pTRAN-gA 2:

5'-CCAAAACAGCCAAGCTTATCATTTTTTTACATCAAATACCTGTAATAATTGAGCAAC-3' (SEQ ID NO:5) (Gibsen junction complementary region shown by underlining, corresponding to pTrc99A vector sequence, stop codon shown by bold underlining, and complementary partner sequence to xanthine dehydrogenase gene in the body part)

The following primer pairs (pTRAN-gA1 and pTRAN-gS2) were designed to amplify the large fragment sequence between the restriction sites NcoI and Hind III of the pTrc99A plasmid,

the upstream primer pTRAN-gA 1:

(sequences underlined are Gibsen junction complementary regions, in italic identity to the pTrc99A vector sequence)

The downstream primer pTRAN-gS 2:

(sequences underlined are complementary regions for Gibsen ligation, in italic consistent with the pTrc99A vector sequence)

3. Amplification of PCR products

And (2) performing PCR amplification by using the total DNA of the Acinetobacter baumannii genome extracted in the step 1 as a template and using the upstream primer pTRAN-gS1 and the downstream primer pTRAN-gA2 synthesized in the step 2 as primers to obtain a 4918bp PCR product, namely the gene fragment containing the complete xanthine dehydrogenase gene cluster.

25 μ l PCR reaction: 10ng of total genomic DNA, 2.5. mu.l of the forward primer, 2.5. mu.l of the reverse primer, 5. mu.l of 5XQ5Reaction buffer, 5. mu.l of 5x Q5High GC enhancer, 2. mu.l of dNTPs (2.5 mM each), 0.5UQ5DNApolymerase, ddH₂The volume of O is filled to 25 μ l.

The PCR reaction program is: pre-denaturation at 98 deg.C for 1min, pre-denaturation at 98 deg.C for 10s, pre-denaturation at 62 deg.C for 5s, and pre-denaturation at 72 deg.C for 2.5min, circulating for 30 times, and final annealing at 72 deg.C for 10 min.

PCR amplification is carried out by taking plasmid pTrc99A DNA as a template and taking the upstream primer pTRAN-gA1 and the downstream primer pTRAN-gS2 synthesized in the step 2 as primers to obtain a 4092bp PCR product, namely a DNA large fragment containing NcoI and Hind III on the mother nucleus of pTrc 99A.

25 μ l PCR reaction: 1ng plasmid pTrc99A DNA, 2.5. mu.l of forward primer, 2.5. mu.l of reverse primer, 5. mu.l of 5x Q5Reaction buffer, 5. mu.l of 5x Q5High GC enhancer, 2. mu.l of dNTPs (2.5 mM each), 0.5U Q5DNApolymerase, ddH₂The volume of O is filled to 25 μ l.

The PCR reaction program is: pre-denaturation at 98 deg.C for 1min, pre-denaturation at 98 deg.C for 10s, pre-denaturation at 62 deg.C for 5s, and pre-denaturation at 72 deg.C for 2min, circulating for 30 times, and final annealing at 72 deg.C for 10 min.

The PCR products obtained above were each added with 10U of DpnI enzyme, and incubated in a water bath at 37 ℃ for 1 hour to digest off the methylated template plasmid. The xanthine dehydrogenase gene large fragment (4918bp) and pTrc99A large fragment (4092bp) were purified and recovered using a Gel Extraction kit (product code D2500-02 of OMEGA).

4. Construction of recombinant plasmid pTRAN

Using Gibsen

cloning kit (NEB, product code E5510) A recombinant plasmid pTRAN was constructed. A10. mu.l reaction was constructed as follows: 5 μ l of Gibson

The method comprises the steps of uniformly mixing 10 mu l of a reaction system, placing the reaction system at 50 ℃ for reacting for 20min, then directly transforming the reaction system into escherichia coli DH5 α competent cells by adopting conventional molecular biology operation, coating an LB solid culture medium containing ampicillin (100 mu g/ml) on a transformation product, inversely placing the transformation product in a 37 ℃ constant temperature incubator for culturing for 16h, selecting positive clones, extracting plasmids for sequencing, and obtaining a result plasmid, namely replacing a DNA molecule shown by SEQ ID NO. 3 in a sequence table with a DNA fragment between pTrc99A plasmid NcoI and HindIII enzyme recognition sites, wherein the obtained recombinant plasmid is named pTRAN, namely the recombinant plasmid of acinetobacter baumannii bacteria xanthine dehydrogenase, and expressing xanthine dehydrogenase.

