Dendrobium huoshanense glycosyltransferase and application thereof in synthesis of antioxidant flavone disglycoside compounds

Abstract

The application discloses a glycosyltransferase coded by Dendrobium huoshanense UGT70 gene and application of the glycosyltransferase in synthesis of antioxidant flavone disglycosides, and belongs to the technical field of biotechnology. The UGT70 gene nucleotide sequence codes an amino acid sequence as shown in SEQ ID NO. 2. The flavone disglycosides are obtained by using the UGT70 protein to perform enzyme catalysis in vitro by a biosynthesis method, and a new method for synthesizing the flavone disglycosides is provided.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Dendrobium huoshanense glycosyltransferase and its application in the synthesis of antioxidant flavonoid disaccharide compounds. Background Technology

[0002] Flavonoid disaccharides are glycoside derivatives in which two sugar groups are linked by a glycosidic bond at a specific hydroxyl position in the flavonoid nucleus. Typical reported compounds include: Rutin: a flavonol disaccharide widely found in plants such as Rutaceae (Rutaceae), Sophora japonica (Fabaceae), and Malus baccata (Hypericaceae), possessing antioxidant, free radical scavenging, hemostatic, and antihypertensive effects; Naringin: a flavanone disaccharide mainly found in plants such as pomelo and grapefruit (Rutaceae), possessing anti-inflammatory, glucose and lipid metabolism regulating, oxidative stress anti-inflammatory, and myocardial protective effects; Cyanidin-3-morula disaccharide: an anthocyanin disaccharide, morula disaccharide composed of glucose and xylose, naturally found in the fruit of Elderberry; and Isohexin: a flavonoid C-glycoside disaccharide found in grasses such as bamboo leaves, possessing unique anti-inflammatory and analgesic activities.

[0003] Currently, the main methods for synthesizing flavonoid disaccharides include three categories: plant extraction, chemical synthesis, and biocatalysis. Plant extraction involves directly extracting and purifying flavonoid disaccharides from natural plants; typical examples include rutin extracted from Sophora japonica buds and naringin extracted from citrus fruits. Its core limitations are: strong resource dependence (the content of flavonoid disaccharides in natural plants is extremely low, leading to huge raw material consumption and low production efficiency); high purification costs; and unstable quality (significantly affected by plant growth cycles and the environment of the growing region, making it difficult to meet the requirements of industrial production). Chemical synthesis involves activating sugar donors with chemical reagents to achieve glycosylation; representative applications include the chemical synthesis of flavonoid C-glycosides. Its main drawbacks are harsh reaction conditions, poor selectivity, significant environmental hazards, and cumbersome procedures. Biocatalysis utilizes glycosyltransferases to catalyze glycosylation reactions; current technologies mainly rely on enzymes derived from microorganisms or model plants. Biocatalysis has advantages such as mild reaction conditions, environmental and safety friendliness, and strong controllability of efficiency.

[0004] Currently, there are very few enzyme resources capable of catalyzing the synthesis of flavonoid disaccharides, and most of them suffer from problems such as demanding catalytic conditions and the need for specific cofactors. This invention is the first to discover a highly efficient disaccharide synthase from Dendrobium huoshanense, which not only fills the gap in the availability of this type of enzyme from medicinal plants, but also overcomes the application limitations of existing enzymes through a wide suitable reaction temperature range, high catalytic efficiency, and wide pH adaptability, providing key technical support for industrialization. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a glycosyltransferase gene for catalyzing the generation of antioxidant flavonoid disaccharide compounds, as well as a method for biosynthesizing antioxidant flavonoid disaccharide compounds, thereby overcoming the shortcomings of existing methods for preparing antioxidant flavonoid disaccharide compounds.

[0006] Specifically, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides a glycosyltransferase whose amino acid sequence includes the sequence shown in SEQ ID NO.2.

[0008] On the other hand, the present invention provides a glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0009] Alternatively, based on the principle of complementary pairing, the glycosyltransferase gene provided by the present invention can be a sequence encoding a fully complementary pair of nucleotide sequences containing the amino acid sequence shown in SEQ ID NO. 2.

[0010] In a preferred embodiment, the coding sequence of the glycosyltransferase gene described above comprises a nucleotide sequence as shown in SEQ ID NO.1, or a sequence that is completely complementary to the sequence shown in SEQ ID NO.1.

[0011] As is known to those skilled in the art, gene sequences can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequence of the above-mentioned glycosyltransferase gene can also contain introns, promoters, and various regulatory elements.

