Method for synthesizing schaftoside or isoschaftoside

Abstract

The present invention discloses a method for synthesizing schaftoside or isoschaftoside, wherein the method uses a broad-leaved schizonepeta carbon glycosyltransferase with an amino acid sequence such as SEQ ID NO:3 or SEQ ID NO:4 as a catalyst, catalyzes the reaction of 2-hydroxynaringenin and UDP-Arabinose, and then continues to react with UDP-glucose; or catalyzes the reaction of 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose. The present invention finds that only one C-glycosyltransferase is needed to biosynthesize schaftoside or isoschaftoside, which is simple and feasible, reduces energy consumption, and is beneficial to the large-scale utilization of schaftoside or isoschaftoside in medicine, agriculture and food industries.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and more specifically, the invention relates to a method for synthesizing schaftoside or isoschaftoside by utilizing Desmodium sibiricum carbonyl transferase. Background Art

[0002] Schaftoside and isoschaftoside are isomers of a pair of flavonoid sugar two-carbon glycosides, and these two compounds are widely distributed in higher plants. It is reported that schaftoside and isoschaftoside exist in at least 39 families and 184 plant species, of which 82.6% are euphyllophytes, 15.2% are monocots, 1.1% are ferns, and 1.1% are mosses. The largest number of schaftosides is found in Passiflora, Leguminosae, and Asteraceae, followed by Poaceae and Araceae.

[0003] Mice fed with 160 mg / kg of schaftoside from Desmodium truncatum can significantly prevent gallstone formation [Liu MJ, et al. 2017]. Recently, researchers have found that schaftoside can also inhibit papain (PL pro ) and the main protease of SARS-CoV-2 (3CL pro ), thereby inhibiting the new coronavirus with dual targets; in cell experiments (VeroE6), shafotaxime glycosides can be used without significant toxicity (CC 50 >200μM) to inhibit the new coronavirus, and its EC 50 The concentration of 10 μmol / L in schaftoside was 11.83 μmol / L. The researchers used hydrogen deuterium exchange mass spectrometry and quantum mechanics to clarify the binding mechanism between schaftoside and the new coronavirus. Schaftoside has significant anti-inflammatory activity in the acute pneumonia mouse model, inhibiting the inflammatory factors IL-1β, IL-6 and TNF-α in plasma and lung tissue, and significantly alleviating the lung damage of mice. Proteomics studies have shown that schaftoside also exhibits immunomodulation. Based on these research results, schaftoside may be used to treat new coronavirus infection. Flavonoid sugar diglycoside compounds such as isoschaftoside secreted by the roots of plants of the genus Delicata can inhibit the growth of parasitic seed embryos, thereby effectively preventing and controlling the damage of single-legged metal parasitic weeds to crops [Khan ZR, et al. 2010]. In addition, schaftoside and isoschaftoside are important plant defense compounds, and their contents are related to the drought resistance of wheat leaves and the UV resistance of rice seeds [GallandM, et al. 2014]. After brown planthoppers infect rice, the content of (iso)schaftoside in the leaves increases significantly [UawisetwathanaU, et al. 2019]. Within 6 hours and 24 hours after the rice blast fungus invades rice, the content of (iso)schaftoside increases by about 40% [Wang ZL, et al. 2020].

[0004] In 2020, researchers discovered that the biosynthesis of (iso)schaftoside in plants involves a previously unreported di-C-glycosylation pathway, that is, the biosynthesis of (iso)schaftoside is catalyzed by two C-glycosyltransferases (CGTs) in sequence, 2-hydroxyflavanone C-glycosyltransferase (CGTa) catalyzes the C-glucosylation of 2-hydroxynaringenin, followed by C-glucose-2-hydroxyflavanone C-glycosyltransferase (CGTb) C-arabinosylation to produce (iso)schaftoside [Wang ZL, et al. 2020]. In 2021, researchers achieved the preparation of (iso)schaftoside in Escherichia coli by introducing specific CGTa and CGTb (PhUGT708A43 and OsUGT708A1) [Chen Z, et al. 2021]. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a method for synthesizing schaftoside or isoschaftoside, which method only requires one carbon glycosyltransferase CGT to achieve the biosynthesis of schaftoside or isoschaftoside.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, a method for synthesizing schaftoside or isoschaaftoside is provided, comprising the following steps: using Desmodium sibiricum carbon glycosyltransferase as a catalyst to catalyze the reaction between 2-hydroxynaringenin and UDP-Arabinose, and then adding UDP-glucose to continue the reaction; the amino acid sequence of the Desmodium sibiricum carbon glycosyltransferase is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] In a second aspect of the present invention, a method for synthesizing schaftoside or isoschaaftoside is provided, comprising the following steps: using Desmodium sibiricum carbon glycosyltransferase as a catalyst to catalyze the reaction of 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose; the amino acid sequence of the Desmodium sibiricum carbon glycosyltransferase is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0009] The present invention has the following beneficial effects:

