Peucedanum praeruptorum dunn glycosyl transferase PpUGT22 and application thereof

Abstract

The invention discloses an application of oxygen glycosyl transferase coded by a peucedanum praeruptorum PpUGT22 gene in biosynthesis of scopolin, and belongs to the technical field of biology. The nucleotide sequence of the PpUGT22 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO. 2. The scopolamine is obtained by a biosynthesis method for carrying out enzyme catalysis by utilizing the PpUGT22 protein in vitro, and a new method is provided for synthesizing the scopolamine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to peucedanum oxyglycosyltransferase and application thereof. Background Art

[0002] Peucedanum praeruptorum Dunn, also known as Peucedanum praeruptorum Dunn, is a perennial herb mainly distributed in Northeast China, North China and East China. Its roots are often used in traditional Chinese medicine and have certain pharmacological effects and clinical application value. The medicinal part of Peucedanum praeruptorum Dunn is mainly its dried roots, which contain a variety of active ingredients, including but not limited to: ·Coumarins: such as imperatorin and isoimperatorin, which are one of the most important active ingredients in Peucedanum praeruptorum Dunn and have multiple biological activities such as anti-inflammatory, antioxidant and anti-tumor. ·Volatile oil: Contains some ingredients that have therapeutic effects on respiratory diseases. ·Polysaccharides: May have the effect of enhancing immunity. ·Flavones: Have antioxidant and anti-inflammatory effects.

[0003] Peucedanum peucedanum contains a large number of glycosylated coumarin compounds. The process of transferring glycosyl groups to small molecule receptors under the action of proteases is called glycosylation. As one of the most common modifications, glycosylation is usually associated with changes in physiological activity. Some glycosylated metabolites show improved biological activity, stability and solubility. Glycosyltransferases (GTs) are mainly responsible for glycosylation reactions, transferring glycosyl donors to glycosyl receptors to form glycosidic bonds. According to the connection pattern of glycosylation, they are divided into O-, N-, C- and S-glycosyltransferases. O-glycosyltransferase is the key enzyme, but there are few reports on the O-glycosyltransferase of Peucedanum peucedanum. If the new enzyme O-glycosyltransferase can be cloned and verified, it can provide a new method for the enzyme-catalyzed biosynthesis of glycosylated coumarin compounds. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides an oxygen-glycosyltransferase gene for catalyzing the production of glycosylated coumarin compounds, and a method for biosynthesizing glycosylated coumarin compounds, so as to solve the shortcomings of the existing chemical synthesis of scopolamine.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides an oxygen-glycosyltransferase, whose amino acid sequence comprises the sequence shown in SEQ ID NO.2.

[0007] In another aspect, the present invention provides an oxyglycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0008] Or based on the principle of complementary pairing, the oxyglycosyltransferase gene provided by the present invention can be a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene comprises a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0010] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements, and thus the nucleotide sequence of the oxygen-glycosyltransferase gene may also contain introns, promoters and various regulatory elements.

[0011] In another aspect, the present invention provides a recombinant expression vector encoding the aforementioned oxyglycosyltransferase.

[0012] In another aspect, the present invention provides a transgenic recombinant bacterium encoding the aforementioned oxyglycosyltransferase.

[0013] On the other hand, the present invention provides an application of an oxygen-glycosyltransferase gene in the synthesis of scopoletin, wherein the nucleic acid sequence of the oxygen-glycosyltransferase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0014] In a preferred embodiment, the above-mentioned oxyglycosyltransferase gene sequence is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is completely complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0015] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene is as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0016] In another aspect, the present invention provides use of the aforementioned oxyglycosyltransferase in the synthesis of scopoletin.

[0017] In another aspect, the present invention provides a method for synthesizing scopoletin, the method comprising the following steps:

[0018] 1) obtaining the aforementioned oxygen-glycosyltransferase;

[0019] 2) Using the oxygen-glycosyltransferase in step 1) to catalyze the synthesis of scopoletin in an enzyme activity reaction system.

[0020] In a preferred embodiment, the above oxygen-glycosyltransferase is obtained by prokaryotic expression.

[0021] In a preferred embodiment, the above-mentioned oxyglycosyltransferase is obtained by chemical synthesis.

[0022] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned oxygen-glycosyltransferase, aglycone, sugar donor and buffer.

