CN114350646B - UDP-sugar epimerase PsUGE2 and application thereof in synthesis of arabinoside - Google Patents
UDP-sugar epimerase PsUGE2 and application thereof in synthesis of arabinoside Download PDFInfo
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Abstract
The invention relates to UDP-sugar epimerase PsUGE2 and application thereof in synthesis of arabinoside, belonging to the field of bioengineering and technology. The amino acid sequence of the UDP-sugar epimerase PsUGE2 is shown in SEQ ID NO. 1. Based on the UDP-sugar epimerase PsUGE2, the invention also provides a method for synthesizing UDP-arabinose, which adopts the UDP-sugar epimerase PsUGE2 with an amino acid sequence shown as SEQ ID NO.1 or a gene sequence shown as SEQ ID NO.2 to catalyze a reaction on a substrate. The invention establishes a UDP-arabinose circulation system and a synthesis system of pentacyclic triterpene arabinoside, realizes the synthesis of pentacyclic triterpene arabinoside, improves the regeneration of the UDP circulation system, improves the reaction rate, and simultaneously takes sucrose as an initial sugar donor, thereby greatly reducing the cost of synthesizing the arabinoside compound.
Description
Technical Field
The invention belongs to the field of bioengineering and technology, and relates to UDP-sugar epimerase PsUGE2 and application thereof in synthesis of arabinoside.
Background
UDP-glucose epimerase (USE) is one of the most conserved enzymes, and UDP-glucose epimerase (UDP-glucose epimerase, UGE) and UDP-xylose epimerase (UXE) are two of the branches, both in eukaryotic and prokaryotic organisms. Bioinformatics analysis shows that the USE family shares two common conserved domains, the N-terminal GxxGxxG sequence and the Ser/Thr-Tyr-Lys triplet of the catalytic center. The reported USEs have different substrate specificities and are capable of catalyzing UDP-glucose/UDP-galactose, as well as UDP-arabinose/UDP-xylose, and the substrate selection mechanism is not clear. PsUGE1 derived from pea, atUGE1 and AtUGE3 derived from Arabidopsis thaliana, and OcUGE1 and OcUGE2 derived from Rohdea japonica all have bifunctional UDP-glucose/UDP-xylose epimerase activities. Has important significance for synthesizing expensive UDP-arabinose.
Pentacyclic triterpene has antiviral, antitumor, antidiabetic, antiinflammatory and antibacterial activities, but has problems of poor water solubility, poor permeability, poor bioavailability, and serious side effects. Glycosylation is used as a natural modification method, so that the problems can be solved, the pharmacological activity of pentacyclic triterpene can be enriched, and the arabinoside has excellent cytotoxicity and wide application prospect in the aspect of anticancer drugs. Compared with chemical glycosylation, the enzymatic method has the advantages of high specificity, good stereoselectivity, simple reaction conditions, environmental friendliness and the like.
However, UDP-glycosyltransferase (UGT) relies on expensive UDP-arabinose (5 mg, 450$) as a sugar donor, and the cost of large-scale production is hard to bear, so that the large-scale synthesis of the arabinoside of pentacyclic triterpene by direct in vitro addition is not possible. When the multi-enzyme cascade in-vitro reaction is established, and the natural product is subjected to glycosylation modification to synthesize the glycoside compound with high added value, the sucrose which is low in cost and easy to obtain is used as an initial sugar donor, and the UDP circulation system of the coupled sucrose synthase and UDP-glycosyltransferase (UGT) solves the problem that the UDP-sugar is high in cost to a great extent, and can effectively relieve the inhibition of accumulation of UDP on UGT. This circulatory system has now evolved to allow UDP-Glc to be regenerated up to about 220 times. Based on this, researchers have developed regeneration systems for various expensive sugar donors such as UDP-Rha, UDP-Gal, UDP-GlcA, etc.
At present, the UDP-arabinose synthesis in plants involves more enzymes, the pathway is longer, and the research on UDP-glucose dehydrogenase, UDP-glucuronic acid decarboxylase and UDP-sugar epimerase of plant sources is little, and the enzymes which have been verified to function and characterized are fewer. Although researchers have proposed a series of UDP-sugar regeneration systems to reduce the cost of sugar donors in UGT reactions, the regeneration systems of UDP-arabinose involving sucrose synthase, UDP-glucose dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-sugar epimerase have not been reported. These factors limit the synthesis of pentacyclic triterpene arabinoside.