[0012] On the other hand, the present invention provides an expression cassette, recombinant vector, or recombinant cell containing the aforementioned glycosyltransferase gene.

[0013] On the other hand, the present invention provides the application of the aforementioned glycosyltransferase or the aforementioned glycosyltransferase gene or the aforementioned expression cassette, recombinant vector or recombinant cell in the synthesis of flavonoid disaccharide compounds.

[0014] In a preferred embodiment, the flavonoid disaccharide compound is rutin.

[0015] On the other hand, the present invention provides a method for synthesizing flavonoid disaccharide compounds, characterized in that the method comprises the following steps:

[0016] 1) Obtain the aforementioned glycosyltransferase;

[0017] 2) The glycosyltransferase from step 1) is used to catalyze the synthesis of flavonoid disaccharide compounds in an enzyme-active reaction system.

[0018] In a preferred embodiment, the glycosyltransferase is obtained in step 1) by microbial synthesis or chemical synthesis.

[0019] In a preferred embodiment, the flavonoid disaccharide compound in step 2) is rutin.

[0020] In a preferred embodiment, isoquercitrin is the substrate that serves as the glycosyl acceptor in the enzyme activation reaction system in step 2).

[0021] In a preferred embodiment, the glycosyl donor in the enzyme activation reaction system in step 2) is UDP-Rha.

[0022] In a preferred embodiment, the enzyme activation reaction system in step 2) contains the glycosyltransferase, UDP-Rha, and isoquercitrin described in step 1).

[0023] In a preferred embodiment, the enzyme activation reaction system in step 2) also contains a buffer solution.

[0024] More preferably, the buffer solution is a Na2HPO4-NaH2PO4 buffer solution.

[0025] In a preferred embodiment, the enzyme activation reaction system in step 2) also contains divalent cations.

[0026] Further preferably, the enzyme activation reaction system in step 2) also contains Mg. 2+ Ba 2+ And one or more of EDTA.

[0027] In a preferred embodiment, the reaction temperature of the enzyme activation reaction system in step 2) is 4-60°C or the pH of the enzyme activation reaction system in step 2) is 5.0-10.0.

[0028] More preferably, the reaction temperature of the enzyme activation reaction system in step 2) is 20-55℃, for example, it can be 20℃, 30℃, 40℃, 45℃, 50℃, 55℃, or any two of the above values.

[0029] More preferably, the pH of the enzyme activation reaction system in step 2) is 6.0-10.0, for example, the pH can be 6, 7, 8, 9, 10, or any two of the above values.

[0030] The present invention has the following advantages and effects compared with the prior art:

[0031] 1) This invention provides a novel glycosyltransferase and a novel method for synthesizing antioxidant flavonoid disaccharide compounds. Compared with the prior art, this method utilizes enzyme-catalyzed biosynthesis to obtain flavonoid disaccharide compounds, which has advantages such as simpler steps, less pollution, and more singular products compared with chemical synthesis methods.

[0032] 2) The glycosyltransferase in this invention has a wide suitable reaction temperature range (4~60℃).

[0033] 3) The glycosyltransferase in this invention has a wide suitable pH range (5-10).

[0034] 4) The glycosyltransferase in this invention can almost completely catalyze isoquercitrin to rutin. Attached Figure Description

[0035] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Figure 1 This is the cloned gene fragment. M is a 2000bp DNA marker, and lane 1 is UGT70.

[0037] Figure 2 This is an SDS-PAGE gel electrophoresis image of the pCold1-UGT70 fusion protein. M is the protein marker (17-180kDa), and lane 1 is the crude UGT70 enzyme.

[0038] Figure 3 This is a liquid phase diagram of the reaction between UGT70 and the substrate.

[0039] Figure 4 This is the mass spectrum of the product of the reaction between UGT70 and the substrate.

[0040] Figure 5 The results show the enzymatic properties of UGT70 catalyzing the conversion of isoquercitrin to rutin. A represents the pH condition, B represents the metal ion condition, C represents the temperature condition, and D represents the enzyme kinetic parameters. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] Example 1 Cloning of the target glycosyltransferase gene

[0044] 1. Total RNA extraction kit: Total RNA was extracted from Dendrobium huoshanense, and single-stranded cDNA was generated using a reverse transcription kit with gDNA wiper. This cDNA was then used as a template for polymerase chain reaction (PCR) amplification of the full-length UGT70 gene fragment. Specific primers are shown in Table 1.

