A recombinant purine nucleoside deaminase and its use

Abstract

The present application provides a kind of recombinant purine nucleoside deaminase and its application, the recombinant purine nucleoside deaminase is the gene sequence shown in SEQ ID NO.1 is connected with PET carrier and increases HIS label to obtain, the amino acid sequence of the recombinant purine nucleoside deaminase is as shown in SEQ ID NO.2.The present application is by constructing the engineering bacteria of expressing a kind of recombinant purine nucleoside deaminase, and attempts to take guanine nucleoside as substrate, the substrate is catalyzed by engineering enzyme, and then obtains xanthine nucleoside.The process method of the present application has the advantages of low cost, high conversion rate, simple process, safe and environment-friendly green compared with chemical synthesis, can greatly expand the application space of xanthine nucleoside.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic engineering, and particularly 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. However, this enzyme is lacking in mammals and microorganisms. This enzyme has good catalytic activity towards 2'-deoxyguanosine, guanosine and 2'-deoxymethylguanosine, and can promote the conversion of amino and carbonyl groups. In 2013, Dahncke et al. [1] This enzyme was first identified and cloned in the model organism Arabidopsis thaliana. In 2018, Schroeder et al. [2] It was found that mutations in this enzyme can delay seed germination. [3] High-resolution methods were used to determine the free and ligand-bound structures of this enzyme from Arabidopsis thaliana. Unlike guanine deaminase, this enzyme utilizes a single-proton shuttling mechanism for catalysis, resulting in structural rearrangements of both the substrate and the enzyme. The final segment of the enzyme folds back and seals the active site, while the substrate rotates during the reaction, both of which are required for deamination.

[0003] Purine nucleosides / deoxynucleosides are a general term for nucleosides with a purine nucleoside as the base part, including adenine nucleoside (adenosine), guanosine (guanosine), xanthine nucleoside (xanthosine), inosine (inosine) and the corresponding purine deoxynucleosides. Their chemical structure is composed of ribose or deoxyribose and purine bases. They can serve as an energy source in cells and participate in the synthesis of nucleic acids and proteins. They also have the effects of regulating cell signaling, anti-inflammatory, antioxidant and immune regulation, and enhancing freshness. Therefore, they play an important role in drug research, clinical and food applications. As a member of the purine nucleoside family, xanthine nucleoside has been shown to play an important role in the proliferation of breast stem cells, bone marrow transplant cells and tumor treatment. At the same time, as an upstream molecule of xanthine, caffeine and theobromine, it has a large application space. Currently, the preparation of this product mainly relies on 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 on environmental protection treatment or dangerous preparation process, which leads to high product prices and greatly limits the application expansion of the product. 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, multiple unsafe factors and the like in the chemical synthesis of xanthosine nucleosides, the present invention provides a recombinant purine nucleoside deaminase and its application.

[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 to 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 xanthosine, 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 xanthosine.

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

[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 then catalyzed by the engineered enzyme to produce xanthosine. Compared with chemical synthesis, the process of the present invention offers the advantages of low cost, high conversion rate, simple process, safety, and environmental friendliness, significantly expanding the application of xanthosine nucleosides. 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, respectively; M: Marker.

[0023] Figure 2 The SDS-polyacrylamide gel electrophoresis results 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 as pH changes.

[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 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 technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical 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 will be further described in detail below with reference to the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0030] The guanine nucleoside substrate structural formula 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] Example 1:

[0033] The present invention obtained the Vitis riparia (riverbank grape) purine nucleoside deaminase gene (KEGG Reference Sequence: 117931280) from KEGG and constructed a recombinant purine nucleoside deaminase based on this gene. By connecting this purine nucleoside deaminase gene to a PET vector and adding a HIS tag to the N-terminus to promote protein expression, an engineered bacterium with high activity was 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 was sent to Guangzhou Qingke Biotechnology for sequence optimization and whole 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. Heat shock the recombinant plasmid into 50 μL of BL21(DE3) competent cells, plate onto a kanamycin-resistant LB plate, and incubate at 37°C for 16 hours. Select well-growing single clones for PCR (polymerase chain reaction) amplification using universal primers (PET-F and PET-R, as shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively). Select positive clones to obtain engineered bacteria, expand them, and then maintain them for seed preservation.

