CN116445442A - UDP-glycosyltransferase and application thereof in synthesis of alpha-hederacoside glycoside compound - Google Patents
UDP-glycosyltransferase and application thereof in synthesis of alpha-hederacoside glycoside compound Download PDFInfo
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- CN116445442A CN116445442A CN202310024443.3A CN202310024443A CN116445442A CN 116445442 A CN116445442 A CN 116445442A CN 202310024443 A CN202310024443 A CN 202310024443A CN 116445442 A CN116445442 A CN 116445442A
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- CN
- China
- Prior art keywords
- alpha
- udp
- hederacoside
- glycosyltransferase
- rhamnosyl
- Prior art date
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Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01017—Glucuronosyltransferase (2.4.1.17)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to an artificially calculated UDP-glycosyltransferase UGT91H_A1 gene and a protein encoded by the same. According to the invention, through performing ancestral enzyme sequence reconstruction on UDP-glycosyltransferase UGT91H subfamily, a novel UDP-glycosyltransferase gene UGT91H_A1 and encoded protein thereof are obtained through calculation, heterologous expression is successfully performed in escherichia coli cells, the function of catalyzing and synthesizing hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose, hederagenin 3-O-beta-D xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose, hederagenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose is verified, and meanwhile, a sucrose synthase GuSUS 1-delta 9, UDP-Rha synthase AtM 2, a bifunctional Rh Li Tangge enzyme AtNRS/transferase and UDP-glycosyl UGT 1_UGT 91H are coupled, so that a system can be regenerated and a system can be constructed. The invention fills the blank of 2' -OH glycosylation research on the pentacyclic triterpene alpha-hederacoside C-3 parent nucleus, provides reference and basis for the subsequent related research on synthesizing novel alpha-hederacoside glycoside compounds with potential clinical value, and simultaneously the constructed UDP-Rha cyclic regeneration system also greatly reduces the preparation cost of hederacoside 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose.
Description
Technical field:
the invention relates to UDP-glycosyltransferase obtained by manual calculation and a coding gene, and also relates to a method for synthesizing various alpha-hederacoside derivatives by coupling the UDP-glycosyltransferase with sucrose synthase from liquorice, UDP-rhamnose (UDP-Rha) synthase from Arabidopsis thaliana and bifunctional mouse Li Tangge enzyme, belonging to the field of bioengineering and technology.
The background technology is as follows:
alpha-hederagenin is a pentacyclic triterpene compound derived from plants such as hedera helix, fructus akebiae, pulsatillae radix and the like, and is a candidate molecule as a clinical drug because of its pharmacological activity of inducing apoptosis, interfering glycolysis of cells, inhibiting cell growth and the like in the treatment of various cancers such as liver cancer, lung cancer, gastric cancer, colorectal cancer, esophageal cancer and the like. However, the strong hydrophobicity of the carbon skeleton of α -hederacoside greatly affects its water solubility and bioavailability, limiting further popularization and application of α -hederacoside. Therefore, strategies for developing novel alpha-hederacoside glycosyl derivative medicines with low toxicity and high drug effect by taking alpha-hederacoside as a precursor are paid attention to. Glycosyl modification is an important means for modifying natural products, and by introducing glycosyl into a specific site, the hydrophobic part of the glycosyl has more hydrophilic carboxyl and hydroxyl, so that more effective glycoside derivatives are obtained, and the glycosyl becomes a research hot spot in recent years.
Compared with the chemical synthesis method for realizing the alpha-hederacoside, the method has the advantages of complex reaction steps, harsh reaction conditions, various toxic and harmful chemical reagents involved and the like, and the biological enzyme method has the advantages of strong substrate specificity, mild reaction conditions, environmental friendliness and the like. UDP-Glycosyltransferase (UGT) is used as a high-efficiency biocatalyst and can catalyze a plurality of natural product glycosyl modifications. However, there is no report currently on UGTs that can catalyze glycosyl modification at the 2"-OH site of α -hederacoside. Therefore, the excavation has important significance for UGT with catalytic activity of alpha-hederacoside.