SEQ ID NO: 3 from position 1 to position 3877, and from position 3884 to position 4867, the DNA molecule is a gene encoding xanthine dehydrogenase, and the amino acid sequence of the xanthine dehydrogenase chaperone is shown in sequence 8 and has a size of 37 kDa.

In the encoding gene of the xanthine dehydrogenase, the 1 st to 1500 th positions of a sequence 3 are encoding gene sequences of a small subunit of the xanthine dehydrogenase, and the amino acid sequence of the small subunit is shown as a sequence 1 and has the size of about 56 kDa; the 1502 th to 3877 th sites of the gene sequence of the large subunit of the xanthine dehydrogenase are shown as the sequence 2, and the size of the large subunit is 87 kDa.

Purification of xanthine dehydrogenase

1. And transforming the pTRAN obtained above into Escherichia coli DH5 α to obtain a recombinant strain, selecting a single colony of the recombinant strain, inoculating the single colony of the recombinant strain into 5mL of LB liquid culture medium containing 100 mu g/mL ampicillin, culturing overnight at 37 ℃ and 220r/min, transferring the overnight-cultured bacterial solution into 500mL of LB liquid culture medium containing 100 mu g/mL ampicillin according to the inoculum concentration of 1%, culturing at 37 ℃ and 220r/min until the bacterial solution is cultured until OD600 is about 0.6, adding an IPTG inducer with the final concentration of 1mmol/L, and continuing to perform induction culture for 16h under the same condition.

2. Centrifuging the bacterial solution obtained in the step 1 at 9000r/min and 4 ℃, collecting precipitates to obtain engineering bacteria for induced expression, washing the precipitates once by using 50mmol/L phosphate buffer solution (pH 7.5), suspending the precipitates in 100ml of the same buffer solution to obtain bacterial suspension, breaking the bacterial suspension by using a high-pressure cell breaker JN-10HC (Guangzhou energy-gathering biotechnology, Inc.) at the flow rate of 10L/H and the pressure of 4 ℃ of 150MPa, centrifuging the cell breaking solution at 12000 r/min and 4 ℃ for 30min, and taking the supernatant as crude enzyme solution.

3. Purification of xanthine dehydrogenase by metal chelate chromatography

(1) Adding NaCl with the final concentration of 300mmol/L and imidazole with the final concentration of 5mmol/L into the crude enzyme solution obtained in the step 2, and filtering by using a filter membrane with the diameter of 0.22 mu m to obtain filtrate;

(2) the filtrate, having a total volume of 40ml, was loaded onto

And (3) washing the hybrid protein by using a nickel column affinity resin, sequentially washing the hybrid protein by using 50mmol/L pH 7.5 phosphate buffer solution containing 10mmol/L, 30mmol/L and 50mmol/L imidazole, wherein the volume of each elution is at least 20 column volumes, then eluting by using 50mmol/L pH 7.5 phosphate buffer solution containing 120mmol/L imidazole, collecting eluent as the target recombinase, and the flow rates of the above elution are all 2.5 ml/min.

(3) And (3) passing the eluted target recombinase through a Millipore ultrafiltration tube with a molecular cut-off of 10KDa, repeatedly exchanging and washing the eluted target recombinase for three times by using 50mmol/L pH 7.5Tris hydrochloric acid buffer solution to remove salt and imidazole components to obtain concentrated enzyme solution, and then suspending the concentrated enzyme solution in 50mmol/L pH 7.5Tris hydrochloric acid buffer solution to obtain the purified xanthine dehydrogenase.