[0023] In a preferred embodiment, the aforementioned aglycone is scopoletin.

[0024] In the embodiment, the reaction system of step 3) also contains other necessary components known in the art for synthesizing coumarin compounds.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1) The present invention provides a novel oxygen-glycosyltransferase.

[0027] 2) The enzyme provided by the present invention has no ion dependence.

[0028] 3) The enzyme provided by the present invention can quickly reach the optimal catalytic efficiency.

[0029] 4) Compared with the prior art, the present invention uses an enzyme-catalyzed biosynthesis method to obtain scopolamine, which has the advantages of simpler steps, less pollution, and a more uniform product compared to chemical synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0031] Figure 1 This is the electrophoresis result of amplifying the target gene fragment using Peucedanum peucedanum cDNA as a template, with the DNA Marker (2k bp) on the left and the target gene fragment on the right.

[0032] Figure 2 This is the SDS-PAGE gel electrophoresis diagram of the purified protein, with the Marker on the left and the target protein on the right.

[0033] Figure 3 This is the liquid phase detection result of the catalytic reaction of PpUGT22.

[0034] Figure 4 Mass spectrometry results of the catalytic reaction solution of PpUGT22

[0035] Figure 5 This is the result diagram of enzyme kinetic parameters, where A is the divalent metal ion, B is the reaction pH, C is the reaction temperature, D is the reaction time, and E is the Michaelis constant. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0038] Example 1

[0039] Cloning of target oxygen-glycosyltransferase gene

[0040] 1. Total RNA extraction kit was used to extract total RNA from Peucedanum praeruptorum, and a reverse transcription kit with gDNA wiper was used to generate single-stranded cDNA, which was used as a template for polymerase chain reaction (PCR) amplification of the full-length fragment of the PpUGT22 gene. The specific primer information is shown in Table 1:

[0041] Table 1 Primers sequence

[0042]

[0043] The PCR reaction system is: cDNA 1μL, 10μmol·L-1 upstream and downstream primers 1μL each, high-fidelity enzyme 2×PhantaMax Master Mix 12.5μL, ddH2O to 25μL. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1.15min, 30 cycles; 72℃ extension for 5min; 4℃ storage. 1.0% agarose gel electrophoresis was used to detect PCR products, and DNA gel recovery kit was used to recover PCR products. Electrophoresis diagram is shown in Figure 1 A clear band appeared at about 1 500 bp, which was basically consistent with the expected gene size.

[0044] 2. Use homologous recombinase to connect the PCR product with the pET-28a linear vector double-digested with Nde I and BamH I to obtain a recombinant plasmid, which is then transformed into DH 5α chemically competent cells and sequenced after resistance screening.

[0045] The nucleotide sequence and the encoded amino acid sequence obtained by sequencing are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively. The gene sequence contains 1365 bases and encodes 454 amino acids.