[0010] The inventors of the present invention found during the performance analysis of C-glycosyltransferase that C-glycosyltransferase GsCGT1 or GsCGT2 can catalyze the arabinosylation of 2-hydroxynaringenin and UDP-Arabinose to generate C-arabinose-2-hydroxynaringenin, and then continue to glucosylate with UDP-glucose to generate di-C-glucose-arabinose-2-hydroxynaringenin (i.e., two-step carbon glycosylation occurs continuously); C-glycosyltransferase GsCGT1 or GsCGT2 can also catalyze 2-hydroxynaringenin with UDP-Arabinose and UDP-glucose to directly generate di-C-glucose-arabinose-2-hydroxynaringenin; di-C-glucose-arabinose-2-hydroxynaringenin is an important precursor compound of schaftoside or isoschaftoside, and can generate schaftoside or isoschaftoside after in vitro hydrochloric acid dehydration treatment.

[0011] Compared with the existing method that requires the serial use of two enzymes, C-glucose-2-hydroxyflavanone C-glycosyltransferase (CGTa) and C-arabinose-2-hydroxyflavanone C-glycosyltransferase (CGTb), to catalyze 2-hydroxynaringenin in order to synthesize schaftoside or isoschaftoside, the present invention only needs to use one C-glycosyltransferase to biosynthesize schaftoside or isoschaftoside, and has the characteristics of reduced energy consumption and environmental friendliness; it provides a simple and feasible method for the future high-efficiency system production of schaftoside or isoschaftoside by microorganisms (Escherichia coli, yeast) or plants (tomato, tobacco), thereby facilitating the large-scale utilization of schaftoside or isoschaftoside in the medicine, agriculture and food industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The present invention provides a roadmap for the current biosynthesis of schaftoside and the biosynthesis of schaftoside of the present invention.

[0013] Figure 2 This is the spectrum of the catalytic product of GsCGT6 detected by HPLC in Example 3 of the present invention.

[0014] Figure 3 This is the spectrum of the catalytic product of GsCGT4 detected by HPLC in Example 3 of the present invention.

[0015] in, Figure 2 and Figure 3 In the figure, a: di-C-glucose-2-hydroxynaringenin; b: di-C-glucose-C arabinose-2-hydroxynaringenin; c: di-C-arabinose-2-hydroxynaringenin; d: mono-C-glucose-2-hydroxynaringenin; e: mono-C-arabinose-2-hydroxynaringenin. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0018] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0019] Figure 1 Figure 1 is a roadmap for the current biosynthesis of schaftoside and the biosynthesis of schaftoside of the present invention. The green arrow in the figure indicates the conventional method for the current biosynthesis of schaftoside, which requires continuous catalysis of 2-hydroxynaringenin to produce schaftoside by two different carbon glycosyltransferases, CGTa and CGTb; the yellow arrow indicates the method for the biosynthesis of schaftoside of the present invention, which only requires the use of one enzyme, carbon glycosyltransferase CGT4 or CGT6, to catalyze 2-hydroxynaringenin to produce schaftoside, thereby providing an energy-saving and efficiency-enhancing method for producing schaftoside using microbial cells.

[0020] In some embodiments of the present invention, a method for synthesizing schaftoside or isoschaftoside is disclosed, the method comprising the steps of: using Herba Lysimachiae carbon glycosyltransferase as a catalyst to catalyze the reaction between 2-hydroxynaringenin and UDP-Arabinose, and then adding UDP-glucose to continue the reaction; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0021] In some embodiments, the usage ratio of the 2-hydroxynaringenin to the Desmodium sibiricum carbonyl glycosyltransferase is 100 μM: 45-55 ug.