The results of detecting xanthine dehydrogenase by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) are shown in FIG. 1, and indicate that xanthine dehydrogenase is composed of two subunits, a small subunit (AbXDHA) with a size of about 56kDa and a large subunit (AbXDHB) with a size of about 87 kDa. The purified enzyme did not contain chaperone proteins of approximately 37 kDa.

Example 2 characterization of AbXDH as a xanthine dehydrogenase

1. Method for measuring xanthine dehydrogenase activity

Based on the fact that xanthine dehydrogenase catalyzes the following reactions (reaction formula 1 and reaction formula 2), the generation of NADH and the catalytic process of the enzyme have strict stoichiometric relation, so that the enzyme activity can be accurately characterized through the generation speed of the product NADH. Since NADH has a specific absorption at 340nm, the present invention measures the enzyme activity by spectrophotometry.

The reaction system for measuring the activity of 2mL xanthine dehydrogenase is shown in Table 1.

TABLE 1 measurement of xanthine dehydrogenase Activity 2mL reaction System composition

The reagents except the xanthine dehydrogenase aqueous solution in the reaction system of 2mL in Table 1 were mixed uniformly and placed in a water bath at 40 ℃ for 5min, and then 100. mu.L of xanthine dehydrogenase aqueous solution was added to start the reaction. Recording the change of the OD340 absorbance value within 3-5min of the reaction under the condition of 40 ℃, calculating the time change rate (delta OD340/min) of the absorbance of the initial linear part of the reaction curve, wherein the time change rate (delta OD340/min) of the absorbance reflects the generation rate of NADH of the xanthine dehydrogenase aqueous solution.

The enzyme activity and specific enzyme activity of the xanthine dehydrogenase detected were calculated according to the following formulas.

Enzyme activity (U/ml) ═ Δ OD₃₄₀/min × 3.21543 × df (formula 1)

Specific enzyme activity (U/mg) ═ U/ml x 1/C (equation 2)

3.21543 in the formula 1 is a calculation coefficient for converting the change of absorbance of NADH in the above 2ml reaction system into molar concentration by using an extinction coefficient method, and the calculation method of the coefficient is as follows:

wherein: vt is the total volume of the reaction (2.0ml), Vs is the volume of the enzyme solution in the reaction system (0.1ml), 6.22 is the molar extinction coefficient of NADH under the measurement conditions (cm 2/. mu.mol), and 1.0cm is the optical path length of the measurement cuvette.

In formula 1, df is the dilution factor of the enzyme solution.

In the formula 2, C is the concentration of the enzyme solution and is in mg/ml.

Definition of enzyme activity unit (U): the amount of enzyme required to convert 1. mu. mol NADH per minute under the conditions of measured temperature and pH.

2. Determination of optimum temperature and temperature tolerance

Whether the AbXDH prepared in example 1 has xanthine dehydrogenase activity and its optimum temperature and temperature tolerance were examined.

The xanthine dehydrogenase in the method of 1 above was replaced with AbXDH prepared in example 1, respectively.

The optimum temperature is determined by respectively placing 200 mul of reaction system in the table 1 in the range of 25-80 ℃ to determine the enzyme activity, the method is the same as the step 1, only the water bath temperature is replaced by the range of 25-80 ℃, the relative enzyme activity is represented by the percentage of the residual enzyme activity relative to the maximum enzyme activity, and the optimum temperature is represented by the temperature corresponding to the maximum value of the relative enzyme activity.

The AbXDH activity curve with respect to the reaction temperature prepared in example 1 is shown in FIG. 2A, which indicates that the optimum reaction temperature is 40 ℃ to 45 ℃.

The temperature tolerance was determined by treating the AbXDH prepared in example 1 at different temperatures in the interval 25-80 ℃ for 30min, and then determining the residual enzyme activity by the method in step 1, the relative enzyme activity being expressed as the percentage of the residual enzyme activity relative to the enzyme activity of the untreated xanthine dehydrogenase. Taking the relative residual enzyme activity as a vertical coordinate and the processing temperature as a horizontal coordinate, performing S-shaped curve (Sigmoidal) curve fitting, calculating the corresponding processing temperature when the relative residual enzyme activity is 50%, and marking as a semi-inactivation temperatureDegree of rotationTo characterize temperature tolerance.