Disclosure of Invention
Based on the above-mentioned shortcomings in the art, the present invention provides a UDP-sugar epimerase PsUGE2 and its use in the synthesis of UDP-arabinose and pentacyclic triterpene arabinoside compounds.
The technical scheme of the invention is as follows:
the UDP-sugar epimerase PsUGE2 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
The gene sequence of the UDP-sugar epimerase PsUGE2 is shown in SEQ ID NO. 2.
A method for synthesizing UDP-arabinose is characterized in that UDP-sugar epimerase PsUGE2 with an amino acid sequence shown as SEQ ID NO.1 or a gene sequence shown as SEQ ID NO.2 is adopted to catalyze a reaction on a substrate.
The catalytic reaction system comprises: 1Mm substrate, 0.5 μg/μLUDP-sugar epimerase PsUGE2, and buffer solution for the rest;
preferably, the substrate is UDP-xylose; the buffer solution is Tris-HCl buffer solution with the pH value of 8.5;
preferably, the catalytic reaction conditions are 35 ℃ for 10min.
A set of enzymes for arabinose modification of pentacyclic triterpene compounds, comprising: the UDP-sugar epimerase PsUGE2 with the amino acid sequence shown in SEQ ID NO.1 or the gene sequence shown in SEQ ID NO.2, the sucrose synthase GuSuS 1-delta 9 with the amino acid sequence shown in SEQ ID NO.3 or the gene sequence shown in SEQ ID NO.4, the UDP-glucose dehydrogenase AtUGDH3 with the amino acid sequence shown in SEQ ID NO.5 or the gene sequence shown in SEQ ID NO.6, the UDP-glucuronic acid decarboxylase AtUXS3 with the amino acid sequence shown in SEQ ID NO.7 or the gene sequence shown in SEQ ID NO.8, the glycosyltransferase UGT99D1 with the amino acid sequence shown in SEQ ID NO.9 or the gene sequence shown in SEQ ID NO. 10.
The pentacyclic triterpene compound is selected from the group consisting of: oleanolic acid and/or betulinic acid.
A method for carrying out arabinose modification on pentacyclic triterpene compounds is characterized in that UDP-glucose epimerase PsUGE2 with an amino acid sequence shown as SEQ ID NO.1 or a gene sequence shown as SEQ ID NO.2, sucrose synthase GuSuS 1-delta 9 with an amino acid sequence shown as SEQ ID NO.3 or a gene sequence shown as SEQ ID NO.4, UDP-glucose dehydrogenase AtUGDH3 with an amino acid sequence shown as SEQ ID NO.5 or a gene sequence shown as SEQ ID NO.6, UDP-glucuronic acid decarboxylase AtUXS3 with an amino acid sequence shown as SEQ ID NO.7 or a gene sequence shown as SEQ ID NO.8, and glycosyltransferase UGT99D1 with an amino acid sequence shown as SEQ ID NO.9 or a gene sequence shown as SEQ ID NO.10 are adopted, and the pentacyclic triterpene compounds are used as substrates for catalytic reaction.
The catalytic reaction comprises the following steps:
(1) Sucrose synthase GuSuS 1-delta 9, UDP, substrate, glycosyl donor and NAD + Reacting the reaction system;
(2) After regulating the pH value, adding UDP-glucose dehydrogenase AtUGDH3 into the reaction system of the step (1) for reaction;
(3) Adding UDP-glucuronic acid decarboxylase AtUXS3 into the reaction system in the step (2) for reaction;
(4) Adding UDP-sugar epimerase PsUGE2 into the reaction system of the step (3) for reaction;
(5) Adding glycosyltransferase UGT99D1 into the reaction system in the step (4) for reaction;
preferably, in step (1), the reaction system comprises: 0.17. Mu.g/. Mu.L sucrose synthase GuSuS 1-. DELTA.9, 667. Mu.M UDP, 83. Mu.M substrate, 6667. Mu.M glycosyl donor, 1333. Mu.M NAD + The method comprises the steps of carrying out a first treatment on the surface of the The reaction condition is 50 ℃ for 30min;
preferably, in the step (2), the pH is adjusted to 8.5 by KOH, the concentration of UDP-glucose dehydrogenase AtUGDH3 in the reaction system is 0.17 mug/. Mu.L, and the reaction condition is 35 ℃ for 30min;
preferably, in the step (3), the concentration of UDP-glucuronic acid decarboxylase AtUXS3 in the reaction system is 0.17 mug/. Mu.L, and the reaction condition is 35 ℃ for 30min;
preferably, in step (4), the concentration of UDP-sugar epimerase PsUGE2 in the reaction system is 0.17. Mu.g/. Mu.L, and the reaction conditions are 25℃for 30min;
preferably, in step (5), the concentration of glycosyltransferase UGT99D1 in the reaction system is 0.17 mug/mu L, and the reaction condition is 35 ℃ for 6 hours;
preferably, the glycosyl donor is sucrose; the substrate is pentacyclic triterpene compound;
the pentacyclic triterpene compound is selected from the group consisting of: oleanolic acid and/or betulinic acid.