[0022] In some embodiments, the molar ratio of 2-hydroxynaringenin to UDP-Arabinose is 1:4-6, preferably 1:4.5-5.5, and more preferably 1:5.

[0023] In some embodiments, the molar ratio of 2-hydroxynaringenin to UDP-glucose is 1:4-6, preferably 1:4.5-5.5, and more preferably 1:5.

[0024] In some embodiments, the reaction temperature is 34-36° C., and the reaction time is 4-6 min.

[0025] In some embodiments, after the reaction is completed, the step of subjecting the reaction product to in vitro hydrochloric acid dehydration treatment is further included.

[0026] In some embodiments, a method for synthesizing schaftoside or isoschaftoside comprises the steps of: preparing a reaction system comprising Herba Lysimachiae carbon glycosyltransferase, 2-hydroxynaringenin, UDP-Arabinose and Na2HPO4-NaH2PO4, reacting at 34-36°C for 4-6 minutes; adding UDP-glucose and continuing to react at 34-36°C for 4-6 minutes, terminating the reaction with methanol, and subjecting the reaction product to in vitro hydrochloric acid dehydration treatment; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4; the molar ratio of the 2-hydroxynaringenin to UDP-glucose is 1:4-6; the molar ratio of the 2-hydroxynaringenin to UDP-Arabinose is 1:4-6; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM:45-55 ug.

[0027] In other embodiments of the present invention, a method for synthesizing schaftoside or isoschaftoside is disclosed, the method comprising the steps of: using Desmodium sibiricum carbon glycosyltransferase as a catalyst to catalyze the reaction of 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose; the amino acid sequence of the Desmodium sibiricum carbon glycosyltransferase is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0028] In some embodiments, the usage ratio of the 2-hydroxynaringenin to the Desmodium sibiricum carbonyl glycosyltransferase is 100 μM: 45-55 ug.

[0029] In some embodiments, the molar ratio of 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose is 1:4-6:4-6, preferably 1:4.5-5.5:4.5-5.5, and more preferably 1:5:5.

[0030] In some embodiments, the reaction temperature is 34-36° C., and the reaction time is 8-12 min.

[0031] In some embodiments, after the reaction is completed, the step of collecting C-glucose-C-arabinose-2-hydroxynaringenin in the reaction product and then performing an in vitro hydrochloric acid dehydration treatment is further included.

[0032] In some embodiments, a method for synthesizing schaftoside or isoschaftoside comprises the steps of: preparing a reaction system comprising Herba Lysimachiae carbon glycosyltransferase, 2-hydroxynaringenin, UDP-Arabinose, UDP-glucose and Na2HPO4-NaH2PO4, reacting at 34-36°C for 8-12 minutes, terminating the reaction with methanol, collecting C-glucose-C arabinose-2-hydroxynaringenin in the reaction product, and performing in vitro hydrochloric acid dehydration treatment; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4; the molar ratio of the 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose is 1:4-6:4-6; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM:45-55 ug.

[0033] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1 Construction of a recombinant expression vector with GsCGT6 or GsCGT4 inserted

[0035] The following steps are involved:

[0036] 1. Synthesis of Desmodium sibiricum cDNA

[0037] Take the root, stem and leaf samples of Desmodium strychnifolia stored at -80°C, grind them into powder with liquid nitrogen and extract RNA using the Plant RNA Dual Column Kit (Cat. No.: R4151-03C, Brand: Magen / Meiji Biotech). TM The first strand of cDNA was synthesized according to the instructions of the II 1st Strand cDNA Synthesis Kit (Cat. No.: 6210A, Brand: Takara / Takara Bio).

[0038] 2. Amplification of GsCGT6 and GsCGT4 sequences

[0039] According to the CDS sequences of GsCGT6 and GsCGT4 obtained by sequencing the genome of Desmodium strychnifolium, primers such as SEQ ID NO: 5-6 (SEQ ID NO: 5: aagtgcatcatcatcatcatcatatgATGTCTGGTTCAGAACATGTTG, SEQ ID NO: 6: atctagactgcaggtcgacaagcttTCAAGTGGCTTTAACATTCTTC) and SEQ ID NO: 7-8 (SEQ ID NO: 7: aaagtgcatcatcatcatcatcatatgATGTCTCCTTCAGAGCATGATG, SEQ ID NO: 8: tatctagactgcaggtcgacaagcttCTAGCAAGTGGCTTTAACATTC) were designed. Max DNA Polymerase high-fidelity enzyme (Cat. No.: R045B, Brand: TaKaRa) was used to amplify the cDNA sequences of GsCGT6 and GsCGT4.