On the other hand, the UDP-glycosyl donor required for glycosyltransferase catalytic substrates is generally expensive, greatly limiting its large-scale industrial production in the synthesis of pentacyclic triterpene glycoside derivatives. The coupling sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase construct a UDP cyclic regeneration system, so that the continuous supply of UDP-Rha driven by using low-cost and easily obtained sucrose as an initial glycosyl donor can be realized, and the production cost of rhamnose glycoside derivatives is greatly reduced. Similar research work has been increasingly attracting attention, but no research on synthesizing alpha-hederacoside derivatives by using a UDP cycle regeneration system has been reported.
Therefore, searching for a suitable UGT to realize the glycosyl modification of the alpha-hederacoside greatly enriches the molecular structure of the compound, and simultaneously utilizes a multienzyme system coupled with sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase to catalyze the alpha-hederacoside to generate the Li Tangtang-glycosylated derivative of the alpha-hederacoside, thereby being beneficial to promoting the large-scale industrial production of the alpha-hederacoside compound.
The invention comprises the following steps:
the invention aims to computationally synthesize and characterize a novel UDP-glycosyltransferase which can catalyze 2' -OH multiple glycosyl modifications of alpha-hederacoside, and simultaneously provides a UDP-Rha cyclic regeneration system coupled with sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and UDP-glycosyltransferase for synthesizing alpha-hederacoside rhamnoside compounds at low cost.
In a first aspect, the invention provides a UDP-glycosyltransferase UGT91H_A1 synthesized by calculation, and the amino acid sequence of the UDP-glycosyltransferase UGT91H_A1 is shown in SEQ ID No.1 in a sequence table; a gene for coding UDP-glycosyltransferase UGT91H_A1 is a nucleotide sequence shown in SEQ ID No.2 in a sequence table.
In a second aspect, the invention provides a UDP-glycosyltransferase ugt91h_a1 for use in the synthesis of an α -hederagenin Li Tangtang-glycosylated derivative: hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose (structure shown as formula I), and alpha-hederacoside xylose glycosylated derivative: hederagenin 3-O-beta-D xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose (structure shown as formula II), and alpha-hederagenin glucosylated derivative: hederagenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose (structure is shown as formula III).
In a third aspect, the present invention provides an in vitro enzyme catalytic system coupled to sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase for synthesizing hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose (structure shown in formula one) at low cost.
Description of the drawings:
FIG. 1 is a SDS-PAGE map of sucrose synthase GuSUS1- Δ9, UDP-mouse Li Tangge enzyme AtRHM2, bifunctional mouse Li Tangge enzyme AtNRS/ER, UGT91H_A1 protein expression in examples 3 and 6 of the present invention. Lanes from left to right in the figure are protein Marker (M), guSUS1- Δ9 (1), atRHM2 (2), atNRS/ER (3), UGT91H_A1 (4), respectively.
FIG. 2 is a high performance liquid chromatogram of glycosyltransferase UGT91H_A1 in example 4 of the invention for catalyzing alpha-hederacoside to generate hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose, 3-O-beta-D-xylose (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose and 3-O-beta-D-grape-yl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose respectively. In the diagram a: an alpha-hederagenin standard; UGT167H2_A1 catalyzes the reaction product of alpha-hederacoside and UDP-Rha; UGT167H_A1 catalyzes the reaction product of alpha-hederacoside and UDP-xylose (UDP-Xyl); UGT167H_A1 catalyzes the reaction product of alpha-hederacoside and UDP-glucose (UDP-Glc).
FIG. 3 is a schematic diagram of the process of the multi-enzyme catalytic system of example 5 of the present invention coupled with sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase for synthesizing a-hederagenin-rat Li Tangtang-glycosylated derivatives.
FIG. 4 is a high performance liquid chromatogram of an in vitro enzyme catalytic system coupled with sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase in example 5 of the present invention. A alpha-hederagenin standard substance in the figure A; and B, coupling a reaction product of the catalytic system.
FIG. 5 shows the result of HPLC-MS analysis of hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6.
FIG. 6 shows the result of HPLC-MS analysis of 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the present invention.