The results of measuring the temperature tolerance of the recombinant xanthine dehydrogenase prepared in example 1 are shown in FIG. 2B, which indicates that the recombinant xanthine dehydrogenase is stable at 25-60 ℃ or lower, and the corresponding compounds

The value was 59 ℃.

3. Determination of optimum pH and pH tolerance

The optimum pH value is represented by the pH value corresponding to the maximum enzyme activity by measuring the relative enzyme activity within the pH range of 4.0-11.0. Specifically, the final concentration of 0.05M in the reaction system shown in Table 1, pH 8.5 Tris-HCl buffer solution was replaced with 0.05M acetic acid buffer system of pH4.0-5.8, 0.05M phosphoric acid buffer system of pH 5.8-8.0, 0.05M Tris-HCl buffer system of pH 7.5-9.0, and 0.05M sodium carbonate buffer system of pH 9.0-11.0, respectively, to perform the measurement. Wild-type xanthine dehydrogenase was used as a control.

As shown in FIG. 3A, the pH optimum of xanthine dehydrogenase was 8.5 to 9.0.

The pH tolerance was determined by placing xanthine dehydrogenase in 0.05M buffer solutions (0.05M acetate buffer at pH 4.0-5.8; 0.05M phosphate buffer at pH 5.8-8.0; Tris HCl buffer at pH 7.5-9.0; sodium carbonate buffer at pH 9.0-11.0) at varying pH values between pH4.0 and 11.0 and allowing to stand at room temperature for 16 h. The residual enzyme activity was then determined by the method in step 1, and the relative enzyme activity was expressed as the percentage of the residual enzyme activity relative to the enzyme activity of the untreated xanthine dehydrogenase.

The results are shown in FIG. 3B, which shows that xanthine dehydrogenase is stable at pH 6.0-11.0, and the enzyme activity is substantially unchanged under alkaline conditions up to pH 11.0.

4. Parameters of enzymatic kinetics

The enzymatic activity of the AbXDH of example 1 was determined according to the method of step 1. The initial reaction rate of AbXDH degradation of xanthine and hypoxanthine was in accordance with the Michaelis equation at an optimum pH of 8.5 and an optimum temperature of 40 ℃ in the concentration range of 0.001-1 mM.

As shown in FIG. 4, A is a graph showing the initial reaction rate as a function of the concentration of xanthine, and B is a graph showing the initial reaction rate as a function of the concentration of hypoxanthine. The results of nonlinear fitting according to the Michaelis equation show that K is obtained when xanthine is used as a substrate_mThe value was 23.66. + -. 1.56. mu.M, and the conversion number was 76.36. + -. 1.07s^-1The maximum specific activity can reach 32.12 +/-0.45U/mg protein, and the catalytic efficiency is 3.23s^-1.μM^-1(ii) a When hypoxanthine is used as a substrate, K_mThe value was 22.48. + -. 1.53. mu.M, and the conversion number was 98.85. + -. 1.31s^-1The maximum specific activity can reach 41.58 +/-0.55U/mg protein, and the catalytic efficiency is 4.40s^-1.μM^-1。

Example 3 use of xanthine dehydrogenase for the degradation of xanthines and hypoxanthine and for the treatment of materials containing such substrates

Referring to the construction method of the xanthine dehydrogenase reaction system in table 1, three groups of reaction systems a) to c) were constructed as follows:

a)50mM Tris-HCl buffer (pH 7.5) containing 1mM EDTA, 0.1mM Potassium Oxonate, 0.1mM nicotinamide adenine dinucleotide (NAD +), 0.1mM xanthine and 0.216mg/L xanthine dehydrogenase prepared in example 1, respectively;

b)50mM Tris-HCl buffer solution (pH 8.5) containing EDTA at a final concentration of 1mM, 0.1mM oteracil potassium, 0.1mM nicotinamide adenine dinucleotide (NAD +), 0.1mM xanthine, and 0.216mg/L xanthine dehydrogenase prepared in example 1, respectively;