A novel anti-DLD-1 drug characterized in that the active ingredients thereof comprise: 3-oxo-arabinose-betulinic acid.
Use of 3-oxo-arabinose-betulinic acid in the preparation of an anti-DLD-1 medicament.
A preparation method of a novel anti-DLD-1 drug is characterized in that the method for carrying out arabinose modification on betulinic acid by adopting the method for carrying out arabinose modification on pentacyclic triterpene compounds is adopted to obtain an active ingredient of the novel anti-DLD-1 drug.
A preparation method of the anti-MCF-7 medicine is characterized in that the method for carrying out arabinose modification on pentacyclic triterpene compounds is adopted to carry out arabinose modification on oleanolic acid, so as to obtain the active ingredient of the anti-MCF-7 medicine.
In a first aspect, the invention provides a UDP-sugar epimerase PsUGE2, the amino acid sequence of which is shown in a sequence table I, and a gene encoding the UDP-sugar epimerase PsUGE2, the sequence of which is shown in a sequence table II.
In a second aspect, the present invention provides a UDP-arabinose recycling system, configured to synthesize UDP-arabinose by coupling sucrose synthase (SuSy), UDP-glucose dehydrogenase (UGDH), UDP-glucuronic acid decarboxylase (UXS), UDP-sugar epimerase (USE), having the structural formula one;
In a third aspect, the present invention provides a synthesis system for synthesizing 3-oxo-arabinose-oleanolic acid (structure shown as formula two) and 3-oxo-arabinose-betulinic acid (structure shown as formula three), wherein the synthesis system is shown as fig. 2, and the synthesis system is exemplified by oleanolic acid.
The invention provides UDP-sugar epimerase with a brand new sequence, and also provides efficient UDP-sugar epimerase PsUGE2 and a protein coded by the efficient UDP-sugar epimerase, and simultaneously provides a UDP-arabinose circulating system and an arabinoside synthesizing system which are used for synthesizing various pentacyclic triterpene arabinoside compounds.
The invention provides UDP-sugar epimerase and a coding gene, and simultaneously relates to coupling glycosyltransferase UGT99D1, sucrose synthase GuSuS 1-delta 9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3 and UDP-sugar epimerase PsUGE 1. The invention provides an all-new UDP-sugar epimerase (USE), and provides new applications of UDP-glucose dehydrogenase, UDP-glucuronic acid decarboxylase and UDP-glycosyltransferase, wherein a UDP-arabinose circulation system and a pentacyclic triterpene arabinoside synthesis system can be established by combining the UDP-glucose dehydrogenase, the UDP-glucuronide decarboxylase and the UDP-glycosyltransferase, the pentacyclic triterpene arabinoside synthesis is realized, the regeneration of the UDP circulation system is improved, the reaction rate is improved, and meanwhile, sucrose is used as an initial sugar donor, so that the cost for synthesizing an arabinoside compound is greatly reduced.
Drawings
FIG. 1 shows agarose gel electrophoresis of genes of sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2 and glycosyltransferase UGT99D1 in experimental example 1 of the present invention. M in fig. 1: DNA molecular weight standard (DNA Marker); 1-5 are in turn sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2, glycosyltransferase UGT99D1.
FIG. 2 is a SDS-PAGE chart showing purification of protein expression of sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2 and glycosyltransferase UGT99D1 in example 3 of the present invention in experimental example 3 of the present invention. M in fig. 2: protein molecular weight standard (protein Marker), 1-5 are respectively: sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2, glycosyltransferase UGT99D1.