[0040] PCR reaction system: 25μL 2×Prime Star Max, F 2μL, R 2μL, template DNA 1μL, ddH2O 20μL.

[0041] PCR reaction procedure: 98℃ pre-denaturation for 5min; 98℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 33 cycles in total; 72℃ extension for 5min. The PCR product was purified by agarose gel and the target fragment was recovered using a DNA gel recovery kit, namely GsCGT6 or GsCGT4, whose nucleotide sequences are shown in SEQ ID NOs: 1-2, and amino acid sequences are shown in SEQ ID NOs: 3-4, respectively.

[0042] SEQ ID NO:1

[0043]

[0044] SEQ ID NO:2

[0045] ATGTCTCCTTCAGAGCATGATGTTCATTTGGTTTTCCTCCCAAGTGCTGGCA

[0046] TGGGACATCTTAACCCATGTCTTAGAATAGCAACAATGTTTCTACGCCATGG

[0047] TTGCAAAGTCACTCTTATCACTCCCAAACCCACTGTCTCTATTGCTGAGTCT

[0048] AACCTCATCTCTCGCTTTTGTTCTTCTTTTCCTCATCAAGTTACTCAAATAG

[0049] ACTTAAATCTCATAACTTTGGATCCAACCACAGTTAACACCGATGACCCTTT

[0050] TTGGCTTCAGTTTGAAACTATTCGTCGTTCGGTTCACCTTGTAGGTCCAATA

[0051] CTATCTTCACTCTCATCAACAGTACCCCCTGTCTCAGCTTTCATCTTCGATG

[0052] TTAGCTTGATCTCGCCTCTAATTCCAATCATTGAGAAACTATCTTGTCCGTC

[0053] TTACATTTACTTCACCTCATCAGCTAGAATGCTCTCTTTTTTTGCACTCACG

[0054] TCTGTTCTTGCTTCTTCCAATCCAGGTGAAAAACCTCATTCATTCATTGGTG

[0055] ATGGTGTTAAAATCCCAGGCGTAGCATCACCAATACCAAGATCTTCTGTCC

[0056] CTAGTATGGTTCTTAAGCCCAACTCTCTCTTTGAGAGCATATTCATGGAGGA

[0057] CAGTGCCAAACTCACTAAGCTTAATGGGGTTTTCATCAACTCGTTTCAAGA

[0058] ATTGGAAGGGGAGGCACTGGCAGCACTCAACGAAGGAAAAGTGGTTGAA

[0059] GGGCTGCCCCCAGTGTATGGTATTGGTCCCTTAATGCCGTGTGAGTTTGAG

[0060] AAGGTGGATCAAGTTCAAAAGGGTGGTTCCATGAGATCAATATTGAAGTG

[0061] GCTAGATGAACAAGCTAAAGAGTCTGTGGTATATGTTTGCTTGGGGAATAG

[0062] GACGGAGACAAGAAAAGAGCAAATAAAAGACACGGCTTTGGGGTTGATA

[0063] GAAAGTGGGTATAGTTTTTTGTGGGTGGTGAAGTTGAAGGCTGTTGATAGA

[0064] GAAGAGGAGGAGGATGTGGAGGATGTGTTGGGGAATGAGTTGATGAATAA

[0065] AGTGAGGGAAAAAGGTTGGTTGTGAAAGAATATGTGGATCAGATGGGAA

[0066] TTCTGGCTCACCCTGCAGTAGGGGGGTTTGTGAATCACGGAGGGTGGAAC

[0067] TCAATAACTGAGACTGTGTGGGAAGGAGTTCCTATTTTGACATGGCCTCAT

[0068] CATGGGGATCAGAAGATAACCTCAGAGTCAGTGCGGTTAAGTGGGGTGGGAATTTGGCCTGAGGAGTGGGGGTGGGGTACAGAAGAGGTGGTGAAAGGGAAGGAGATTGCGAAGAGAATCAAAGAGTTGATGAGC AATGAATCTTTAAGGGTCAAAACTGCAGAAATGAAGGAGGCAGCTAGGAAGGCTGCAGGTGTTGGTGGGAGTTGTGAGGTTATTGTTAAGAGGCTAATTGAGGAGTGGAAGAAGAATGTTAAAGCCACTTGCTAG

