High-temperature-resistant uridine kinase and application thereof

Abstract

The present application is based on the amino acid of wild type uridine kinase (EC 2.7.1.213) from Thermus thermophilus, the uridine kinase mutants with improved optimum reaction temperature are screened by changing the amino acid sequence of the surface of the wild type protein, the modified uridine kinase can catalyze the reaction at a higher temperature, significantly improves the catalytic efficiency, and meets the demand of industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a heat-resistant uridine kinase and its applications. Background Technology

[0002] In biocatalysis, high-temperature environments above 45°C are beneficial for increasing reaction rates, thereby improving catalytic efficiency. However, laboratory studies have shown that when the temperature reaches 45°C, the activity of uridine kinase (UDK, EC2.7.1.213) decreases by more than 40%, affecting the catalytic effect. To increase the enzyme's ability to withstand high-temperature catalytic environments, it is necessary to construct a uridine kinase with high-temperature tolerance to achieve high-efficiency uridine acid production in the preparation of uridine acid. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a high-temperature resistant uridine kinase, which addresses the shortcomings of the prior art.

[0004] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned high-temperature resistant uridine kinase.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A heat-resistant uridine kinase M1, the amino acid sequence of which is shown in SEQ ID NO:3.

[0007] The nucleotide sequence encoding the thermostable uridine kinase M1 is shown in SEQ ID NO:4.

[0008] Furthermore, another heat-resistant uridine kinase M2 is provided, the amino acid sequence of which is shown in SEQ ID NO:5.

[0009] The nucleotide sequence encoding the thermostable uridine kinase M2 is shown in SEQ ID NO:6.

[0010] A recombinant expression vector, wherein the recombinant expression vector comprises the above-mentioned nucleotide sequence encoding thermostable uridine kinase M1 and the nucleotide sequence encoding thermostable uridine kinase M2, respectively.

[0011] The expression vector is pET-28a (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0012] A recombinant expression transformant is constructed by transforming the above-mentioned recombinant expression vector into host cells.

[0013] The host cell was Escherichia coli BL21(DE3) (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0014] The application of the above-mentioned high-temperature resistant uridine kinase M1 and high-temperature resistant uridine kinase M2 in the preparation of uridine acid is also within the scope of protection of this invention.

[0015] Among them, the heat-resistant uridine kinase M1 and heat-resistant uridine kinase M2 can withstand temperatures of 40~50℃.

[0016] Beneficial effects: Compared with the prior art, the high-temperature resistant uridine kinase M1 and high-temperature resistant uridine kinase M2 of the present invention have a stronger ability to adapt to high-temperature environments than wild-type uridine kinase, and can withstand temperatures of 40~50℃. They can be better applied to the preparation of uridine acid, and achieve high-efficiency generation of uridine acid. Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0018] Figure 1 This is a diagram showing the construction of the recombinant expression plasmid for the thermostable uridine kinase mutant M1 in this case.

[0019] Figure 2 This is a diagram showing the construction of the recombinant expression plasmid for the thermostable uridine kinase mutant M2 in this case. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0022] In the following embodiments, the terms recombinant expression plasmid and recombinant expression vector have the same meaning.

[0023] In the following examples, the terms recombinant Escherichia coli and recombinant expression transformant have the same meaning.

[0024] Example 1: Modification of thermostable uridine kinase M1 and thermostable uridine kinase M2

[0025] (1) The target gene was synthesized through whole-genome synthesis (by GenScript).

[0026] (2) The target gene and vector are prepared by the corresponding restriction endonucleases (EcoRI, Hind) Enzyme digestion treatment;

[0027] (3) The target gene is ligated into the vector to obtain the expression plasmid, and the recombinant plasmid is transformed into competent Escherichia coli cells by calcium chloride method (according to Molecular Cloning Handbook).

[0028] Wild-type uridine kinase UDK from *Thermus thermophilus* was synthesized in its entirety (by GenScript) according to the corresponding sequence described in NCBI.