[0046] SEQ ID NO.1

[0047] ATGGAGAAGAGCAAAAAATGTCCTCATGCTCTGGTTCTTCCTTTCCCTACACAAGGCCACATAAACCCCATGCTCCAGTT

[0048] TTGCAAACGCTTGGTCTCGAAAGGCATCAAAGTCACACTTGCAAACACTATTCTCATCTCCAAATCCATGCATGCTGACC

[0049] CTCAAAGCCCCGTCTCTCTCGAAACTTACTCAGATGGTTTTGATCAAGTTGCAGGCTATGCTCAAGCAGGAAGCACTGAT

[0050] ATCTACCTCTCCAGTCTACGCAATGTCGGCTCTCAAACACTGGGCAATCTTGTCAAGAAACTTGATGAGGCTGGCAACCC

[0051] TGTGACTGTGCTCATTTACGATAGTTTGTTGCCTTGGGCTTTTGATGTTGCTAACAACTTTGGACTAAAGAAAGCTGTGT

[0052] TTTTTACTCAGTCATGTGCTGTGAACACTATATATTATCATGTTTATAAGGGTCTTATCTCTCTCCCACCGCGAGATGAC

[0053] GAATCAACAGTTATTTCGATTCCTGGATTGCCCCTGTTTAAATCATGGGAAGCACCAGATCCTCGTGACTACAAAGATAT

[0054] TCTAATAAATCAGTTCTCTAATGTTGATGATGTAGATTGGGTCTTTGTTAACACATTCCACAAATTGGAGGAAGAGGAGC

[0055] TTAAGTATGTGCCAAAATGTGGAAACTGAGGACAATAGGACCAACACTTCCATCTATGTACCTAGACCAGAGACTTGAA

[0056] GATGATAAAGGTTATGGTATTAATATTCAAGCCAGATAATGGGGTTTGTATGAAGTGGCTAAGTGAAAAACCTAGTGG

[0057] CTCAGTCATATATGTTTCGTTTGGAAGCTTCGTACAAGTTGAAGCAGAACAATTGGAAGAAGTAGCATGGGGGCTCAAGG

[0058] ACAGCAACTTCAACTTTTTGTGGGTGGTAAGGGAATCGGAAAAGCCAAAGCTTCCAAAATAACGTCATTAATGAGATATCT

[0059] GATAAGGGTCTGGTTGTGACATGGAGTCCACAGTTGGAGATATTAATACATGAGTCAATAGTTGTTTCGTGACACACTG

[0060] TGGTTTCAAATTCTGTTCTTGAAGCCATAAGTTTGGGAGTGCCTATGGTGGGGATACCGCAGTGGACTGATCAACCTACCA

[0061] ATGGCAAGTATATGGAGGATGTTTGGGGCGTGGGGGTCAGAGCTAGACCAGACGAAGGGAATTTGTGAAACGAAATGTT

[0062] ATAAAATCATCGATAAAAGAGGTAATGGAGGGAGAAAGGGGAAACAATCAAGAAAAATGCTGCCAAGTGGAAGAATTT

[0063] GGCTACAGAAGCTCTGGCTGAAGGTGGAACTTCTGATAAGAATATTAACGAGTTTGCAGCTGAATTGTTGCACTCTGTTG

[0064] NO

[0065] SEQ ID NO.2

[0066] MEKSKKCPHALVLPPFTQGHINPMLQFCKRLVSKGIKVTLANTIFISKSMHADPQSPVSLETYSDGFDQVAGYAQAGSTD

[0067] IYLSSLRNVGSQTLGNLVKKLDEAGNPVTVLIYDSLLPWAFDVANNFGLKKAVFFTQSCAVNTIYYHVYKGLISLPPRDD

[0068] ESTVISIPGLPLFKSWEAPDPRDYKDILINQFSNVDDVDWVFVNTFHKLEEEELKYVPKLWKLRTIGPTLPSMYLDQRLE

[0069] DDKGYGINIFKPDNGVCMKWLSEKPSGSVIYVSFGSFVQVEAEQLEEVAWGLKDSNFNFLWVVRESEKPKLPNNVINEIS

[0070] DKGLVVTWSPQLEILIHESIGCFVTHCGFNSVLEAISLGVPMVGIPQWTDQPTNGKYMEDVWGVGVRARPDEKGIVKRNV

[0071] IKSSIKEVMEGEKGETIKKNAAKWKNLATEALAEGGTSDKNINEFAAELLHSVE

[0072] Example 2 Induced expression and purification of PpUGT22 protein

[0073] Induction of PpUGT22 protein

[0074] (1) Add 10 μL of Amp to 100 mL of liquid LB medium, and then add 1 mL of BL21-Psj8-MBP bacterial solution obtained by expansion culture, and culture in a shaker at 37°C and 200 rpm for 2-3 h.

[0075] (2) Cultivate until OD 600 When the concentration reaches about 0.6-0.8, take it out from the shaker, add 100mM of inducer IPTG to make the final concentration 0.5mM, and continue shaking and culturing in a shaker at 16°C and 200rpm for 16h.

[0076] Protein extraction

[0077] (1) The induced bacterial solution was divided into two 50 mL centrifuge tubes, balanced to a weight difference of less than 0.2 g, and centrifuged at 4500 rpm for 10 min at 4°C.

[0078] (2) Discard the supernatant, add 15 mL of Tris-HCl buffer to wash the bacterial pellet, and mix by pipetting.

[0079] (3) Repeat steps (1) and (2) once.

[0080] (4) Centrifuge again at 4°C, 4500 rpm for 10 min, discard the supernatant, add 7 mL of Tris-HCl buffer, and transfer to two 5 mL centrifuge tubes.

