Recombinant purine nucleoside deaminase and application thereof

Abstract

The invention provides a recombinant purine nucleoside deaminase and application thereof, the recombinant purine nucleoside deaminase is obtained by connecting a gene sequence as shown in SEQ ID NO.1 with a PET carrier and adding an HIS tag, and the amino acid sequence of the recombinant purine nucleoside deaminase is as shown in SEQ ID NO.2. According to the invention, the xanthine nucleoside is obtained by constructing and expressing an engineering bacterium of a recombinant purine nucleoside deaminase and trying to take guanine nucleoside as a substrate and catalyzing the substrate by an engineering enzyme. Compared with chemical synthesis, the process method disclosed by the invention has the advantages of low cost, high conversion rate, simple process, safety and environmental friendliness, and the application space of xanthine nucleoside can be greatly expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to a recombinant purine nucleoside deaminase and an application thereof. Background Art

[0002] This type of purine nucleoside deaminase is an enzyme containing zinc ions and belongs to the cytidine deaminase family. It is widely present in plants and algae and participates in the body's purine metabolism and regulates seed germination, but it is lacking in mammals and microorganisms. This enzyme has good catalytic activity for 2'-deoxyguanosine, guanosine and 2'-deoxymethylguanosine, and can promote the conversion of amino and carbonyl groups. In 2013, Dahncke et al. [1] The enzyme was first identified and cloned in the model organism Arabidopsis thaliana. [2] It was found that mutations in this enzyme can delay seed germination. [3] The free and ligand-bound structures of the enzyme in Arabidopsis thaliana were resolved using high-resolution methods. Unlike guanine deaminase, this enzyme uses a single proton shuttling mechanism for catalysis, and both the substrate and the enzyme undergo structural rearrangements. The last segment of the enzyme folds back and closes the active site, while the substrate rotates during the reaction, which are all necessary for deamination.

[0003] Purine nucleosides / deoxynucleosides are the general term for nucleosides with purine nucleosides as the base part, including adenine nucleosides (adenosine), guanosine nucleosides (guanosine), xanthine nucleosides (xanthine), inosine nucleosides (inosine) and corresponding purine deoxynucleosides. Their chemical structure is composed of ribose or deoxyribose and purine bases. They can be used as energy sources in cells, participate in the synthesis of nucleic acids and proteins, and also have the functions of regulating cell signal transduction, anti-inflammatory, antioxidant and immune regulation, and enhancing freshness. Therefore, it plays an important role in drug research, clinical and food applications. As a member of the purine nucleoside family, xanthine nucleosides have been shown to play an important role in breast stem cells, bone marrow transplant cell proliferation and tumor treatment. At the same time, as an upstream molecule of xanthine, caffeine and theobromine, they have a large application space. At present, the preparation of this product is mainly achieved by chemical reactions such as diazotization / hydrolysis or amino reduction. However, the chemical synthesis process has unfavorable factors such as poor selectivity, many impurities, low yield, high pressure of environmental treatment or dangerous preparation process, which leads to high product prices and greatly limits the application expansion of products. New production processes are urgently needed to solve this situation.

[0004] References:

[0005] [1]K.Dahncke and CPWitte,Plant Cell,2013,25,4101-4109.

[0006] [2]RYSchroeder,A.Zhu,H.Eubel,K.Dahncke and CPWitte,New Phytol.,2018,217,233-244

[0007] [3] Qian Jia, Hui Zeng, Huanxi Li, Nan Xiao, Jing Tang, Shangfang Gao, Jinbing Zhang and Wei Xie, Chem. Commun, 2021, 57, 9748 Summary of the invention

[0008] In order to solve the problems of complex process, low yield, many unsafe factors and the like in the method of chemically synthesizing xanthine nucleosides, the present invention provides a recombinant purine nucleoside deaminase and application thereof.

[0009] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0010] In a first aspect, the present invention provides a recombinant purine nucleoside deaminase, wherein the recombinant purine nucleoside deaminase is obtained by connecting the gene sequence shown in SEQ ID NO.1 with a PET vector and adding a HIS tag, and the amino acid sequence of the recombinant purine nucleoside deaminase is shown in SEQ ID NO.2.

[0011] In a second aspect, the present invention provides a purine nucleoside deaminase gene recombinant vector, characterized in that the gene sequence shown in SEQ ID NO.1 is connected to a PET vector, and the recombinant vector has a nucleotide sequence shown in SEQ ID NO.3.