FIG. 7 shows the result of HPLC-MS analysis of 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the present invention.
FIG. 8 shows the synthesis of Chun-tenninol 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the present invention 1 H spectrum.
FIG. 9 is a diagram of the synthesis of Chun-tenninol 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the present invention 13 C spectrogram.
FIG. 10 is a DEPT-135 spectrum of the Chun Tenggenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 11 is a HSQC spectrum of Chun Tenggenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 12 is a HMBC pattern of Chun-tengenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 13 is a diagram of the synthesis of Chun Tenggenin 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the invention 1 H spectrum.
FIG. 14 shows the synthesis of Chun-tenninol 3-O-beta-D-xylosyl (1-2) -alpha-L-mouse according to example 6 of the inventionGlycosyl (1-2) -alpha-L-arabinose 13 C spectrogram.
FIG. 15 is a DEPT-135 spectrum of the Chun Tenggenin 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 16 is a HSQC spectrum of Chun Tenggenin 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 17 is a HMBC pattern of the synthetic Chun Teng aglycone 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the present invention.
FIG. 18 is a diagram showing the synthesis of Chun-tenninol 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the present invention 1 H spectrum.
FIG. 19 is a diagram showing the synthesis of Chun-tenninol 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose according to example 6 of the present invention 13 C spectrogram.
FIG. 20 is a DEPT-135 spectrum of Chun Tenggenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 21 is a HSQC spectrum of Chun Tenggenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
FIG. 22 is a HMBC pattern of Chun-tengenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose synthesized in example 6 of the invention.
The specific embodiment is as follows:
the following describes the embodiments of the present invention in further detail with reference to examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1: acquisition of glycosyltransferase UGT91H_A1, sucrose synthase GuSuS1- Δ9, UDP-Rha synthase AtRHM2 and bifunctional murine Li Tangge enzyme AtNRS/ER
A. Acquisition of glycosyltransferase UGT91H_A1 Gene
UGT91H subfamily protein sequences are obtained according to NCBI database (https:// www.ncbi.nlm.nih.gov), UGT91H_A1 protein sequences are obtained by utilizing ancestral enzyme sequence reconstruction analysis, and the glycosyltransferase UGT91H_A1 gene fragments after chemical synthesis optimization are subjected to codon optimization, and are shown as SEQ ID No. 2. The amino acid sequence of the fragment expression protein shown in SEQ ID No.2 is shown in SEQ ID No. 1.
B. Acquisition of sucrose synthase GuSuS1- Δ9, UDP-Rha synthase AtRHM2, bifunctional murine Li Tangge enzyme AtNRS/ER Gene
According to the Gene sequences of sucrose synthase GuSuS 1-delta 9, UDP-Rha synthase AtRHM2 (NCBI Gene ID: 841785) and bifunctional mouse Li Tangge enzyme AtNRS/ER (NCBI Gene ID: 842603), the optimized sucrose synthase GuSuS 1-delta 9 Gene fragment is chemically synthesized through codon optimization, as shown in SEQ ID No.4, and the UDP-Rha synthase AtRHM2 Gene fragment is shown in SEQ ID No.6 and the AtNRS/ER Gene fragment is shown in SEQ ID No. 8. The amino acid sequence expressed by the fragment shown in SEQ ID No.4 is shown in SEQ ID No.3, the amino acid sequence expressed by the fragment shown in SEQ ID No.6 is shown in SEQ ID No.5, and the amino acid sequence expressed by the fragment shown in SEQ ID No.8 is shown in SEQ ID No. 7.
Example 2: construction of E.coli engineering bacteria expressing glycosyltransferase UGT91H_A1, sucrose synthase GuSuS 1-delta 9, UDP-Rha synthase AtRHM2, and bifunctional mouse Li Tangge enzyme AtNRS/ER
To construct E.coli engineering bacteria expressing glycosyltransferase UGT91H_A1, sucrose synthase GuSuS1-delta 9, UDP-Rha synthase AtRHM2 and bifunctional mouse Li Tangge enzyme AtNRS/ER genes, serial primers were designed for PCR amplification to obtain glycosyltransferase UGT91H_A1 gene fragment (shown as SEQ ID No. 2), sucrose synthase GuSuS1-delta 9 gene fragment (shown as SEQ ID No. 4), UDP-Rha synthase AtRHM2 gene fragment (shown as SEQ ID No. 6), bifunctional mouse Li Tangge enzyme AtNRS/ER gene fragment (shown as SEQ ID No. 8) and enzyme cleavage sites BamHI, xhoI and protective bases.
