NAD kinase coding gene with high conversion rate and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a high-conversion-rate NAD kinase coding gene and application thereof. The invention provides an NAD (Nicotinamide Adenine Dinucleotide) kinase coding gene with high conversion rate. The NAD kinase coding gene comprises a nucleotide sequence as shown in SEQ ID NO: 1. According to the invention, a gene library of the NAD kinase is constructed, an NAD kinase coding gene (NADK gene) is screened, and the NAD kinase obtained by expressing the NAD kinase coding gene can improve the conversion efficiency of converting NAD into NADP (Nicotinamide Adenine Dinucleotide Phosphate). The result of the embodiment shows that the NAD kinase coded by the NAD kinase coding gene can improve the conversion efficiency of converting NAD into NADP to 97.41%, which is obviously higher than that of NAD kinases coded by other coding genes.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a NAD kinase encoding gene with a high conversion rate and an application thereof. Background Art

[0002] Nicotinamide adenine dinucleotide phosphate (NADP), also known as pyridine triphosphate, participates in cellular redox metabolism and a series of other biochemical reactions. It is an indispensable coenzyme in organisms as a hydrogen carrier in the biological oxidation process. Studies have shown that NADP plays an important role in promoting material metabolism, energy metabolism, resisting cell aging and anti-oxidation.

[0003] At present, the synthesis methods of NADP can be divided into chemical method, biological fermentation method and biological enzyme method. Due to the problems of lengthy process route, harsh reaction conditions, environmental pollution, low yield and high cost in the chemical method, this process route is not suitable for industrial production. The traditional biological fermentation method obtains NADP by separating and extracting yeast or other microorganisms. This method also has the problems of high cost and low yield. The enzyme method uses NAD kinase (NADK) to catalyze the transfer of phosphate groups from ATP to NAD to generate NADP. However, the existing NAD kinase generally has the problem of low conversion rate. Summary of the invention

[0004] The purpose of the present invention is to provide a NAD kinase encoding gene with a high conversion rate and an application thereof, so as to improve the conversion efficiency of NAD to NADP.

[0005] The present invention provides a NAD kinase encoding gene with a high conversion rate. The NAD kinase encoding gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0006] The present invention also provides a biomaterial of NAD kinase with high conversion rate, wherein the biomaterial comprises a recombinant vector and / or a recombinant bacterium;

[0007] The recombinant vector comprises a basic vector and a NAD kinase encoding gene inserted into the basic vector;

[0008] The recombinant bacteria include a basic strain and the NAD kinase encoding gene or the recombinant vector introduced into the basic strain;

[0009] The NAD kinase encoding gene is the NAD kinase encoding gene described in the above technical solution.

[0010] Preferably, the basic vector comprises pET28a;

[0011] The basic strain includes Escherichia coli BL21 (DE3).

[0012] The present invention also provides a method for expressing NAD kinase with a high conversion rate, characterized in that the strain containing the NAD kinase encoding gene described in the above technical solution is induced to express to obtain NAD kinase.

[0013] Preferably, the induced expression utilizes isopropyl-β-D-thiogalactoside.

[0014] Preferably, the final concentration of the isopropyl-β-D-thiogalactoside is 0.2-1.3 mM.

[0015] Preferably, after the induced expression, the method further comprises: purifying the induced expression product.

[0016] The application of the NAD kinase encoding gene, the biological material or the expression method described in the above technical solution to improving the conversion efficiency of NAD to NADP.

[0017] The present invention also provides a method for improving the conversion efficiency of NAD to NADP, wherein the NAD kinase encoding gene or the biological material or the NAD kinase obtained by the expression method is added to the NAD to NADP conversion system and then reacted.

[0018] Preferably, the concentration of NAD kinase in the system is 0.3 mg / mL; the reaction temperature is 30° C., and the reaction time is 6 to 24 hours.