[0081] (5) Take one of the centrifuge tubes in (4), dilute its liquid until it turns milky white, and then divide it into 2 mL centrifuge tubes.

[0082] (6) Take the 2 mL centrifuge tubes obtained in step (5) and place them in an ice bath for 8 min of ultrasonic cell wall disruption. Centrifuge at 4000 rpm for 10 min at 4°C. Dispense the supernatant and precipitate to obtain the crude enzyme.

[0083] The protein expression was detected by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, and the results showed that ( Figure 2 ).

[0084] Example 3 In vitro enzyme activity detection

[0085] The initial reaction was carried out in a 200 μL mixture of 0.5 mM scopoletin, 5 mM UDP-Glc, 14 mM reducing agent and 100 μL crude enzyme, which was then filled with sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH = 8. The protein inactivated by heating at 100 ° C for 10 min was used as a negative control. The mixture was incubated at 37 ° C for 12 h, terminated by adding 200 μL methanol, centrifuged at 12,000 × g at 4 ° C for 5 min, and then removed by injection through a microporous filter membrane and then detected by UHPLC. The solvent gradient elution conditions are as follows:

[0086] Table 2 HPLC gradient elution conditions

[0087]

[0088] LC-MS conditions:

[0089] Chromatographic column model: Waters ACQUITY UPLC BEH C18 column (2.1×100mm, 1.7μm)

[0090] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0091] Elution gradient: 0-5min: 5-20% B; 5-8min: 20-22% B; 8-17min: 22-25% B; 17-23min: 25-35% B; 23-25min: 35-50% B; 25-32min: 50-95% B; 32-38min: 100% B; 38-40min: 100-5% B.

[0092] Mass spectrometry conditions: electrospray ion source; acquisition mode is AutoMS2; positive ion mode; capillary voltage is 3500V; sheath gas temperature is 280℃, flow rate is 11L / min; drying gas temperature is 320℃, flow rate is 10L / min; mass-to-charge ratio scanning range is 100-1700m / z; collision voltage is 175V; collision energy is 15-50eV.

[0093] Test results: HPLC test results show ( Figure 3 ), by comparing the peak graph with the standard, it was shown that the reaction solution contained scopoletin and scopolamine, which means that scopoletin was generated by the PpUGT22 enzymatic reaction. The conversion efficiency reached 74%. The mass spectrometry results also showed that the product was scopoletin ( Figure 4 ).

[0094] Example 4

[0095] Enzyme kinetic parameters detection

[0096] Using scopoletin as substrate and UDP-Glc as glycosyl donor, the effects of pH, temperature, metal ions and reaction time on the catalytic activity were investigated.

[0097] 1. Temperature: Investigate the change of enzyme activity at different reaction temperatures (20℃, 30℃, 37℃, 40℃, 45℃, 50℃, 60℃ and 70℃). The reaction system contains 100μL crude enzyme, 0.5mM substrate, 5mM UDP-GLC, 14mM β-mercaptoethanol, and is fixed to 200μL with 50mM, pH=8.0 phosphate buffer. The reaction solution is reacted at each temperature for 12h. After the reaction, twice the volume of pre-cooled methanol is immediately added to the reaction system to terminate the reaction. After centrifugation, the supernatant is filtered and tested by HPLC. Three parallel tests are set for each group of reactions. The conversion rate of the substrate is estimated based on the peak area ratio in the chromatogram.

[0098] 2. pH: Investigate the effects of different pH buffers of 50mM: 5.0-6.0 citric acid-sodium citrate buffer; 7.0-8.0, Na2HPO4-NaH2PO4 Buffer; 8.0-9.0, Tris-HCl Buffer; 9.0-10.0, Na2CO3-NaHCO3 Buffer on enzyme activity. The reaction system is: 14mMβ-mercaptoethanol, 5mM UDP-GLC, 100μL crude enzyme, 0.5mM compound scopoletin in a 200μL reaction system, and the corresponding buffer is supplemented to the specified volume. The reaction solution is placed at 37℃ for 12h, and immediately after the reaction, two volumes of pre-cooled methanol are added. After centrifugation, the supernatant is filtered and tested by HPLC. To ensure the reliability of the data, three replicate samples are set up for each group of reactions, and the final conversion rate is calculated by the peak area of ​​the chromatogram.