[0012] Preferably, the PET vector is selected from pET-24a, pET-22b, pET-28a, pET-30a or pET-32a.

[0013] In a third aspect, the present invention provides a microorganism comprising the purine nucleoside deamination gene recombinant vector described in the second aspect.

[0014] Preferably, the microorganism is Escherichia coli, Pichia pastoris, or Bacillus subtilis.

[0015] Further preferably, the microorganism is Escherichia coli BL21 (DE3).

[0016] In a fourth aspect, the present invention provides use of the recombinant purine nucleoside deaminase described in the first aspect or the microorganism described in the third aspect in producing xanthine nucleosides.

[0017] In a fifth aspect, the present invention provides a method for producing xanthoside, comprising the following steps: using guanosine as a reaction substrate, adding the recombinant purine nucleoside deaminase described in the first aspect or the microorganism described in the third aspect to carry out a catalytic deamination reaction, thereby obtaining xanthoside.

[0018] Preferably, sodium chloride solution and Hepes buffer are also added to the system for catalytic deamination reaction.

[0019] More preferably, the concentration of the sodium chloride solution is 1-2 M, and the pH of the Hepes buffer is 7.0-7.5.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention constructs an engineered bacterium expressing a recombinant purine nucleoside deaminase, and attempts to use guanine nucleoside as a substrate, which is catalyzed by the engineered enzyme to obtain xanthine nucleoside. Compared with chemical synthesis, the process of the present invention has the advantages of low cost, high conversion rate, simple process, safety and environmental protection, and can greatly expand the application space of xanthine nucleoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The SDS-polyacrylamide gel electrophoresis results of the crude enzyme solution, wherein 1 to 4 represent the bacterial lysate (crude enzyme solution) after induction expression of the recombinant bacteria in shake flasks No. 1 to 4; M: Marker.

[0023] Figure 2 The results of SDS-polyacrylamide gel electrophoresis of the eluate (purified solution), wherein 1 to 4 represent the eluate after the lysis and purification of the recombinant bacteria in shake flasks No. 1 to 4 after induction expression; M: Marker.

[0024] Figure 3 This is the enzyme activity curve of purine nucleoside hydrolase changing with pH.

[0025] Figure 4 This is the enzyme activity curve of purine nucleoside hydrolase changing with temperature. DETAILED DESCRIPTION

[0026] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0027] To facilitate understanding of the present invention, the present invention will be described more comprehensively and in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0030] The guanine nucleoside substrate structure used in the following reaction is:

[0031] This product is produced and sold by the applicant of the present invention. Guanosine nucleoside supplied by other manufacturers can also be used in the present invention.

[0032] Embodiment 1:

[0033] The present invention obtains the purine nucleoside deaminase gene (KEGG Reference Sequence: 117931280) of Vitis riparia (riverbank grape) on KEGG, and constructs a recombinant purine nucleoside deaminase based on it. By connecting the purine nucleoside deaminase gene with a PET vector, a HIS tag is added to the N-terminus to promote protein expression, and an engineered bacterium with higher activity is obtained. The specific steps are as follows:

[0034] 1. The nucleotide sequence of purine nucleoside hydrolase is SEQ ID NO.1, and the protein sequence is SEQ ID NO.2. The sequence SEQ ID NO.1 is sent to Guangzhou Qingke Biotechnology for sequence optimization and full gene synthesis to construct it into the pET-28a plasmid expression vector to obtain a recombinant plasmid (SEQ ID NO.3).

[0035] SEQ ID NO.1:

[0036] Atggaggatgccaaagtggtggaagcaaaagatggaaccatttctgtagcttctgcattcgctggccatcaggaagttgtacaggatagagaccacaaattcttaacagcagcagttgaggaggcatataaaggggttgaatgtggagatggaggtccattcggtgcggttgttgttcgtaatgatgaagtacttgtgagctgtcacaacatggttctgacaaacaccgatccgactgcacatgcagaggttactgcaataagagaggcatgtaagaagctcaaccaaattgagctatcagactgtgaaatctatgcctcttgtgagccttgcccaatgtgctttggtgctatacatctttcaagaattaagagattggtctatggggccaaagctgaagcagctatagctattggattcgacgattttatagcagatgccttaaggggtactggattttaccaaaaggctcacttggagatcaaaagggctgatggtgatggtgctgaaattgcggaacaagtttttgagaatacaaaagccaagtttcgtatgtat

[0037] SEQ ID NO.2:

[0038] MEDAKVVEAKDGTISVASAFAGHQEVVQDRDHKFLTAAVEEAYKGVECGDGGPFGAVVVRNDEVLVSCHNMVLTNTDPTAHAEVTAIREACKKLNQIELSDCEIYASCEPCPMCFGAIHLSRIKRLVYGAKAEAAIAIGFDDFIADALRGTGFYQKAHLEIKRADGDGAEIAEQVFENTKAKFRMY

[0039] SEQ ID NO.3:

[0040]

[0041] 2. The recombinant plasmid was introduced into 50 μL BL21 (DE3) competent cells by heat shock method, spread on LB solid plate containing kanamycin resistance, and cultured at 37°C for 16 hours. The monoclonal clones with good growth were selected and amplified by PCR (polymerase chain reaction) using universal primers (PET-F and PET-R, as shown in SEQ ID NO.4 and SEQ ID NO.5, respectively), and the positive clones were selected to obtain the engineered bacteria, which were then cultured and preserved.

[0042] SEQ ID NO.4(5'-3'): ATgCgTCCggCgTAgAggAT

[0043] SEQ ID NO.5(5'-3'):gCTAgTTATTgCTCAgCgg

[0044] 3. Inoculate the frozen glycerol engineered strain into kanamycin-resistant LB medium at a 0.2% inoculation rate, and culture at 37°C, 220 rpm for 12 h to activate the strain. Inoculate the activated bacterial solution into fresh LB medium containing 50 μg / ml kanamycin sulfate (4 250 ml shake flasks, labeled 1, 2, 3, and 4) at a 1% inoculation rate, and culture at 37°C, 220 rpm until the bacterial solution OD600 is 0.6-1.0. Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.2 mM, and induce the culture at 30°C for 20 h. Wet cells were collected by centrifugation at 4°C, and the cells were weighed. A 25% bacterial solution was prepared with phosphate buffer (0.1 M, pH 7.2). The cells were broken by ultrasonic cell disruptor precooled at 4°C. The working time was set to 10 s, the interval time was 10 s, and the total time was 10 min. The crude enzyme solution was obtained. The enzyme activity was detected and the protein electrophoresis analysis was performed by SDS-PAGE. The electrophoresis results were as follows: Figure 1 .

[0045] 4. Take 0.35ml of 50% BeyoGold mixed evenly TM Load the column with His-tag Purification Resin (reduction-resistant chelating type), centrifuge at 4°C (1000g×10s), discard the storage solution, then balance with 0.5ml non-denaturing lysis buffer twice, 1000g×1min, and discard the liquid. Add the crude enzyme solution to the column, seal both ends of the column, and shake on ice for about 2h. Open both ends of the column to allow the solution to drain, and wash the column 5 times with non-denaturing washing solution, adding 0.5ml each time. After the last column wash, seal both ends of the column, centrifuge at 1000g for 1min, add 750μl of eluent, collect the eluent in an EP tube, repeat the elution on the column 3 times, centrifuge at 1000g for 1min, and perform enzyme activity detection and protein electrophoresis on the eluents from 4 shake flasks. The electrophoresis results are as follows: Figure 2 .

[0046] Embodiment 2:

[0047] Some properties of the purine nucleoside hydrolase are analyzed as follows:

[0048] (1) Optimal catalytic pH detection of purine nucleoside hydrolases

[0049] Purine nucleoside hydrolase purified by the present invention was subjected to enzymatic reaction under different pH conditions (5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.5) to determine its optimum pH value. The buffer pH used was 7.2 HEPES buffer and sodium chloride solution. The optimum pH of the purified purine nucleoside hydrolase measured in buffer systems of different pH values ​​at a temperature of 37° C. is shown in FIG. Figure 3 As shown: Under the condition of temperature of 37°C, the optimal catalytic pH range of purine nucleoside hydrolase is 7.5-8.0, within which the enzyme can maintain a high enzyme activity.

[0050] (2) Optimal catalytic temperature of purine nucleoside hydrolase

[0051] The purine nucleoside hydrolase purified by the present invention was tested for enzyme activity at different temperatures (22°C-60°C) under the condition of pH 7.5. Figure 4 As shown: the optimal catalytic temperature range of the enzyme is 47-52°C, and it still has more than 80% enzyme activity at 60°C.