The PCR (polymerase chain reaction) reaction system is as follows: 1. Mu.L of template, 2. Mu.L of upstream and downstream primers, 25. Mu.L of PCR polymerase, and 50. Mu.L of double distilled water were used. PCR reaction conditions: pre-denaturation at 98℃for 1min, denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 2min for 30s, circulation for 30 times, 10min at 72℃and preservation at 4 ℃.
The cloned glycosyltransferase UGT91H_A1 gene, sucrose synthase GuSuS1-delta 9 gene, UDP-Rha synthase AtRHM2 gene and bifunctional mouse Li Tangge enzyme AtNRS/ER gene were purified and recovered by using agarose gel DNA recovery kit (Thermo company). The prokaryotic expression vector pET28a is subjected to double digestion by restriction enzymes BamHI and XhoI, and the digestion system is as follows: bamHI 2. Mu.L and XhoI 2. Mu.L, 10 Xdigestion buffer 5. Mu.L, and 30. Mu.L of DNA fragment were used to make up 50. Mu.L of the cleavage system with double distilled water at 37℃for 2h. After cleavage, the cleavage product was recovered using an agarose gel DNA recovery kit.
The Gibson assembly method is used for respectively carrying out seamless connection on glycosyltransferase UGT91H_A1 gene, sucrose synthase GuSuS 1-delta 9 gene, UDP-Rha synthase AtRHM2 gene and bifunctional mouse Li Tangge enzyme AtNRS/ER gene with a linearized vector pET28 a. The ligation product was transformed into competent cells of E.coli BL21 (DE 3), plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L,20g/L agarose) containing 100mg/L kanamycin, and cultured overnight at 37 ℃.
Transformants were identified by colony PCR and sequencing methods. Colony PCR system: the template LB plate was single-colony, 1. Mu.L of each of the upstream and downstream primers, 10. Mu.L of 2 XTaq mix (Beijing Polymer Biotechnology Co., ltd.) and 20. Mu.L of each of the upstream and downstream primers was supplemented with double distilled water. PCR conditions: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 2min for 30s, circulation for 30 times, 10min at 72℃and preservation at 4 ℃. The transformants containing the correct target bands were confirmed by colony PCR for DNA sequencing (Jin Weizhi Biotechnology Co., st. Of Suzhou) and successful transformation of the recombinant plasmids pET28a-UGT91H_A1, pET28a-GuSuS1- Δ9, pET28a-AtRHM2, pET28a-AtNRS/ER into E.coli BL21 (DE 3) was determined.
Example 3: fermentation of E.coli genetically engineered bacteria and purification of glycosyltransferase
A. Fermentation of genetically engineered escherichia coli
(1) The E.coli engineering bacteria with correct identification were picked up and inoculated in 50mL of LB liquid medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L) containing 100mg/L kanamycin, and cultured overnight at 37℃and 200rpm.
(2) Taking overnight cultureInoculating 4mL of cultured bacterial liquid into 400mL of LB liquid medium containing 100mg/L kanamycin, and culturing for 2-3h to OD 600 =0.6, culture conditions 37 ℃,200rpm.
(3) The inducer IPTG was added to a final concentration of 0.1mM,16℃and incubated overnight at 200rpm.
(4) After the obtained fermentation broth was centrifuged at 9000rpm for 3min, the cells were collected.
(5) The cells were resuspended in 15ml PBS buffer (50 mM, pH 7.0) and lysed using a low temperature high pressure cell disrupter.
(6) The lysed cells were centrifuged at 12000g for 10min at 4℃and the pellet was discarded and the supernatant was collected to obtain a crude enzyme solution containing sucrose synthase.