[0099] 3. Metal ions: The receptor molecule of the reaction solution is selected as the compound scopoletin, and different divalent metal ions are investigated: Fe 2+ , Mn 2+ 、Co 2+ 、Zn 2+ , Ba 2+ , Ca 2+ Mg 2+ The effect of EDTA on enzyme activity. A 200μL reaction system contains 14mMβ-mercaptoethanol, 5mM UDP-GLC donor, 100μL crude enzyme, 0.5mM substrate, 5mM divalent metal ions, and 50mM, pH=8.0 phosphate buffer is added to the specified volume. The reaction solution is placed at 37°C for 6h, and immediately after the reaction, two volumes of pre-cooled methanol are added, and centrifuged at 14000rpm, 4°C for 4min. The supernatant is filtered and tested by HPLC. Three replicates are set up for each reaction, and the final conversion rate is calculated by the peak area of ​​the chromatogram.

[0100] 4. Reaction time: 200 μL phosphate buffer reaction system contains 14 mM β-mercaptoethanol, 5 mM UDP-GLC, 100 μL crude enzyme, 0.5 mM substrate, and reacts in a 37 ° C water bath for 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 24h, 26h, 28h. After the reaction, quickly add two volumes of pre-cooled methanol to each reaction solution to terminate the reaction, and centrifuge at 14000rpm and 4°C for 4min. Take the supernatant and filter it for detection on HPLC, and calculate the conversion rate of the substrate. To ensure the accuracy of the data, three parallel tests are set up for each group, and the substrate conversion rate is calculated according to the ratio of the peak area in the HPLC spectrum.

[0101] 5. Michaelis constant Km: The total volume of the enzyme activity reaction system is 200 μL, including: 5 μL crude enzyme and 5 mM UDP-Glc, substrate concentration is 50-900 μM (50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 μM respectively), supplemented with pH=8.0, 50 mM phosphate buffer to 200 μL, reacted at 37°C for 10 min, and then immediately terminated the reaction with an equal volume of pre-cooled methanol after the reaction was completed. After high-speed centrifugation at 14000 rpm for 5 min, 20 μL was taken for HPLC detection, and the liquid phase conditions were as above. The Michaelis constant is calculated by the Michaelis equation and the Lineweaver-Burk plot method.

[0102] Test results:

[0103] Enzyme kinetic studies have shown that ( Figure 4 ), PpUGT22 has the highest enzyme activity at 37°C and has a good conversion rate in the range of 30-40°C. The optimum pH of the reaction solution is 8.0, suitable for reaction in the range of 7.0-8.0 pH, and the catalytic efficiency is highest in phosphate buffer. The enzyme is not dependent on metal ions, Mg 2+ It can significantly increase the catalytic efficiency. The relative catalytic efficiency can reach more than 70 in 12 hours, and the relative conversion rate can reach the maximum catalytic efficiency in 22 hours. Finally, through calculation, the Km value of the above recombinant protein for kaempferol is 356.6mmol / L. The above enzyme kinetics related results are shown in Figure 5 A-5E.

[0104] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An oxygen-glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. An oxygen-glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.

2.

3. A recombinant expression vector encoding the oxyglycosyltransferase according to claim 1.

4. A transgenic recombinant bacterium expressing the oxygen-glycosyltransferase according to claim 1.

5. Use of the enzyme according to claim 1 or the gene according to claim 2 in the synthesis of scopoletin.

6. Use of the vector according to claim 3 or the bacterium according to claim 4 in synthesizing scopoletin.

7. A method for synthesizing scopoletin, the method comprising the following steps: 1) obtaining the oxygen-glycosyltransferase as claimed in claim 1; 2) Using the oxygen-glycosyltransferase in step 1) to catalyze the synthesis of scopolamine in an enzyme activity reaction system.

8. A method for synthesizing scopolamine as claimed in claim 7, characterized in that: In step 1), the oxyglycosyltransferase is obtained by prokaryotic expression or by chemical synthesis.

9. A method for synthesizing scopolamine as claimed in claim 5, characterized in that: In step 2), the enzyme activity reaction system contains scopoletin and UDP-Glc.

10. A method for synthesizing scopolamine according to claim 5, characterized in that: The enzyme activity reaction system in step 2) also contains a buffer.

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