[0045] Table 1 Primer Sequences

[0046]

[0047] The PCR reaction system consisted of: 25 μL of 2 × Phanta Max Master Mix (Dye Plus) (P515, Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of forward and reverse primers, 50-100 ng of template, and dd H2O to a final volume of 50 μL. The PCR amplification program was as follows: ① 95℃ for 3 min; ② 95℃ for 15 s, 60℃ for 45 s, and 72℃ for 60 s, for a total of 35 cycles; ③ 72℃ extension for 5 min. After the reaction, the product size was determined by 1% agarose gel electrophoresis.

[0048] 2. The amplification product was ligated into the pCold1 vector and transformed into E. coli DH5α. Single colonies were selected, cultured in LB medium, and verified by bacterial PCR (verification results are shown in [link to documentation]). Figure 1 M: DL 2000 marker; Lane 1: UGT70 gene), the bacterial culture PCR reaction system was as follows: 12.5 μL Green Taq Mix (P131, Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of forward and reverse primers, 2 μL template (bacterial culture), and dd H2O added to a final volume of 25 μL. Samples with correct bacterial culture PCR results were sent for sequencing.

[0049] The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1. This gene sequence contains 1392 nucleotides and encodes 463 amino acids (as shown in SEQ ID NO.2).

[0050] >SEQ ID NO.1

[0051]

[0052] >SEQ ID NO.2

[0053] MANNDDATARGSLHILMFPFVAFGHISPFLQLSRKLSAGGGIHITFLSTPANLPRISSLLPSSSPIRLHSLPLPSIPGLPAGAESTADLPQQTAELLKLAVDSMQPQVAALLADLR PDLVFFDFAQPWLPSIAHPLGVKTLFFSVFSAAATAYLTVPSRRSKHSIANIAGELIRPPAAFPDSTALSAGVPAYQAADFSYIFRSSGDAGELSVFDRVLTGLTGCSAVVAKTCM EMESPYIQYIESQLGKPVLLAGPVVPESPAGTLGPEWTNFLDRFADGSVVFCSFGSETALSEEGVEELLLGLEMAGMPFLAVLNGATAAVGKASADGSRARLVREGWAPQQLILGH RSVGCFVCHAGMSSLVEAVVSGCKLVLLPQRGDQYLNARLFAGDLGIGVEVEREESGGFKREAVRDAVVRVMEEEKGRESFDKWRGFFMDGEVQMKFLEEFVGKLKELAYGKSSK.

[0054] Example 2: UGT70 protein induction and purification

[0055] 1. Protein-induced expression

[0056] Expand the culture of the target bacterial culture that was correctly sequenced and extract the recombinant plasmid pCold1-UGT70. The obtained plasmid was introduced into BL21(DE3) competent cells according to the instructions and plated (the solid culture plate contained 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 15 g / L agar powder, and antibiotics at a final concentration of 100 μg / mL). The solid culture plate was placed in a water-cooled incubator at 37°C and incubated overnight. The grown single colonies were placed in a culture medium containing 100 μg / mL antibiotics and incubated at 37°C with a shaker at 200 rpm for 6-8 h. After that, the bacterial culture was verified by PCR. The PCR products were detected by 1% agarose gel electrophoresis. The electrophoresis results were detected by a gel imaging system. If the bands were correct, the culture was amplified to 100 mL. After the OD600 = 0.6-0.8, the bacterial culture was cooled to 16°C, and IPTG was added to a final concentration of 0.5 mM. The culture was then incubated at 16°C with a shaker at 200 rpm for 18-20 h.

[0057] 2. Protein purification

[0058] The obtained bacterial culture was collected using a benchtop low-speed centrifuge, and the cells were resuspended in equilibration buffer. The culture was then poured into a beaker, immersed in an ice-water bath, and placed in a cell sonicator for initial disruption for about 10 minutes. The cells were then further disrupted completely in a homogenizer. Before use, the homogenizer was rinsed with pure water to remove any 20% ethanol stored inside, preventing any potential impact on the proteins. Finally, the crude enzyme solution was centrifuged using a low-temperature high-speed centrifuge, and the precipitate was discarded to obtain a large quantity of crude enzyme solution.

[0059] (1) Nickel column purification

[0060] After assembling the gravity column purification apparatus, rinse the column with pure water and then use elution buffer to remove any residual protein from the previous rinse. Rinse the column again with pure water to remove the elution buffer, and then equilibrate the column. Incubate the crude enzyme solution in the packing material for 30 min. Add the flow-through buffer to the column again and incubate for 10 min. Wash away any impurities with buffer and then elute the target protein with elution buffer to complete the initial protein purification.