B. Purification of enzymes
Protein purification was performed using the protein purification system AKTA purifier.
(1) Sample pretreatment: the crude enzyme solution obtained was filtered through a 0.45 μm pore size filter and stored at 4 ℃.
(2) Nickel column pretreatment: the equilibrated nickel column was rinsed at a flow rate of 1mL/min with no less than 10 column volumes of binding buffer (25 mM imidazole, 50mM PBS, pH 7.0).
(3) Loading: the sample was pumped into the nickel column by a constant flow pump at a flow rate of 0.5 mL/min.
(4) Gradient elution: eluting nickel column according to different gradient ratio of eluting buffer (1M imidazole) and binding buffer, with flow rate of 1mL/min, and observing and monitoring OD 280 The value, the eluent corresponding to the protein absorption peak is collected and the target protein is determined after detection by SDS-PAGE.
(5) Protein concentration and concentration determination: proteins were concentrated using ultrafiltration tubes, protein concentration was measured using Nanodrop 2000, and molecular weight of target proteins was determined by SDS-PAGE.
Example 4: characterization of glycosyltransferase ugt169h_a1 catalytic alpha-hederagenin glycosyl modification
Mu.g of the purified enzyme UGT 91H-A1 of example 3 (50 mM PBS buffer, pH 7.0), 1mM UDP-sugar (UDP-Rha, UDP-Xyl or UDP-Glc) was taken and 100. Mu.L of the system was reacted in a metal bath at 35℃for 5 hours, followed by adding 400. Mu.L of methanol to terminate the reaction. The reaction products were detected using a Shimadzu LC-30A ultra high performance liquid chromatograph UHPLC system, column model is Shim-pack GIST-HP C18 (2.1X 150,3 μm). The UHPLC conditions are as follows: mobile phase a: acetonitrile (ACN), mobile phase B: 1%phosphoric acid; 0min,20% ACN;3min,35% ACN;4.0min,50% ACN;8.0min,65% ACN;12.0min,85% ACN;15.0min,95% ACN;16.0min,20% ACN;20min,80% ACN, detection wavelength 203nm.
Example 5: UDP-Rha regeneration System coupled with sucrose synthase, UDP-Rha synthase, bifunctional rhamnose synthase and glycosyltransferase
Alpha-hederacoside is used as a substrate, sucrose synthase GuSuS 1-delta 9, UDP-Rha synthase AtRHM2, bifunctional mouse Li Tangge enzyme AtNRS/ER and glycosyltransferase UGT91H_A1 are added, and Uridine Diphosphate (UDP) and Nicotinamide Adenine Dinucleotide (NAD) are added + ) Sucrose and carrying out water bath reaction for 5 hours at 35 ℃ to realize 2' -OH rhamnosyl modification of alpha-hederacoside and synthesize hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose.
Specifically, 200 μl of the reaction system was prepared: guSuS1- Δ9 pure enzyme solution (final concentration 1.5 mg/mL), atRHM2 pure enzyme solution (final concentration 3 mg/mL), atNRS/ER pure enzyme solution (final concentration 3 mg/mL), UGT91H_A1 pure enzyme solution (final concentration 1 mg/mL), sucrose (6 mM), UDP (0.6 mM), NAD + (1.2 mM), alpha-hederagenin (200. Mu.M) and PBS buffer (50 mM, pH 7.0). Placing in a 35 ℃ water bath for reaction for 5 hours, adding 300 mu L of methanol for uniform mixing, centrifuging at 12000rpm for 10 minutes to remove sediment, and detecting a sample by using a UHPLC.
Example 6: preparation and structural characterization of glycosylated derivatives
(1) A, preparation of hederagenin 3-O-alpha-L-rhamnosyl (1-2) -alpha-L-arabinose
The method comprises the following steps: following the procedure of example 4, 20. Mu.g of the enzyme UGT 91H-A1 purified in example 3, 1mM UDP-Rha and 200. Mu.M alpha. -hederacoside were added to 100. Mu.L of the reaction system (50 mM PBS buffer, pH 7.0), 200 identical reaction systems were reacted in a metal bath at 35℃for 5 hours, and then the reaction system was added to 10mL of methanol to terminate the reaction.