[0029] Its nucleotide sequence is: (SEQ ID NO:2)

[0030] atgagcgcgccgaaaccgtttgtgattggcattgcgggcggcaccgcgagcggcaaaaccaccctggcgcaggcgctggcgcgcaccctgggcgaacgcgtggcgctgctgccgatggatcattattataaagatctgggccatctgccgctggaaga acgcctgcgcgtgaactatgatcatccggatgcgtttgatctggcgctgtatctggaacatgcgcaggcgctgctgcgcggcctgccggtggaaatgccggtgtatgattttcgcgcgtatacccgcagcccgcgccgcaccccggtgcgcccggcgc cggtggtgattctggaaggcattctggtgctgtatccgaaagaactgcgcgatctgatggatctgaaagtgtttgtggatgcggatgcggatgaacgctttattcgccgcctgaaacgcgatgtgctggaacgcggccgcagcctggaaggcgtggtg gcgcagtatctggaacaggtgaaaccgatgcatctgcattttgtggaaccgaccaaacgctatgcggatgtgattgtgccgcgcggcggccagaacccggtggcgctggaaatgctggcggcgaaagcgctggcgcgcctggcgcgcatgggcgcggcg

[0031] The amino acid sequence of the corresponding protein is: (SEQ ID NO:1)

[0032] MSAPKPFVIGIAGGTASGKTTLAQALARTLGERVALLPMDHYYKDLGHLPLEERLRVNYDHPDAFDLALYLEHAQALLRGLPVEMPVYDFRAYTRSPRRTPVRPAPVVILEGILVLYPKELRDLMDLKVFVDADADERFIRRLKRDVLERGRSLEGVVAQYLEQVKPMHLHFVEPTKRYADVIVPRGGQNPVALEMLAAKALARLARMGAA

[0033] The enzyme sites were modified to obtain a thermostable uridine kinase with two nucleotide sequences and the following amino acid sequence. The corresponding sequences were obtained through whole-genome synthesis (by GenScript):

[0034] The nucleotide sequence of the thermostable uridine kinase M1 is as follows: (SEQ ID NO:4)

[0035] atgagcgcgccgaaaccgtttgtgattggcattgcgggcggcaccgaaagcggcaaaaccaccctggcgcaggcgctggcgcgcaccctgggcgaacgcgatgcgctgctgccgatggatcattattataaagatctggatcatctgccgctggaagaacgcctgcgcgtgaactatgatcatccggatgattttgatctggcgctgtatctggaacatgcgcaggcgctgctgcgcggcctgccggtggaaatgccggtgtatgattttcgcgcgtatacccgcagcccgcgccgcaccccggtgcgcccggcgccggtggtgattctggaaggcattctggtgctgtatccgaaagaactgcgcgatctgatggatctgaaagtgtttgtggatgcggatgcggatgaacgctttattcgccgcctgaaacgcgatgtgctggaacgcggccgcagcctggaaggcgtggtggcgcagtatctggaacaggtgaaaccgatgcatctgcattttgtggaaccgaccaaacgctatgcggatgaaattgtgccgcgcggcggccagaacccggtggcgctggaaatgctggcggcgaaagcgctggcgcgcctggcgcgcatgggcgcggcg

[0036] The amino acid sequence of the thermostable uridine kinase M1 corresponding protein is: (SEQ ID NO:3)

[0037] MSAPKPFVIGIAGGTESGKTTLAQALARTLGERDALLPMDHYYKDLDHLPLEERLRVNYDHPDDFDLALYLEHAQALLRGLPVEMPVYDFRAYTRSPRRTPVRPAPVVILEGILVLYPKELRDLMDLKVFVDADADERFIRRLKRDVLERGRSLEGVVAQYLEQVKPMHLHFVEPTKRYADEIVPRGGQNPVALEMLAAKALARLARMGAA