c)50mM Tris-HCl buffer (pH 8.5) containing 1mM EDTA, 0.1mM oteracil potassium, 0.1mM nicotinamide adenine dinucleotide (NAD +), 0.1mM hypoxanthine, and 0.216mg/L xanthine dehydrogenase prepared in example 1, respectively;

adding and uniformly mixing all reagents except xanthine dehydrogenase in the reaction systems from a) to c) respectively, placing the reagents in a water bath at 40 ℃ for warm bath for 5min, then adding xanthine dehydrogenase to start a reaction, controlling the reaction temperature at 40 ℃ by using a spectrophotometer with a heating module, recording the change of the absorbance value at 295nm within 3-5min of the reaction while reacting, drawing a relation curve of the increase of the absorbance (delta OD295) along with the change of time, calculating the change rate (delta OD295/min) of the absorbance of the initial linear part of the reaction curve along with the change of time, and detecting the degradation conditions of substrates xanthine and hypoxanthine through the change of product uric acid. The results of the experiment are shown in FIG. 5A.

By the same procedure, b) and c) were incubated at 40 ℃ and then the reaction was initiated by addition of recombinant xanthine dehydrogenase, and the Δ OD was recorded at 40 ℃ under the reaction conditions₃₄₀And (3) detecting the degradation condition of the substrates xanthine and hypoxanthine through the change of the product NADH. The results of the experiment are shown in FIG. 5B.

FIG. 5A shows that xanthine dehydrogenase can efficiently degrade xanthine and hypoxanthine at both pH 7.5 and 40 ℃ and at pH 8.5 and 40 ℃. a) To c) maximum rate of change of absorbance (Δ OD) in the reaction system₂₉₅Min) were 0.0683, 0.0863 and 0.0301, respectively, corresponding to a degradation rate of 0.1093. mu. mol^-1.min^-1，0.1381μmol.L^-1.min^-1And 0.0482. mu. mol.L^-1.min^-1The corresponding specific activities were 0.506U/mg, 0.6394U/mg and 0.2231U/mg, respectively.

FIG. 5B shows the rate of change of absorbance (Δ OD) of the reaction systems in NADH₃₄₀Min) of 0.0313 and 0.0457, respectively, corresponding to a degradation rate of 0.1006. mu. mol^-1.min^-1And 0.1467. mu. mol.L^-1.min^-1The corresponding specific activities were 0.4657U/mg and 0.6792U/mg, respectively.

Example 4 method for measuring hypoxanthine/xanthine content in blood samples Using xanthine dehydrogenase

1) Preparation of a xanthine-containing blood sample

The bovine serum (the "Sijiqing" brand phage-free and low endotoxin fetal bovine serum, production lot No. 150212) preserved by freezing at-20 ℃ was slowly dissolved in a refrigerator at 4 ℃ and bovine serum samples containing 0 to 0.4mmol/L xanthine were constructed according to the following table 2.

TABLE 2 construction of fetal bovine serum samples containing xanthine

2) Construction of enzyme-containing detection System

50 μ L of a mixture of xanthine dehydrogenase assay systems was constructed according to Table 3 below, while assay systems other than xanthine dehydrogenase were constructed as blanks.

TABLE 3 construction of assay System for xanthine dehydrogenase-containing

3) Standard Curve preparation

A50. mu.L standard sample containing 0 to 0.4mmol/L xanthine was prepared according to the following Table 4, and the 50. mu.L standard sample was mixed with the 50. mu.L enzyme-containing detection standard system constructed in 2) respectively, and the mixture was left at room temperature for 30min, and then the change in absorbance at 340nm was measured with a microplate reader (Tecan, model number infinite M200 Pro).

TABLE 4 construction of xanthine-containing Standard samples

And (3) taking the 1# blank xanthine standard as a background contrast, and deducting the 1# background reading value from the reading of the 2# to 6# standard. And (3) constructing a standard curve for measuring the content of the xanthine by a spectrophotometry by taking the content of the xanthine of each standard (unit is nmole/well) as an ordinate (y) and the change value of OD340 after background subtraction as an abscissa (x). The results are shown in fig. 6A below, where the standard curve is y-64.083 x-0.0386 and the correlation coefficient R2 is 0.987.