[0069] SEQ ID NO:3

[0070] MSGSEHVVHLAFLPSAGMGHLNPFLRIAALFLRHGCKVTLITPKPTVSLAESKLISRFCSSFPKQVTQMDLNLIPVDPTTVNTTDPFWLQFETIRRSVHLVGPILSSLSPPLTAFIYDV SLITPLLPITEKLSCPSYIIFTSSARMLSFFAHLSVLAASNPGEHPCSFIDVIEIPGFSSPIPRSSVPPMLLQPNSALETIFMADSPKLTKLNGIFINTFEELEGEALAALNEGKVVKG LPPVLAVGPLMACEFEEEDQGQRSSSMTSILKWLDEQDEASVVYVCLGSRTETRKEQIKDTALGLIESGYRFLWVVKLKMVDREEEEGLEDVLGNGLMSKVREKGVVVKEYVDQLEILS HPAVGGFVSHGGWNSTIETVWEGVPILSWPQHGDQKMSSQTTRISGVGIWPEDWGWGTQDMVKAKEIAKRIKEMMSNESLRVKAAEIKEAARKAAGVGGSCEVIVKRQIEEWKKNVKAT

[0071] SEQ ID NO:4

[0072] MSPSEHDVHLVFLPSAGMGHLNPCLRIATMFLRHGCKVTLITPKPTVSIAESNLISRFCSSFPHQVTQIDLNLITLDPTTVNTDDPFWLQFETIRRSVHLVGPILSSLSSTVPPVSAFIFDVSLISPLIPIIEKLSCPSYIYFTSSARMLSFFALTSVLASSNPGEKPHSFIGDGVKIPGVASPIPRS SVPSMVLKPNSLFESIFMEDSAKLTKLNGVFINSFQELEGEALAALNEGKVVEGLPPVYGIGPLMPCEFEKVDQVQKGGSMRSILKWLDEQAKE SVVYVCLGNRTETRKEQIKDTALGLIESGYSFLWVVKLKAVDREEEEDVEDVLGNELMNKVREKGVVVKEYVDQMGILAHPAVGGFVNHGGWNS

[0073] ITETVWEGVPILTWPHHGDQKITSESVRLSGVGIWPEEWGWGTEEVVKGKEI

[0074] AKRIKELMSNESLRVKTAEMKEAARKAAGVGGSCEVIVKRLIEEWKKNVKA

[0075] TC

[0076] 3. Construction of cloning vector

[0077] The cloning vector was constructed using Fast DNA Assembly Mix (Cat. No.: E0201S, Brand: CISTRO), 5 μL Fast DNA Assembly Mix, 2 μL of the vector after restriction digestion (pcoldⅡ-Nde I+HindⅢ), 3 μL of the PCR amplification product of GsCGT1 or GsCGT2, and reacted at 50°C for 15 minutes to obtain the ligation product.

[0078] Add the competent E. coli DH5α to the sample in an ice bath and let it stand for 5 minutes until the competent E. coli melts. Take the ligation product and add it to 100 μL of the competent E. coli DH5α in an ice-water mixture, flick to mix, then ice bath for 20 minutes; heat shock at 42°C for 60 seconds; let it stand on ice for 3 minutes; add 700 μL of LB liquid culture medium without antibiotics; shake and culture at 200 rpm, 37°C for 1 hour; centrifuge at 10,000×g for 1 minute; discard the supernatant, and take an appropriate amount of the remaining 50 μL of suspended bacteria and apply it to a plate containing 50 mg / L of Lamp. +The cells were incubated on LB solid medium at 37°C overnight until single colonies grew out, and positive clone detection and sequencing confirmation (Qingke Biotechnology Co., Ltd.) were performed to obtain recombinant expression vectors inserted with GsCGT6 or GsCGT4, respectively.