[0019] Beneficial effects:

[0020] The present invention provides a high conversion rate NAD kinase encoding gene, the NAD kinase encoding gene comprises a nucleotide sequence as shown in SEQ ID NO: 1. The present invention constructs a gene library of NAD kinase, screens a NAD kinase encoding gene (NADK gene), and by expressing the NAD kinase encoding gene, the obtained NAD kinase can improve the conversion efficiency of NAD to NADP. The results of the embodiment show that the NAD kinase encoded by the NAD kinase encoding gene of the present invention can improve the conversion efficiency of NAD to NADP to 97.41%, which is significantly higher than the NAD kinase encoded by other encoding genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is a gel image of the NADK protein obtained by the recombinant expression vector 1 in Example 2;

[0023] Figure 2This is the HPLC test result when the NADK protein activity detection reaction obtained by the recombinant expression vector 1 in Example 2 did not occur;

[0024] Figure 3 This is the HPLC test result when the NADK protein activity test reaction obtained by the recombinant expression vector 1 in Example 2 is complete. DETAILED DESCRIPTION

[0025] The present invention provides a NAD kinase encoding gene with a high conversion rate. The NAD kinase encoding gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0026] The present invention also provides a biomaterial of NAD kinase with high conversion rate, the biomaterial comprising a recombinant vector and / or a recombinant bacterium; the recombinant vector comprising a basic vector and an NAD kinase encoding gene inserted into the basic vector; the recombinant bacterium comprising a basic strain and the NAD kinase encoding gene or the recombinant vector introduced into the basic strain; the NAD kinase encoding gene is the NAD kinase encoding gene described in the above technical solution.

[0027] As an embodiment, the strain includes Escherichia coli BL21 (DE3). The present invention uses Escherichia coli BL21 (DE3) for induced expression, which has the advantages of high expression amount and low cost compared with other strains.

[0028] As an embodiment, the strain further comprises a basic vector; the NAD kinase encoding gene is inserted into the basic vector. As an embodiment, the basic vector comprises pET28a. As an embodiment, the NAD kinase encoding gene is inserted between the Nco I and HindIII restriction sites of the pET28a. The present invention uses pET28a as a basic vector, which has the advantages of high expression amount and low cost compared with other basic vectors.

[0029] The present invention also provides a method for expressing NAD kinase with a high conversion rate, wherein a strain containing the NAD kinase encoding gene described in the above technical solution is induced to express to obtain NAD kinase.

[0030] The present invention induces expression of a strain containing the NAD kinase encoding gene described in the above technical solution to obtain an induced expression product; the induced expression product contains NAD kinase.

[0031] As an embodiment, the induced expression utilizes isopropyl-β-D-thiogalactoside (IPTG). As an embodiment, the final concentration of the isopropyl-β-D-thiogalactoside is 0.2-1.3 mM; as another embodiment, the final concentration of the isopropyl-β-D-thiogalactoside is 1 mM.

[0032] As an embodiment, after obtaining the induced expression product, the present invention purifies the induced expression product. As an embodiment, the purification of the present invention uses a Ni column. The present invention has no strict requirements on the specific steps of the purification, and conventional methods in the art can be used.

[0033] The present invention constructs a gene library of NAD kinase, screens a NAD kinase encoding gene (NADK gene), and the nucleotide sequence is shown in SEQ ID NO: 1. By expressing the NAD kinase encoding gene, the obtained NAD kinase can improve the conversion efficiency of NAD to NADP.

[0034] In view of this, the application of the NAD kinase encoding gene or the biological material or the expression method described in the above technical solution in improving the conversion efficiency of NAD to NADP also falls within the protection scope of the present invention.

[0035] The present invention also provides a method for improving the conversion efficiency of NAD to NADP, wherein the NAD kinase encoding gene or the biological material or the NAD kinase obtained by the expression method is added to the NAD to NADP conversion system and then reacted.

[0036] As an embodiment, the concentration of NAD kinase in the system is 0.1-1 mg / mL; as another embodiment, the concentration of NAD kinase in the system is 0.3 mg / mL. As an embodiment, the system includes HEPES 20-500 mM, ATP 5-20 mM, NAD + 5~20mM, MgCl20.5~20M; As another embodiment, the system includes 500mM HEPES, ATP 16.6mM, NAD + 16.6mM, MgCl216.6 mM.