4) 50 mu L of the sample constructed in the step 1) and 50 mu L of the enzyme-containing detection system in the step 2) are uniformly mixed, placed at room temperature for 30min, and the change of absorbance at 340nm is measured by a microplate reader (Tecan corporation, model number is infinite M200 Pro). The OD measured after mixing each standard sample with 50 microliter sample contrast constructed in 2) uniformly and standing at room temperature for 30min₃₄₀Change to blank control. Subtracting the difference value obtained by blank control from the reading of each sample to substitute for a standard curve in 3) to obtain the content of xanthineThe measured value (Sa). The concentration of xanthine (C) in the sample was calculated according to the following formula:

Sa/Sv ═ C (equation 4)

Wherein:

sa-the xanthine/hypoxanthine content (nmole) in the sample, determined from the standard curve;

sv-sample volume, here 50 μ Ι _;

c-concentration of xanthine/hypoxanthine in the sample (nmole/. mu.L).

In order to evaluate the effect of the xanthine dehydrogenase method provided by the present invention in detecting the xanthine content in serum, the real value of the xanthine content in the sample is used as the abscissa, and the detected value is used as the ordinate, and a correlation curve is plotted as shown in fig. 6B below. Therefore, the real value of the content of the xanthine in the fetal bovine serum sample is basically consistent with the detection value, and the linear correlation coefficient (R) of the real value and the detection value²) Is 0.987, and is basically consistent with the detection result of the xanthine aqueous solution.

Example 5 method of Using xanthine dehydrogenase in detection kit

The present example aims to provide a detection kit comprising xanthine dehydrogenase. The kit comprises: XDH enzyme, oxidized coenzyme, xanthine standard, buffer system and stabilizer; the components are as follows:

basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

oxidized coenzyme 0.1-20 mmol/L;

0.001-20mmol/L of xanthine standard;

0.1-80% (total volume) of stabilizer.

The kit is characterized in that:

The pH range of the buffer is pH 7.0-12.0, preferably pH 7.4-9.0, the buffer reagent is one of tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, tris (hydroxymethyl) aminomethane-diaminoethane tetraacetic acid buffer solution, phosphate buffer solution, triethanolamine buffer solution, 2-amino-2-methyl-1-propanol buffer solution, imidazole buffer solution, glycylglycine buffer solution or glycine-hydrochloric acid buffer solution;

the oxidized coenzyme is one of NAD +, NADP, thio-NAD + oxidized nicotinamide coenzyme or derivatives thereof;

reagent I

Basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

oxidized coenzyme 0.1-20 mmol/L;

0.1-80% (total volume) of stabilizer.

Reagent II

The xanthine standard is 0.001-20 mmol/L.

reagent I

Basic xanthine dehydrogenase 40-40000U/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

0.1-80% (total volume) of stabilizer.

Reagent II

Oxidized coenzyme 0.1-20 mmol/L;

10-1000mmol/L buffer solution, preferably 50-200mmol/L buffer solution;

0.1-80% (total volume) of stabilizer.

Reagent III

The xanthine standard is 0.001-20 mmol/L.

A preferred three-dosage form kit is: reagent I-enzyme mixture, reagent II-sample detection buffer solution box, reagent III-xanthine standard substance, specifically composed as follows:

reagent I-enzyme mixtures

1000U/L of alkaline xanthine dehydrogenase;

Tris-Hcl 50mmol/L(pH 8.5)；

EDTA 1mmol/L(pH 8.5)；

NAD 100mmol/L；

glycerol 50% (w/v);

potassium oxonate is 10 mmol/L.

Reagent II-sample detection buffer solution

Tris-Hcl 50mmol/L(pH 8.5)；

EDTA 1mmol/L(pH 8.5)；

NAD 0.1mmol/L；

Potassium oxonate 0.1 mmol/L.

Reagent III-xanthine standard

The xanthine standard is 20 mmol/L.