[0079] Example 2 Target protein induced expression and purification

[0080] The positive monoclonal clone sequenced correctly in Example 1 was selected and inoculated into 5 mL LB (containing 100 mg / L Amp + ) medium, cultured overnight at 37°C, 220 rpm; the bacterial solution was transferred 1:100 to 200 mL LB medium (containing 100 mg / L Amp + ) and cultured at 37°C and 220 rpm until the bacterial solution reached OD 600 = about 0.6; immediately place the bacterial solution in an ice-water mixture to completely cool, then add IPTG to a final concentration of 0.1 mM; continue to culture at 15°C, 160rpm for 18h, and then all operations are carried out at 0-4°C to maintain enzyme activity.

[0081] Under low temperature conditions, collect the bacteria at 4000rpm for 10min, add an appropriate amount of ddH2O to wash once, suspend the bacteria with 20mL Lysis buffer (pH8.0, 50mM Tris HCl, 300mM NaCl, 5mM imidazole), and add PMSF to a final concentration of 1mM. The bacterial suspension was placed in a mixture containing ice water for ultrasonic disruption, with an ultrasonic power of 250W for 30min, 3s for 3s, and 3s for 3s. After the disruption is completed, centrifuge at 4°C, 12000×g for 25 minutes and collect the supernatant. Because the target protein can form a fusion protein with the histidine (His) tag on the expression vector, the target protein crude enzyme solution is affinity purified with nickel filler (Ni-NTAagarose, QIAGEN). The specific steps are as follows:

[0082] Take 1mL Ni-NTA suspension into the purification column, then balance it with Lysis buffer for 3 times, add 20mL crude enzyme solution supernatant obtained by ultrasound into the purification column, and mix it evenly with a rotary oscillator (speed of 200rpm) at 4℃ for 1 hour; transfer the recombinant protein crude enzyme solution and Ni-NTA mixture to an empty centrifuge tube to release the flow-through liquid; wash the Ni-NTA filler 3 times with Washbuffer; elute the target protein 3 times with Elution buffer containing 250mM imidazole, collect the eluate and transfer it to an ultrafiltration tube (brand Millipore, product number UFC901096-1) for subsequent desalting and concentration.

[0083] Desalting and concentration: balance the filter membrane of the Millipore ultrafiltration tube with desalting buffer (pH 8.0, 50 mM Tris-HCl) for 3 times, and pre-cool the ultrafiltration tube on ice; add the collected eluate, centrifuge at 4000g for 25 minutes at 4°C; add 1 mL of desalting buffer and repeat 3 times. After the last desalting, the remaining concentrate is about 500 uL, add DTT with a final concentration of 1 mM, and store the concentrated recombinant protein solution (i.e., C-glycosyltransferase GsCGT6 and GsCGT4) at -20°C.

[0084] Example 3 Method for synthesizing schaftoside using the C-glycosyltransferase of Example 2

[0085] In this example, 100 μM 2-hydroxynaringenin was used as a substrate, 500 μM UDP-Glc and / or UDP-Ara was used as a glycosyl donor, and 50 μg of enzyme was used as a research object to characterize the functional characteristics of GsCGT6 or GsCGT4. The following steps were included:

[0086] 1. Experimental Grouping

[0087] ① GsCGT6 or GsCGT4, 100 μM 2-hydroxynaringenin and 500 μM UDP-Glc, Na2HPO4-NaH2PO4 (pH 8.0) buffer was added to 100 μL, incubated at 35°C for 5 min, and the reaction was immediately terminated by methanol;

[0088] ② GsCGT6 or GsCGT4, 100 μM 2-hydroxynaringenin and 500 μM UDP-Ara, Na2HPO4-NaH2PO4 (pH 8.0) buffer was added to 100 μL, incubated at 35°C for 5 min, and the reaction was immediately terminated by methanol;

[0089] ③ GsCGT6 or GsCGT4, 100 μM 2-hydroxynaringenin and 500 μM UDP-Glc, Na2HPO4-NaH2PO4 (pH 8.0) buffer was added to 100 μL, incubated at 35°C for 5 min, and then 500 μM UDP-Ara was immediately added and incubated for 5 min, and then methanol was immediately used to terminate the reaction;

[0090] ④ GsCGT6 or GsCGT4, 100 μM 2-hydroxynaringenin and 500 μM UDP-Ara, Na2HPO4-NaH2PO4 (pH 8.0) buffer was added to 100 μL, incubated at 35°C for 5 min, 500 μM UDP-Glc was immediately added and incubated for 5 min, and methanol was immediately used to terminate the reaction;

[0091] ⑤GsCGT6 or GsCGT4, 100 μM 2-hydroxynaringenin, 500 μM UDP-Glc and 500 μM UDP-Ara, Na2HPO4-NaH2PO4 (pH 8.0) buffer was added to 100 μL, and the mixture was incubated at 35°C for 10 min, and the reaction was immediately terminated by methanol.