[0038] The nucleotide sequence of the thermostable uridine kinase M2 is: (SEQ ID NO:6)

[0039] atgagcgcgccgaaaccgtttgtgattggcattgcgggcggcaccgaaagcggcaaaaccaccctggcgcaggcgctggcgcgcaccctgggcgaacgcgatgcgctgctgccgatggatcattattataaagatctggatcatctgccgctggaagaacgcctgcgcgaaaactatgatcatccggatgattttgatctggcgctgtatctggaacatgcgcaggcgctgctgcgcggcctgccggtggaaatgccggtgtatgattttcgcgcgtatacccgcagcccgcgccgcaccccggtgcgcccggcgccggtggtgattctggaagatattctggtgctgtatccgaaagaactgcgcgatctgatggatctgaaagtgtttgaagatgcggatgcggatgaacgctttattcgccgcctgaaacgcgatgtgctggaacgcggccgcagcctggaaggcgtggtggcgcagtatctggaacaggtgaaaccgatgcatctgcattttgtggaaccgaccaaacgctatgcggatgaaattgtgccgcgcggcggccagaacccggtggcgctggaaatgctggcggcgaaagatctggcgcgcctggcgcgcatgggcgatgcg

[0040] The amino acid sequence of the thermotolerant uridine kinase M2 corresponding protein is: (SEQ ID NO:5)

[0041] MSAPKPFVIGIAGGTESGKTTLAQALARTLGERDALLPMDHYYKDLDHLPLEERLRENYDHPDDFDLALYLEHAQALLRGLPVEMPVYDFRAYTRSPRRTPVRPAPVVILEDILVLYPKELRDLMDLKVFEDADADERFIRRLKRDVLERGRSLEGVVAQYLEQVKPMHLHFVEPTKRYADEIVPRGGQNPVALEMLAAKDLARLARMGDA

[0042] Primers F (tgcagaattcatgagcgcgccgaa) and R (actgaagcttcgccgcgcccatgc) were designed and synthesized. PCR amplification was performed using the synthesized uridine kinase and M1 and M2 mutant genes as templates. The PCR reaction system included: 2.5 μL 10× Buffer (Mg... 2+ The following reagents were added: 2 μL dNTP Mixture (2.5 mM), 3 μL MgCl2 (25 mM), 0.1 μL each of primers F and R, 1 μL corresponding gene template, 0.5 μL high-fidelity enzyme, and sterile double-distilled water to a final volume of 25 μL. The PCR reaction parameters were: denaturation at 95℃ for 5 minutes, annealing at 55℃ for 30 seconds, extension at 72℃ for 1 minute, 20 cycles, incubation at 72℃ for 15 minutes, and then incubation at 16℃ for 1 hour.

[0043] The target gene and pET-28a expression vector were expressed by the corresponding restriction endonucleases (EcoRI, Hind) respectively. Enzyme digestion followed by ligation into the pET-28a expression vector yielded recombinant expression plasmids pET28a-uridine kinase M1 and pET28a-uridine kinase M2. Figure 1 and Figure 2 The recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells using the calcium chloride method (according to the Molecular Cloning Handbook). Ten single colonies from LB agar plates were picked and inoculated into 20 mL test tubes containing 5 mL of LB liquid medium, and cultured at 30°C and 220 rpm for 12 hours. Plasmids were extracted using a plasmid extraction kit (purchased from Shanghai Shenneng Bocai Biotechnology Co., Ltd.), and verified by double enzyme digestion. The enzyme digestion system was as follows: 8.4 μL recombinant expression plasmid, 0.3 μL EcoRI, 0.3 μL Hind. 1 μL of 10× buffer was added. After incubating at 37℃ for 3 hours, the target fragment was separated by 1% agarose gel electrophoresis, and the fragment size was observed under a gel imaging system. Plasmids that were verified by enzyme digestion were sent to Nanjing GenScript Technology Co., Ltd. for sequencing. The sequencing results were compared with the target gene using the gene alignment software BioXM. The plasmids with the correct results were the catalytic recombinant E. coli elements in this case.