[0052] Embodiment 3:

[0053] Application of purine nucleoside hydrolase in the preparation of xanthine nucleoside

[0054] Accurately weigh 2.5 g of guanosine nucleoside, add pH 7.5 Hepes buffer, 1.5 M sodium chloride solution and tap water to a final volume of 50 ml to prepare a 5% guanosine nucleoside suspension, add 1 g of wet bacteria of purine nucleoside deaminase induced and expressed in 4 different batches of shake flasks, stir evenly, and carry out catalytic reaction at 37°C. Samples are taken after 3 hours of reaction, and 1 M / L hydrochloric acid is added at a ratio of 1:5 to terminate the reaction. The conversion of guanosine and xanthosine is detected by diluting 200 times. The specific detection method is as follows: 1) Chromatographic conditions: C18 column, flow rate 1.0 mL / min, injection volume 10 μL, column temperature, 25°C, detection wavelength, 262 nm; 2) Mobile phase: Mobile phase A 0.01 mol / L ammonium acetate solution (pH 5.0, take 0.77 g of ammonium acetate, dissolve in water and make up to 1000 ml, adjust pH to 5.0 with glacial acetic acid, and filter). Mobile phase B: 100% methanol solution. The gradient elution conditions are as shown in Table 1:

[0055] Table 1 Gradient elution conditions

[0056] Time (minutes) Mobile phase A (%) (0.01 mol / L ammonium acetate solution pH 5.0) Mobile phase B (%) (methanol) 0 95 5 15 95 5 25 70 30 26 95 5 35 95 5

[0057] 3) Prepare standard guanosine and xanthine nucleoside (0.2 mg / ml); 4) Pass the diluted sample to be tested through a 0.2 μm filter membrane and apply it to the machine; 5) Refer to Table 2 below for the specific retention time of each component and calculate the conversion rate of each group.

[0058] Table 2 Retention time of each component

[0059]

[0060]

[0061] The specific results are shown in Table 3.

[0062] Table 3 Transformation results of 4 shake flask strains

[0063] serial number Guanosine dosage 200mM Hepes 1.5M NaCl Tap water Wet bacteria Reaction 3h conversion rate 1 2.5g(5％) 5ml 5ml 40ml 1g 99.2％ 2 2.5g(5％) 5ml 5ml 40ml 1g 98.9％ 3 2.5g(5％) 5ml 5ml 40ml 1g 97.5％ 4 2.5g(5％) 5ml 5ml 40ml 1g 98.6％

[0064] In summary, after 3 h of reaction between each batch of enzyme and substrate, the conversion rate of guanine nucleoside to xanthine nucleoside was above 95%, and the substrate was basically completely converted, which has potential industrial application value.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A recombinant purine nucleoside deaminase, characterized in that: The recombinant purine nucleoside deaminase is obtained by connecting the gene sequence shown in SEQ ID NO.1 with a PET vector and adding a HIS tag. The amino acid sequence of the recombinant purine nucleoside deaminase is shown in SEQ ID NO.

2.

2. A purine nucleoside deaminase gene recombinant vector, characterized in that: The gene sequence shown in SEQ ID NO.1 is connected to the PET vector, and the recombinant vector has the nucleotide sequence shown in SEQ ID NO.

3.

3. The purine nucleoside deaminase gene recombinant vector according to claim 2, characterized in that: The PET vector is selected from pET-24a, pET-22b, pET-28a, pET-30a or pET-32a.

4. A microorganism, characterized in that It comprises the purine nucleoside deamination recombinant vector according to claim 2 or 3.

5. The microorganism according to claim 4, characterized in that The microorganism is Escherichia coli, Pichia pastoris, or Bacillus subtilis.

6. The microorganism according to claim 4 or 5, characterized in that The microorganism is Escherichia coli BL21 (DE3).

7. Use of the recombinant purine nucleoside deaminase according to claim 1 or the microorganism according to any one of claims 4 to 6 in producing xanthine nucleosides.

8. A method for producing xanthine riboside, characterized in that: The method comprises the following steps: taking guanosine nucleoside as a reaction substrate, adding the recombinant purine nucleoside deaminase according to claim 1 or the microorganism according to any one of claims 4 to 6 to carry out catalytic deamination reaction, thereby obtaining xanthine nucleoside.

9. The method according to claim 8, characterized in that Sodium chloride solution and Hepes buffer are also added to the catalytic deamination reaction system.

10. The method according to claim 9, characterized in that The concentration of the sodium chloride solution is 1-2M, and the pH of the Hepes buffer is 7.0-7.5.

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