[0092] 2. HPLC detection of reaction products

[0093] Gradient elution was performed using two mobile phases, phase A (water containing 0.1% formic acid) and phase B (methanol). The gradient elution conditions were: 0-3 min, 15%-27% B; 3-19 min, 27%-65% B; 20-25 min, 60% B; 25-29 min, 60%-67% B; 30-34 min, 95% B; 35-38 min, 15% B; liquid chromatography column InertSustain C18 (4.6×150 mm, 5 μm), flow rate was 1 mL / min; detection wavelengths were 290 nm and 335 nm, column temperature was 35°C.

[0094] 3. Experimental results

[0095] Figure 2 It is the catalytic product of GsCGT6 detected by HPLC. Figure 2 It can be learned that:

[0096] ① After GsCGT6 and 500 μM UDP-Glc were reacted for 5 minutes, mono-C-glucose-2-hydroxynaringenin (d) and di-C-glucose-2-hydroxynaringenin (a) could be detected;

[0097] ②After CGT6 and 500μM UDP-Ara reacted for 5 minutes, mono-C-arabino-2-hydroxynaringenin (e) and a small amount of di-C-arabino-2-hydroxynaringenin (c) could be detected.

[0098] ③ After CGT6 and 500 μM UDP-Glc were reacted for 5 minutes, UDP-Ara was added and reacted for 5 minutes, and only di-C-glucose-2-hydroxynaringenin (a) was detected.

[0099] ④ After CGT6 and 500mM UDP-Ara were reacted for 5 minutes, UDP-Glc was added and reacted for 5 minutes. The main product detected was di-C-glucose-arabinose-2-hydroxynaringenin (b), indicating that CGT6 can use mono-C-arabinose-2-hydroxynaringenin (e) as a substrate and continue to undergo C-glucosylation to produce di-C-glucose-arabinose-2-hydroxynaringenin (b).

[0100] ⑤CGT6 was reacted with 500mM UDP-Glc and 500mM UDP-Ara for 10min, and di-C-glucose-2-hydroxynaringenin (a), di-C-glucose-arabinose-2-hydroxynaringenin (b) and mono-C-glucose-2-hydroxynaringenin (d) could be detected.

[0101] di-C-glucose-arabinose-2-hydroxynaringenin (b) is an important precursor compound of schaftoside and can be converted into schaftoside after in vitro hydrochloric acid dehydration treatment.

[0102] Figure 3 The catalytic product of GsCGT4 was detected by HPLC, and the results were similar to those of the catalytic reaction of GsCGT6. Figure 3 It can be learned that:

[0103] ① After GsCGT4 and 500 μM UDP-Glc were reacted for 5 minutes, mono-C-glucose-2-hydroxynaringenin (d) and di-C-glucose-2-hydroxynaringenin (a) could be detected;

[0104] ②After CGT4 and 500μM UDP-Ara reacted for 5 minutes, mono-C-arabino-2-hydroxynaringenin (e) and a small amount of di-C-arabino-2-hydroxynaringenin (c) could be detected.

[0105] ③ After CGT4 and 500 μM UDP-Glc were reacted for 5 min, UDP-Ara was added and reacted for 5 min, and only di-C-glucose-2-hydroxynaringenin (a) was detected.

[0106] ④ After CGT4 and 500mM UDP-Ara were reacted for 5 minutes, UDP-Glc was added and reacted for 5 minutes. The main product detected was di-C-glucose-arabinose-2-hydroxynaringenin (b), indicating that CGT4 can use mono-C-arabinose-2-hydroxynaringenin (e) as a substrate and continue to undergo C-glucosylation to produce di-C-glucose-arabinose-2-hydroxynaringenin (b).

[0107] ⑤CGT4 was reacted with 500mM UDP-Glc and 500mM UDP-Ara for 10min, and di-C-glucose-2-hydroxynaringenin (a), di-C-glucose-arabinose-2-hydroxynaringenin (b) and mono-C-glucose-2-hydroxynaringenin (d) could be detected.