[0061] (2) Further purification by nickel column

[0062] The purified protein solution obtained after initial purification using a nickel column was concentrated using an ultrafiltration tube to approximately 2 mL, and then injected onto an Akta gel. A linear gradient elution program was used, and the protein was collected based on the UV detection signal. Finally, the molecular weight and purity of the target protein were determined by 10% SDS-PAGE electrophoresis. Figure 2 As shown (M: protein marker; Lane 1: UGT70 purified protein), this indicates that the protein was successfully expressed and the purified protein can be used for subsequent experiments.

[0063] Example 3 In vitro enzyme activity detection

[0064] 1. Enzyme activity reaction system: Using isoquercitrin as the substrate and UDP-rhamnose (UDP-Rha) as the sugar donor, in vitro enzyme activity verification was performed. The reaction system consisted of 10 μg purified protein, 0.5 mM substrate, 5 mM UDP-Rha, 14 mM β-mercaptoethanol, and Tris-HCl buffer (pH 7.5) to a final volume of 200 μL. The reaction was carried out at 37°C for 12 h. After the reaction, 200 μL of pre-cooled methanol was added to terminate the reaction. The mixture was centrifuged at 14,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm microporous membrane. The samples were analyzed by HPLC and UPLC-MS.

[0065] 2. HPLC conditions

[0066] Chromatographic column: Waters ACQUITY UPLC HSS T3 column, 1.8 µm, 2.1 x 100 mm.

[0067] Mobile phase A was 1% (v / v) formic acid in water, mobile phase B was acetonitrile, the mobile phase flow rate was 0.8 mL / min, the injection volume was 20 μL, the column temperature was 30℃, and the gradient elution system was as follows:

[0068] Table 2 HPLC gradient elution conditions

[0069]

[0070] 3. LC-MS conditions:

[0071] The detection mode is negative ion mode; the mobile phase composition is consistent with HPLC; the detection source is an electrospray ionization source; and the acquisition mode is AutoMS. 2 Capillary voltage 3500 V; sheath gas temperature 350℃, flow rate 11 L / min; dry gas temperature 325℃, flow rate 8 L / min; mass-to-charge ratio scan range 100-1700 m / z; collision voltage 175 V; collision energy 15-50 eV.

[0072] 4. Test Results:

[0073] See results Figure 3 In the enzymatic reaction using isoquercitrin as a substrate, UGT70 was used to compare the retention times of the standard and the reaction solution. Figure 3 ), and the mass spectrum of the catalytic product ( Figure 4 They discovered that the product of the enzymatic reaction was rutin.

[0074] Example 4 Enzymatic properties of UGT70 catalyzing the conversion of isoquercitrin to rutin

[0075] Using isoquercitrin as a glycosyl acceptor and UDP-Rha as a glycosyl donor, the effects of pH, temperature, metal ions, and reaction time on catalytic activity were investigated.

[0076] 1. pH conditions

[0077] The total reaction volume was 100 μL, containing 10 μg purified protein, 0.5 mM isoquercitrin, and 5 mM UDP-Rha. The volume was replenished to 100 μL with the appropriate buffer solutions, and the reaction was carried out at 37 °C for 8 min. The buffer solutions included: Citric acid-sodium citrate buffer (pH 5.0–6.0); Na₂HPO₄-NaH₂PO₄ buffer (pH 6.0–9.0); Tris-HCl buffer (pH 7.0–9.0); and Na₂CO₃-NaHCO₃ buffer (pH 9.0–10.0). Immediately after the reaction, an equal volume of pre-cooled methanol was added to terminate the reaction. After centrifugation at 14,000 rpm at 4 °C for 5 min, the supernatant was collected, filtered, and analyzed by HPLC. To ensure data reliability, three replicates were performed for each reaction. The final conversion rate was calculated from the peak area of ​​the chromatogram.

[0078] 2. Temperature conditions

[0079] The reaction system contained 10 μg of purified protein, 0.5 mM isoquercitrin, and 5 mM UDP-Rha, supplemented to 100 μL with 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 8.0), and reacted for 8 min. The reaction solution was reacted at various temperatures (4℃, 20℃, 30℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 8 min each. Immediately after the reaction, an equal volume of pre-cooled methanol was added to terminate the reaction. After centrifugation at 14,000 rpm at 4℃ for 5 min, the supernatant was collected, filtered, and analyzed by HPLC. To ensure data reliability, three replicates were set up for each reaction. The final conversion rate was calculated from the peak area of ​​the chromatogram.