The second method is as follows: the procedure of example 5 is followed, firstly, by the addition of the sucrose synthase GuSuS1-Δ9, UDP-Rha synthase AtRHM2, bifunctional murine Li Tangge enzyme AtNRS/ER and glycosyltransferase UGT91H_A1, sucrose (6 mM), UDP (0.6 mM), NAD were added to the mixed enzyme solution + (1.2 mM), alpha-hederacoside (200 mu M) (added in multiple times, UHPLC detection is sampled every 2h after the reaction, the conversion rate of the alpha-hederacoside is calculated, and if the alpha-hederacoside is completely converted, the alpha-hederacoside can be supplemented), and the reaction is carried out for 10h by a water bath shaking table at 35 ℃ and 170 rpm.
B. Preparation of hederagenin 3-O-beta-D-xylosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose with hederagenin 3-O-beta-D-glucosyl (1-2) -alpha-L-rhamnosyl (1-2) -alpha-L-arabinose: following the procedure of example 4, 20. Mu.g of the enzyme UGT 91H-A1 purified in example 3, 1mM UDP-Xyl or UDP-Glc and 200. Mu.M alpha. -hederagenin were added to 100. Mu.L of the reaction system (50 mM PBS buffer, pH 7.0), 200 identical reaction systems were reacted in a metal bath at 35℃for 5 hours, and then the reaction system was added to 10mL of methanol to terminate the reaction.
(2) The reaction product was centrifuged at 15000rpm for 10min, the pellet was resuspended well with 10mL of methanol by vortexing and filtered using a 0.22 μm pore size organic filter membrane to make a sample.
(3) The product was isolated and purified using the semi-preparative liquid phase as follows:
mobile phase ratio, A: acetonitrile, B: 1%formic acid aqueous solution, A: B=65:35; the mobile phase solution is filtered through a 0.22 mu m pore size filter membrane and degassed by ultrasonic vibration. The flow rate was 3mL/min and the detection wavelength was 203nm. The liquid chromatography column was a Shimadzu C18 silica gel column (20X 250mm,5 μm). Taking 2mL of the sample obtained in the step 1, separating by a machine, collecting liquid from each peak in UV rays, and detecting by a UHPLC (ultra high Performance liquid chromatography) to judge the retention time corresponding to different alpha-hederacoside glycosylated derivatives and judge the purity. After all the prepared samples were collected, they were concentrated and dried to a powder using a vacuum concentrator and stored at 4 ℃.
(4) Three alpha-hederacoside glycosylated derivative powders are dissolved in methanol, high performance liquid chromatography-mass spectrometry is used for molecular weight identification, and simultaneously, the product is dissolved in deuterated methanol and is carried out by a Bruker Assetnd 700M nuclear magnetic resonance spectrometer 1 H spectrum, 13 C spectrum, DEPT-135 spectrum, HSQC spectrum, HMBC spectrum analysis, and determinationAnd (5) fixing structure information.
Claims (4)
1. The UDP-glycosyltransferase UGT167H_A1 is characterized in that the amino acid sequence of the UDP-glycosyltransferase UGT167H_A1 is shown in SEQ ID NO. 1.
2. The UDP-glycosyltransferase of claim 1, wherein the nucleotide sequence of the gene encoding UDP-glycosyltransferase UGT91H_A1 is shown in SEQ ID NO. 2.
3. Use of UDP-glycosyltransferase ugt167h_a1 according to claim 1 for the synthesis of an α -hederacoside compound, characterized in that the α -hederacoside rhamnoside compound has the structure shown in formula 1, the α -hederacoside xyloside compound has the structure shown in formula 2 and the α -hederacoside glucoside compound has the structure shown in formula 3.
4. Use according to claim 3, characterized in that the reaction system for the preparation of the alpha-hederacoside rhamnoside compound (structure represented by formula 1) comprises sucrose synthase, UDP-glycosyltransferase, UDP-rhamnose synthase, bifunctional rhamnose synthase, sucrose, uridine diphosphate, nicotinamide adenine dinucleotide and alpha-hederacoside.
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