[0108] di-C-glucose-arabinose-2-hydroxynaringenin (b) is an important precursor compound of schaftoside and can be converted into schaftoside after in vitro hydrochloric acid dehydration treatment.

[0109] Based on the above results, it can be seen that after CGT6 or CGT4 catalyzes the reaction of 2-hydroxynaringenin and UDP-Ara alone, UDP-Glc is added to continue the catalytic reaction, or CGT6 or CGT4 catalyzes the reaction of 2-hydroxynaringenin, UDP-Ara and UDP-Glc simultaneously, schaftoside or isoschaftoside can be synthesized.

[0110] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0111] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for synthesizing schaftoside or isoschaftoside, It is characterized in that The following steps are involved: Using Herba Lysimachiae carbon glycosyltransferase as a catalyst, catalyzing the reaction between 2-hydroxynaringenin and UDP-Arabinose, and then adding UDP-glucose to continue the reaction; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4; the molar ratio of the 2-hydroxynaringenin and UDP-Arabinose is 1:4-6, and the molar ratio of the 2-hydroxynaringenin and UDP-glucose is 1:4-6; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM:45-55 ug; the reaction temperature is 34-36°C, and the reaction time is 4-6 min.

2. The method for synthesizing schaftoside or isoschaftoside according to claim 1, It is characterized in that The molar ratio of the 2-hydroxynaringenin to UDP-Arabinose is 1:4.5-5.5; and / or the molar ratio of the 2-hydroxynaringenin to UDP-glucose is 1:4.5-5.

5.

3. The method for synthesizing schaftoside or isoschaftoside according to claim 1 or 2, It is characterized in that After the reaction is completed, the method further includes a step of subjecting the reaction product to an in vitro hydrochloric acid dehydration treatment.

4. The method for synthesizing schaftoside or isoschaftoside according to claim 1, It is characterized in that The following steps are involved: Formulated with Desmodium sibiricum C-glycosyltransferase, 2-hydroxynaringenin, UDP-Arabinose and Na 2 HPO 4 -NaH 2 PO 4 The invention relates to a reaction system, reacting at 34-36° C. for 4-6 min; adding UDP-glucose and continuing to react at 34-36° C. for 4-6 min, terminating the reaction with methanol, and subjecting the reaction product to in vitro hydrochloric acid dehydration treatment; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4; the molar ratio of the 2-hydroxynaringenin to UDP-Arabinose is 1:4.5-5.5; the molar ratio of the 2-hydroxynaringenin to UDP-glucose is 1:4.5-5.5; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM:45-55 ug.

5. A method for synthesizing schaftoside or isoschaftoside, It is characterized in that The following steps are involved: Use Herba Lysimachiae carbon glycosyltransferase as a catalyst to catalyze the reaction of 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO: 3 or SEQ ID NO: 4; the molar ratio of the 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose is 1: 4-6: 4-6; the reaction temperature is 34-36° C., and the reaction time is 8-12 min; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM: 45-55 ug.

6. The method for synthesizing schaftoside or isoschaftoside according to claim 5, It is characterized in that The molar dosage ratio of the 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose is 1:4.5-5.5:4.5-5.

5.

7. The method for synthesizing schaftoside or isoschaftoside according to claim 5, It is characterized in that After the reaction is completed, the method further includes collecting C-glucose-C-arabinose-2-hydroxynaringenin in the reaction product and then performing an in vitro hydrochloric acid dehydration treatment.

8. The method for synthesizing schaftoside or isoschaftoside according to claim 5, It is characterized in that The following steps are involved: Formulated with Desmodium sibiricum carbonyl transferase, 2-hydroxynaringenin, UDP-Arabinose, UDP-glucose and Na 2 HPO 4 -NaH 2 PO 4 The invention relates to a reaction system, reacting at 34-36° C. for 8-12 min, terminating the reaction with methanol, collecting C-glucose-C-arabinose-2-hydroxynaringenin in the reaction product, and performing in vitro hydrochloric acid dehydration treatment; the amino acid sequence of the Herba Lysimachiae carbon glycosyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4; the molar ratio of the 2-hydroxynaringenin, UDP-Arabinose and UDP-glucose is 1:4.5-5.5:4.5-5.5; the dosage ratio of the 2-hydroxynaringenin to the Herba Lysimachiae carbon glycosyltransferase is 100 μM:45-55 ug.

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