[0037] As an embodiment, the reaction temperature is 30° C. As an embodiment, the reaction time is 6 to 24 hours; as another embodiment, the reaction time is 6 to 12 hours; as another embodiment, the reaction time is 6 to 10 hours; as another embodiment, the reaction time is 6 to 8 hours.

[0038] To further illustrate the present invention, a high conversion rate NAD kinase encoding gene and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] The inventor team constructed a gene library of NAD kinase and screened a NAD kinase-encoding gene (NADK gene), the nucleotide sequence of which is: 5'-ATGAAAAAAATTGGCGTGATTACCAACCGT GAAAAAGATAAAGGCCTGAAATATACCAACCAGCTGGTGGAAAGCATTGAAAAACATGGCGGCCAGGCGGTGCTGCCGACCTATGATGGCAGCTTTCAGATGGATGATATTGATAACCAGGTGGTGGAAATTTGCAACAACTGCGATATGGTGATTTGCCTGGGCGGCGATGGCACCTTTCTGCGTACCGCGCGTACCGCGTATCTGTATGGCCTGCCGATGCTGGGCATTAACCTGGGCAGCCTGGGCTTTCTGACCGATGTGGAAAAAGGCGAAATTGATAAAGCGGTGGAAAACATTCTGAACAACCGTTTTTGCCTGGAAGATCGTATTATGCTGACCAGCAAACTGTATAAAGATGGCAAACTGATTGCGCGTGATGTGGCGATTAACGATATTGTGATTAGCCGTGGCGGCATTCCGCGTATTCTGCATCTGAGCACCTATATTGATAACAACCTGGTGGAAATGTTTCCGGGCGATGGCATTGTGGTGGCGACCCCGACCGGCAGCACCGCGTATAGCCTGAGCGCGGGCGGCCCGATTGTGGAACCGACCAGCGGCCTGATTCTGATTACCCCGATTTGCCCGCATATTCTGAGCAGCCGTGCGCTGATTACCAGCGATATGCGTAAAATTAAAATTTGCGTGAGCCAGGGCTTTGAACATAAAGCGACCGTGACCGTGGATGGCCAGAAAAACCTGGAAATTACCGGCGGCGATTATCTGGAAATTGAAAAAGCGAACAGCACCGTGAAAATTATTCGTGTGAACAGCAAAAACTTTTTTACCGTGCTGCGTAGCAAAATTTATGAACGTAAAGAAGAATGA-3' (SEQ ID NO:1),The amino acid sequence of the encoded NAD kinase is MKKIGVITNREKDKGLKYTNQLVESIEKHGGQAVLPTYDGSFQMDDIDNQVVEICNNCDMVICLGGDGTFLRTARTAYLYGLPMLGINLGSLGFLTDVEKGEIDKAVENILNNRFCLEDRIMLTSKLYKDGKLIARD VAINDIVISRGGIPRILHLSTYIDNNLVEMFPGDGIVVATPTGSTAYSLSAGGPIVEPTSGLILITPICPHILSSRALITSDMRKIKICVSQGFEHKATVTVDGQKNLEITGGDYLEIEKANSTVKIIRVNSKNFFTVLRSKIYERKEE(SEQ IDNO:2). ,

[0041] Example 2

[0042] 1. Implementation Materials

[0043] (1) Expression vector: pET28a (Kana), IPTG induction;

[0044] (2) Expression host: Escherichia coli BL21 (DE3);

[0045] (3) PBS buffer (pH 7.2-7.4): 2 mM disodium hydrogen phosphate, 0.4 mM potassium dihydrogen phosphate, 0.54 mM potassium chloride, 27.4 mM sodium chloride and 250 mM imidazole buffer (containing PBS buffer, pH 8.0).