[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% inoculum size 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% inoculum size, and culture at 37°C, 220 rpm until the bacterial solution OD600 is 0.6-1.0. Add IPTG (isopropyl-β-D-thiogalactopyranoside) 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 and prepared into a 25% bacterial solution with phosphate buffer (0.1 M, pH 7.2). The cells were disrupted using an ultrasonic cell disruptor that had been pre-cooled at 4°C. The working time was set to 10 s, the rest time to 10 s, and the total time to 10 min. The crude enzyme solution was obtained, and the enzyme activity was tested and analyzed by SDS-PAGE protein electrophoresis. The electrophoresis results were as follows: Figure 1 .

[0045] 4. Take 0.35ml of 50% BeyoGold TM The column was loaded with His-tag Purification Resin (reduction-resistant chelating type), centrifuged at 4°C (1000g×10s), and the storage solution was discarded. Then, the column was balanced with 0.5ml of non-denaturing lysis solution twice, 1000g×1min, and the liquid was discarded. The crude enzyme solution was added to the column, both ends of the column were sealed, and the column was shaken on ice for about 2h. Open both ends of the column to allow the solution to flow out, and the column was washed 5 times with non-denaturing washing solution, adding 0.5ml each time. After the last column wash, both ends of the column were sealed, centrifuged at 1000g for 1min, 750μl of eluent was added, and the eluent was collected in an EP tube. The column elution was repeated 3 times, and centrifuged at 1000g for 1min. The eluents from the 4 shake flasks were subjected to enzyme activity detection and protein electrophoresis. The electrophoresis results are as follows: Figure 2 .

[0046] Example 2:

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

[0048] (1) Detection of the optimal catalytic pH of purine nucleoside hydrolases

[0049] The 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 optimal pH value. The buffer used was a pH of 7.2 HEPES buffer and sodium chloride solution. The optimal pH values ​​of the purified purine nucleoside hydrolase were determined in buffer systems of different pH values ​​at a temperature of 37°C. Figure 3 As shown in the figure: under the condition of temperature of 37℃, 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 in the figure: the optimum catalytic temperature range of the enzyme is 47-52°C, and it still has more than 80% enzyme activity at 60°C.

[0052] Example 3:

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

[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 suspension, add 1 g of wet bacteria of purine nucleoside deaminase induced by shaking flasks from 4 different batches, 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 to terminate the reaction at a ratio of 1:5. The conversion of guanosine and xanthosine is detected by dilution 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 it in water and make up to 1000 ml, adjust the pH to 5.0 with glacial acetic acid, and filter). Mobile phase B: 100% methanol solution. 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 guanosine and xanthosine standards (0.2 mg / ml); 4) Pass the diluted sample through a 0.2 μm filter and load onto the analyzer; 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 below.

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

[0063] serial number Guanosine dosage 200mM Hepes 1.5M NaCl Tap water Wet bacteria Conversion rate after 3h reaction 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 guanosine to xanthine nucleoside was above 95%, and the substrate was basically completely converted, which has potential industrial application value.

[0065] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a recombinant purine nucleoside deaminase or a microorganism comprising a recombinant purine nucleoside deaminase gene vector in the production of xanthosine nucleosides, wherein the amino acid sequence of the recombinant purine nucleoside deaminase is as shown in SEQ ID NO. 2; or, the recombinant purine nucleoside deaminase gene vector is a vector comprising the gene sequence as shown in SEQ ID NO. 1 linked to a pET vector, wherein the recombinant vector has the nucleotide sequence as shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The microorganism is Escherichia coli.

3. The use according to claim 1 or 2, characterized in that The microorganism is Escherichia coli BL21 (DE3).

4. A method for producing xanthoside, characterized in that: The method comprises the following steps: using guanosine as a reaction substrate, adding a recombinant purine nucleoside deaminase or a microorganism containing a recombinant vector of a purine nucleoside deaminase gene to carry out a catalytic deamination reaction, thereby obtaining xanthine nucleoside; The amino acid sequence of the recombinant purine nucleoside deaminase is shown in SEQ ID NO. 2; or, The purine nucleoside deaminase gene recombinant vector is obtained by connecting the gene sequence shown in SEQ ID NO.1 with a PET vector. The recombinant vector has a nucleotide sequence shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that The microorganism is Escherichia coli.

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

7. The method according to claim 4, characterized in that Sodium chloride solution and Hepes buffer are also added to the catalytic deamination reaction system.

8. The method according to claim 7, characterized in that The concentration of the sodium chloride solution is 1-2 M, and the pH of the Hepes buffer is 7.0-7.5.

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