[0044] Recombinant *E. coli* was induced to express its contents using 100 mM IPTG. Recombinant *E. coli* bacteria were streaked onto LB agar plates containing 50 mg / L kanamycin resistance (containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 2% agar powder) and incubated at 37 °C for at least 8 h until single colonies could be isolated. Single colonies were picked and transferred to 50 ml centrifuge tubes (containing 10% v / v LB liquid medium with 50 mg / L kanamycin resistance, containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride) and incubated at 37 °C, 220 rpm for 12 h. A 5% inoculum was then added to 1 L shake flasks (containing 25% v / v LB liquid medium with 50 mg / L kanamycin resistance) and incubated at 37 °C, 220 rpm until OD (out of control) was reached. 600 Add IPTG to a final concentration of 0.2 mM (0.6-0.8). Lower the temperature to 30 °C, incubate at 200 rpm for another 7 h to induce expression. Centrifuge and collect the cells, then freeze and incubate at -20 °C for 24 h for subsequent catalytic reactions.

[0045] Example 2: Effect detection of wild-type uridine kinase and its mutant thermostable uridine kinases M1 and M2

[0046] The activities of wild-type uridine kinase WT and the thermostable uridine kinases M1 and M2 prepared in Example 1 were measured. The enzyme activity assay system of 50 ml contained: 30 mM uridine, 100 mM MgCl2·6H2O, 10 mM ATP, 10 g / L xylene, and 20 g / L WT / M1 / M2. The reaction mixture was stirred at 750 rpm for 1 h at 32 °C and the pH of the reaction was maintained at 7.5 by adding 5 mol / L alkaline solution. The enzyme activities of wild-type uridine kinase WT, thermostable uridine kinase M1 and M2 were measured at 32, 40, 45 and 50 °C. One enzyme activity unit U was defined as the weight of recombinant Escherichia coli dry bacteria required to convert 1 µmol UMP in 1 min.

[0047] Table 1. Differences in the activity of uridine kinases of WT, M1, and M2 at different temperatures.

[0048]

[0049] The experimental results are shown in Table 1. The results indicate that, under normal temperature conditions, the activities of the two uridine kinase mutants, M1 and M2, are similar to those of the wild-type uridine kinase WT. However, when the temperature increases, the activity of WT is significantly inhibited, and the two uridine kinase mutants, M1 and M2, exhibit higher activities than the wild-type uridine kinase WT under all conditions. At 45℃, the enzyme activities of uridine kinase mutants M1 and M2 are 43% and 67% higher than those of the wild-type uridine kinase WT, respectively. At 50℃, the enzyme activities of uridine kinase mutants M1 and M2 are 67% and 55% higher than those of the wild-type uridine kinase WT, respectively. Therefore, the modified uridine kinase is suitable for high-temperature catalytic environments, and its enzyme activity is significantly improved under high-temperature conditions, which is beneficial for improving catalytic efficiency.

[0050] This invention provides a concept and method for heat-resistant uridine kinase and its applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A heat-resistant uridine kinase, characterized in that, The heat-resistant uridine kinase described herein has an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:

5.

2. The heat-resistant uridine kinase according to claim 1, characterized in that, The corresponding nucleotide sequence encoding the thermostable uridine kinase is shown in SEQ ID NO:4 or SEQ ID NO:

6.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleotide sequence of claim 2.

4. A recombinant expression transformant, characterized in that, The recombinant expression vector described in claim 3 is constructed by transforming it into host cells.

5. The application of the heat-resistant uridine kinase according to claim 1 or 2 in the preparation of uridine acid.

6. The application according to claim 5, characterized in that, The aforementioned heat-resistant uridine kinase can withstand temperatures of 40-50°C.

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