[0046] 2. Construction of recombinant expression vector

[0047] (1) inserting the coding gene obtained in Example 1 between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining a recombinant expression vector 1;

[0048] (2) inserting the NAD kinase encoding gene (NCBI accession number: B5EFY8) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining recombinant expression vector 2;

[0049] (3) inserting the NAD kinase encoding gene (NCBI accession number: Q03QF3) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining recombinant expression vector 3;

[0050] (4) inserting the NAD kinase encoding gene (NCBI accession number A9KMB6) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining recombinant expression vector 4;

[0051] (5) inserting the NAD kinase encoding gene (NCBI accession number: WP_094426973.1) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining the recombinant expression vector 5;

[0052] (6) inserting the NAD kinase encoding gene (NCBI accession number A5V6G8) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining the recombinant expression vector 6;

[0053] (7) inserting the NAD kinase encoding gene (NCBI accession number: B8FN99) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining recombinant expression vector 7;

[0054] (8) inserting the NAD kinase encoding gene (NCBI accession number: Q6A7W9) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining the recombinant expression vector 8;

[0055] (9) inserting the NAD kinase encoding gene (NCBI accession number: Q661U4) between the Nco I and HindIII restriction sites of the expression vector, screening positive clones, and obtaining the recombinant expression vector 9;

[0056] (10) The NAD kinase encoding gene (NCBI accession number: O30297) was inserted between the Nco I and HindIII restriction sites of the expression vector, and positive clones were screened to obtain the recombinant expression vector 10.

[0057] 3. Construction of recombinant expression bacteria

[0058] The recombinant expression vectors 1 to 10 in step 2 were transformed into Escherichia coli BL21 (DE3) (purchased from Sangon Biotech (Shanghai) Co., Ltd.), respectively, and the positive cell lines were screened to obtain the recombinant expression bacteria 1 to 10.

[0059] 4. Inducible Expression

[0060] In 500 mL of LB medium (containing the corresponding resistance), inoculate the seed solution of recombinant expression bacteria 1 to 10 in step 3 at a 1% inoculum volume and culture on a shaker (37°C, 220 rpm) for 3 to 5 h until the OD 600 About 0.6-0.8; add IPTG inducer to a final concentration of 1mM and induce at 30℃ overnight.

[0061] 5. Purification

[0062] The fermentation broth obtained in step 4 was centrifuged at 6000 rpm for 30 min, and the supernatant was discarded to retain the bacterial cells; PBS buffer (pH 7.2-7.4) was added to the bacterial cells, and the bacterial cells were broken by a high-pressure homogenizer; the broken liquid was centrifuged at 15000 rpm for 30 min to remove bacterial fragments; the broken liquid after centrifugation was further filtered through a 0.22 μm filter membrane to further remove large particle impurities, and 10 kinds of filtered crude enzyme solutions were obtained; Ni column purification was performed according to the following steps:

[0063] (1) Equilibrate the column: wash with PBS (pH 7.2-7.4) buffer;

[0064] (2) Loading: directly load the filtered crude enzyme solution;

[0065] (3) Equilibration of the column: washing with 10 mM imidazole in PBS buffer;

[0066] (4) Obtaining the target protein: gradient elution was performed using PBS buffer with different concentrations of imidazole. The elution procedure was as follows: solution A was PBS, and solution B was PBS buffer with 500 mM imidazole; when the total volume was 135 mL, solution B was 100%, and the elution rate was 3 mL / min.

[0067] (5) Column storage: After obtaining the protein, rinse with ultrapure water and store in 20% ethanol solution.

[0068] 6. Concentration

[0069] During the elution process, a peak graph appeared at about 12 minutes, and the corresponding eluate was collected for SDS detection. The gel graph of the NADK protein obtained by the recombinant expression vector 1 in Example 2 is as follows: Figure 1 As shown, the size of NADK protein (NAD kinase) is about 31KDa. A 10KDa ultrafiltration tube was selected to remove the imidazole eluate by centrifugation and to concentrate the protein, and the protein concentration was detected by Bradford kit.