Example 6 detection of xanthine content in human serum with detection kit

The three-dosage form assay kit prepared in example 5 was used to assay human serum containing xanthine in the same manner as in example 4. The specific operation steps are as follows,

1) preparation of human serum samples containing xanthine

Human serum samples containing 0-0.4 mmol/L xanthine were prepared according to Table 2 by dissolving human serum (Solarbio, product code Solarbio SA45, LotNo. 20150106) frozen at-20 ℃ slowly in a refrigerator at 4 ℃ in place of fetal bovine serum in Table 2 and in place of Tris-HCl buffer (0.1mM, pH 8.5) with solution II.

2) Construction of enzyme detection System

mu.L of reagent I and 48. mu.L of reagent II are taken to form an enzyme detection system for detecting samples (same as example 4, Table 3), and 50. mu.L of reagent II is taken as a blank control for sample detection.

3) Preparation of Standard Curve

50. mu.L of a standard substance containing 0 to 0.4mmol/L xanthine was prepared using reagent II in place of Tris-HCl buffer (0.1mM, pH 8.5) in Table 4, and reagent III. Using the same procedure as in example 4, a plot of xanthine content y (100 μ L per well) versus the change in absorbance x at 340nm was obtained, as shown in fig. 7A, with y being 63.467x-0.3694 and the correlation coefficient R2 being 0.9947.

4) The same measurement method as in example 4 was used to measure the content of xanthine in human serum in 1). The relationship between the obtained detection value and the actual value is shown in FIG. 7B, and it can be seen that the detection value of the content of xanthine in serum is very consistent with the actual value, and the detection kit provided by the invention has good detection effect.

The invention adopts a commercial xanthine/hypoxanthine detection kit (Sigma company, product code MAK186) to carry out comparative study on the content of xanthine in human serum. The Sigma kit is based on cow's milk xanthine oxidase as the main detection enzyme. The detection results are shown in fig. 7C: as can be seen, the linear range of the Sigma kit for detecting the content of the xanthine (whether the content of the xanthine is in a standard xanthine solution or human serum) is only in the range of 0-12 nmole/well, the corresponding concentration is 0.24mmol/L, and no reaction basically occurs beyond the range. This phenomenon is probably due to the requirement for O for the oxidation of xanthine by xanthine oxidase₂With the addition of (A) at room temperature₂Solubility in water solution is not more than 0.21mmol/L. Besides, the detection kit based on xanthine oxidase needs to be coupled with horseradish peroxidase for detecting H₂O₂Indirectly determining the amount of xanthine. Therefore, the xanthine dehydrogenase kit provided by the invention uses less reagents, has fewer reaction steps and is used for direct measurement.

Example 7 detection of xanthine content in human urine with detection kit

A human urine sample containing 0-0.4 mmol/L xanthine is prepared from fresh urine by the method for preparing human serum containing xanthine in example 6. Then, the content of xanthine was measured by the three-dosage form assay kit prepared in example 5 using the same assay method as in example 6. Meanwhile, the measurement results of a commercial xanthine/hypoxanthine detection kit (Sigma Co., product code MAK186) are compared. As shown in FIG. 8, the assay results of the assay kit based on xanthine dehydrogenase provided by the present invention are broader in assay range and better in linearity. The results are consistent with the results of human serum xanthine content determination.

Claims

1. A protein consisting of subunit a and subunit b 2:

the amino acid sequence of the subunit A is a sequence 1 in a sequence table;

the amino acid sequence of the subunit B is a sequence 2 in a sequence table.

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

3. The DNA molecule of claim 2, wherein: the DNA molecules comprise a DNA molecule coding for the subunit A and a DNA molecule coding for the subunit B.

4. The DNA molecule of claim 2 or 3, wherein:

the DNA molecule is at least one of the following 1) to 2):

2) the coding region is a DNA molecule shown by nucleotide of a sequence 3 in a sequence table.

5. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a DNA molecule according to any one of claims 2 to 4.