[0080] 3. Metal ion conditions

[0081] Investigating different divalent metal ions (Ca 2+ Mn 2+ Ba 2+ Co 2+ Zn 2+ Mg 2+ Cu 2+The effect of EDTA on the catalytic efficiency of the reaction was investigated. The reaction system contained 10 μg of purified protein, 0.5 mM isoquercitrin, 5 mM UDP-Rha, and 5 mM divalent metal ions, and was supplemented to 100 μL with 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8.0). The control reaction system did not contain divalent metal ions. The reaction solution was placed at 37 ℃ for 8 min. After the reaction was completed, an equal volume of pre-cooled methanol was added to the reaction system to terminate the reaction. After centrifugation at 14,000 rpm and 4 ℃ for 5 min, the supernatant was collected, filtered, and analyzed by HPLC. To ensure the reliability of the data, three replicate samples were set up for each reaction. The final conversion rate was calculated by the peak area of ​​the chromatogram.

[0082] 4. Enzyme kinetic parameters

[0083] The total volume of the enzyme activation reaction system was 100 μL. 100 μL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (50 mM, pH=9.0) contained: 10 μg of purified protein, different concentrations of isoquercitrin, and 5 mM UDP-Rha. The reaction temperature was 50 ℃. After the reaction was completed in 40 min, an equal volume of pre-cooled methanol was immediately added to the reaction system to terminate the reaction. After centrifugation at 14,000 rpm and 4 ℃ for 5 min, the supernatant was collected, filtered, and analyzed by HPLC. To ensure the reliability of the data, three replicate samples were set up for each reaction. The conversion rate was calculated by the peak area of ​​the chromatogram, and the Michaelis constant was obtained by calculating the Michaelis equation.

[0084] 5. Test Results

[0085] The results showed that ( Figure 5 The optimal pH for the enzymatic reaction is 9.0, and it is suitable for reaction within a pH range of 5.0–10.0. Its catalytic activity in Na₂HPO₄-NaH₂PO₄ buffer is higher than in other buffers. UGT70 exhibits high conversion rates within the temperature range of 20–55℃. Within the temperature range of 4–50℃, its catalytic activity gradually increases with increasing temperature; above 50℃, its catalytic activity gradually decreases with increasing temperature. This enzyme is not metal ion dependent; among divalent metal ions, Mg… 2+ Ba 2 + Both Mg and EDTA can increase enzyme activity, among which Mg 2+ The best promoting effect was achieved by using UDP-Rha as the glycosyl donor at a constant concentration of 5 mM. Enzyme kinetics of isoquercitrin were investigated with different substrate concentrations, and the product was determined to be rutin. By fitting the Michaelis-Menten curve, the results showed that the Km of UGT70 for isoquercitrin was 72.57 μM.

[0086] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of a glycosyltransferase or a gene encoding said glycosyltransferase or an expression cassette containing said gene, a recombinant vector, or a recombinant cell in the synthesis of flavonoid disaccharide compounds, characterized in that, The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.2; the flavonoid disaccharide compound is rutin, and the synthesis of the flavonoid disaccharide compound uses isoquercitrin as a glycosyl acceptor and UDP-rhamnose as a glycosyl donor to synthesize rutin.

2. A method for synthesizing flavonoid disaccharide compounds, characterized in that, The method includes the following steps: 1) Obtain a glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO.2; 2) The glycosyltransferase from step 1) is used to catalyze the synthesis of flavonoid disaccharide compounds in an enzyme-active reaction system; The flavonoid disaccharide compound is rutin, and the synthesis of the flavonoid disaccharide compound uses isoquercitrin as a glycosyl acceptor and UDP-rhamnose as a glycosyl donor to synthesize rutin.

3. The method for synthesizing flavonoid disaccharide compounds as described in claim 2, characterized in that, In step 1), the glycosyltransferase is obtained through microbial synthesis or chemical synthesis.

4. The method for synthesizing flavonoid disaccharide compounds as described in claim 2, characterized in that, The enzyme activation reaction system in step 2) also contains Mg. 2+ Ba 2+ And one or more of EDTA.

5. The method for synthesizing flavonoid disaccharide compounds as described in claim 2, characterized in that, The reaction temperature of the enzyme activation reaction system in step 2) is 4-60℃.

6. The method for synthesizing flavonoid disaccharide compounds as described in claim 2, characterized in that, The pH of the enzyme activation reaction system in step 2) is 5.0-10.0.

Images