[0070] Example 3

[0071] Activity detection

[0072] (1) The NADK protein obtained by different recombinant expression vectors in Example 2 was used as the test sample for activity detection, wherein the activity detection system was: 500mM HEPES, 16.6mM ATP, NAD + 16.6mM, MgCl216.6 mM, test sample 0.3mg / mL, pH 8.0; reaction temperature is 30℃.

[0073] (2) 0.5 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM NADP were prepared for liquid phase detection, and the peak area and injection volume were plotted to obtain a standard curve; during the reaction of step (1), samples were taken at fixed points (reaction time 1 h, 3 h, 6 h, and 24 h), and high performance liquid chromatography (HPLC) detection was performed to detect the NADP peak area, which was used to calculate the amount of NADP generated, and the conversion rate of NAD to NADP was calculated according to the following formula. The conversion rate after the reaction was complete is shown in Table 1. The HPLC detection results of the NADK protein obtained by using the recombinant expression vector 1 in Example 2 before and after the reaction were as shown in Table 1. Figures 2-3 shown.

[0074] Conversion rate (%) = (NADP production / NAD + The amount of reaction) × 100%;

[0075] Table 1 Conversion rate of NAD to NADP

[0076] NADK protein Conversion rate Complete response time Recombinant expression vector 1 97.41％ 6h Recombinant expression vector 2 19.55％ 6h Recombinant expression vector 3 2.3％ 6h Recombinant expression vector 4 31.37％ 6h Recombinant expression vector 5 10.92％ 6h Recombinant expression vector 6 8.3％ 6h Recombinant expression vector 7 12.62％ 6h Recombinant expression vector 8 10.37％ 6h Recombinant expression vector 9 9.97％ 6h Recombinant expression vector 10 3.37％ 6h

[0077] It can be seen from the above content that the NAD kinase encoded by the NAD kinase encoding gene of the present invention can improve the conversion efficiency of NAD to NADP, which is significantly higher than the NAD kinase encoded by other encoding genes.

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high conversion rate NAD kinase encoding gene, characterized in that: The NAD kinase encoding gene comprises the nucleotide sequence shown in SEQ ID NO:

1.

2. A biomaterial with high conversion rate of NAD kinase, characterized in that: The biological material includes a recombinant vector and / or a recombinant bacterium; The recombinant vector comprises a basic vector and a NAD kinase encoding gene inserted into the basic vector; The recombinant bacteria include a basic strain and the NAD kinase encoding gene or the recombinant vector introduced into the basic strain; The NAD kinase encoding gene is the NAD kinase encoding gene according to claim 1.

3. The biomaterial according to claim 2, characterized in that: The basic vector includes pET28a; The basic strain includes Escherichia coli BL21 (DE3).

4. A method for expressing NAD kinase with high conversion rate, characterized in that: The strain containing the NAD kinase encoding gene according to claim 1 is induced to express to obtain NAD kinase.

5. The expression method according to claim 4, characterized in that: The induced expression utilized isopropyl-β-D-thiogalactoside.

6. The expression method according to claim 5, characterized in that: The final concentration of the isopropyl-β-D-thiogalactoside is 0.2-1.3 mM.

7. The expression method according to any one of claims 4 to 6, characterized in that: After the induced expression, the method further comprises: purifying the product of the induced expression.

8. Use of the NAD kinase encoding gene according to claim 1, the biological material according to claim 2 or 3, or the expression method according to any one of claims 4 to 7 in improving the conversion efficiency of NAD to NADP.

9. A method for improving the conversion efficiency of NAD to NADP, characterized in that: The NAD kinase encoding gene according to claim 1, the biological material according to claim 2 or 3, or the NAD kinase obtained by the expression method according to any one of claims 4 to 7 is added to the system for converting NAD to NADP and then reacted.

10. The method according to claim 9, characterized in that The concentration of NAD kinase in the system is 0.3 mg / mL; the reaction temperature is 30° C. and the reaction time is 6 to 24 hours.

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