6. Use of the protein of claim 1 as a xanthine dehydrogenase for non-disease diagnosis and therapy.

7. Use according to claim 6, characterized by the use in the degradation of hypoxanthine-or xanthine-containing materials, for non-disease diagnostic and therapeutic applications.

8. Use of a DNA molecule according to any one of claims 2 to 4 or a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 5 for the preparation of xanthine dehydrogenase.

9. Use of the protein of claim 1 or the DNA molecule of any one of claims 2 to 4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 5 for the preparation of a product having xanthine dehydrogenase activity.

10. Use of the protein of claim 1 or the DNA molecule of any one of claims 2 to 4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 5 for the non-disease diagnosis and treatment of degradation of xanthine or hypoxanthine.

11. Use according to claim 7 or 8, characterized in that: the xanthine dehydrogenase has at least one of the following characteristics 1) to 3):

1) the subunit configuration of the xanthine dehydrogenase is (αβ)₂Molding;

2) the optimal reaction temperature of the xanthine dehydrogenase is 40-45 ℃;

3) the xanthine dehydrogenase is tolerant to alkaline environmental conditions of pH 7.0-11.0.

12. The use of claim 11, wherein the xanthine dehydrogenase is tolerated at a pH optimum of 8.5 to 9.0.

13. A method for preparing xanthine dehydrogenase, comprising the steps of: inducing and expressing the recombinant bacterium of claim 5 to obtain the protein of claim 1.

14. Use according to claim 6, characterized in that: the xanthine dehydrogenase reacts with a substrate in a sample, and the content of xanthine/hypoxanthine in the sample is detected according to the change of the product, which comprises the following two steps:

1) mixing the sample with a reagent consisting of alkaline xanthine dehydrogenase, an oxidized coenzyme and a buffer reagent, and allowing at least one of the following reactions to occur:

2) detecting the increase of uric acid product at 295nm of main wavelength or the increase of NADH at 340nm, and calculating the hypoxanthine/xanthine content in the sample;

wherein, the sample in the step 1) is serum, urine, muscle tissue, or fish muscle tissue, or fruit and vegetable plant tissue extracted by conventional physical examination;

the xanthine dehydrogenase is alkaline xanthine dehydrogenase and comprises the following components: subunit A coded by SEQ ID NO. 1 and subunit B coded by SEQ ID NO. 2;

the pH value of the detection system is 7.5-10.5, the reaction conditions are that the temperature is 25-40 ℃, and the reaction time is 1-60 min;

the ratio of sample to reagent is between 1/2 and 1/200;

the step 2) is a method for putting the final reactant on an ultraviolet/visible spectrophotometry analyzer or a semi-automatic biochemical analyzer and quantifying the enzyme activity through the product change, wherein the method is to detect the variation of uric acid through the absorbance change at 295nm or detect the variation of NADH at 340 nm;

15. A detection kit comprising a xanthine dehydrogenase, characterized in that: the kit comprises xanthine dehydrogenase, oxidized coenzyme, a xanthine standard, a buffer system and a stabilizer; the components are as follows:

basic xanthine dehydrogenase 40-40000U/L;

50-200mmol/L buffer solution;

oxidized coenzyme 0.1-20 mmol/L;

0.001-20mmol/L of xanthine standard;

0.1-80% of the total volume of the stabilizer;

wherein, the xanthine dehydrogenase comprises the following components: subunit A coded by SEQ ID NO. 1 and subunit B coded by SEQ ID NO. 2;

the pH range of the buffer is 7.0-12.0, and the buffer reagent is one of tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, tris (hydroxymethyl) aminomethane-diaminoethane tetraacetic acid buffer solution, phosphate buffer solution, triethanolamine buffer solution, 2-amino-2-methyl-1-propanol buffer solution, imidazole buffer solution, glycylglycine buffer solution or glycine-hydrochloric acid buffer solution;

the oxidized coenzyme is one of NAD +, NADP and thio-NAD + oxidized nicotinamide coenzyme;

the stabilizer is at least one of ethylene glycol, propylene glycol, glycerol, trehalose, glycan, polyalcohol, ammonium sulfate, Bovine Serum Albumin (BSA) and oteracil potassium.