FIG. 3 is a high performance liquid chromatogram of UDP-sugar epimerase PsUGE2 in Experimental example 4 of the present invention for catalyzing UDP-xylose to UDP-arabinose, wherein UDP-Ara represents UDP-arabinose, UDP-Xyl represents UDP-xylose, psUGE2+ UDP-Xyl represents UDP-sugar epimerase PsUGE2 for catalyzing UDP-xylose to UDP-arabinose.
FIG. 4 shows the UDP-arabinose circulating system and the arabinoside synthesizing system established in Experimental example 5 of the present invention.
FIG. 5 is a high performance liquid chromatography mass spectrometry chart of 3-oxo-arabinose-oleanolic acid synthesized in experimental example 5 of the present invention.
FIG. 6 is a high performance liquid chromatography mass spectrum of 3-oxo-arabinose-betulinic acid synthesized in experimental example 5 of the present invention.
FIG. 7 is a hydrogen spectrum of 3-oxo-arabino-oleanolic acid prepared in experimental example 6 of the present invention.
FIG. 8 is a carbon spectrum of 3-oxo-arabino-oleanolic acid prepared in experimental example 6 of the present invention.
FIG. 9 is a hydrogen spectrum of 3-oxo-arabinose-betulinic acid prepared in experimental example 6 of the present invention.
FIG. 10 is a carbon spectrum of 3-oxo-arabinose-betulinic acid prepared in experimental example 6 of the present invention.
FIG. 11 is a high performance liquid chromatography mass spectrometry chart of the synthesis of 3-oxo-arabinose-oleanolic acid from the head of an engineered yeast 1 in Experimental example 7 of the present invention.
FIG. 12 is a high performance liquid chromatography mass spectrometry chart of experimental example 7 of the present invention for synthesizing 3-oxo-arabinose-betulinic acid from the head by using engineering yeast 2.
FIG. 13 is a graph showing the inhibition of MCF-7 by oleanolic acid at various concentrations of 3-oxo-arabinose-oleanolic acid in Experimental example 8 of the present invention; wherein, OA represents oleanolic acid, bA-Ara represents 3-oxygen-arabinose-oleanolic acid; the horizontal axis represents the concentration of 3-oxygen-arabinose-oleanolic acid and oleanolic acid, and the vertical axis represents the inhibition rate.
FIG. 14 is a graph showing the inhibition of DLD-1 by betulinic acid at different concentrations of 3-oxo-arabinose-betulinic acid in experimental example 8 of the present invention. Wherein BA represents betulinic acid and BA-Ara represents 3-oxo-arabinose-betulinic acid; the horizontal axis shows the concentrations of 3-oxygen-arabinose-betulinic acid and betulinic acid, and the vertical axis shows the inhibition rate.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples. The following examples illustrate the invention without limiting its scope.
Sources of biological materials
The vector pET28a, E.coli BL21 (DE 3) competent cells used in experimental example 2 were commercially available.
Herein, UDP refers to uridine diphosphate (uridine diphosphate, UDP), NAD + Refers to nicotinamide adenine dinucleotide. MCF-7 has the conventional technical meaning commonly understood by those skilled in the art and refers to a human breast cancer cell; DLD-1 has the conventional technical meaning commonly understood by those skilled in the art and refers to a human colorectal adenocarcinoma epithelial cell.
Group 1 example UDP-sugar epimerase PsUGE2 of the present invention
This set of examples provides a UDP-sugar epimerase PsUGE2. All embodiments of this group share the following common features: the amino acid sequence of the UDP-sugar epimerase PsUGE2 is shown in SEQ ID NO. 1.
In a specific embodiment, the gene sequence of the UDP-sugar epimerase PsUGE2 is shown in SEQ ID NO. 2.
Group 2 example, method of the invention for synthesizing UDP-arabinose
This set of examples provides a method for synthesizing UDP-arabinose. All embodiments of this group share the following common features: the UDP-sugar epimerase PsUGE2 with the amino acid sequence shown as SEQ ID NO.1 or the gene sequence shown as SEQ ID NO.2 is adopted to catalyze the reaction of the substrate.
In a specific embodiment, the catalytic reaction system comprises: 1Mm substrate, 0.5 μg/μLUDP-sugar epimerase PsUGE2, and buffer solution for the rest;
preferably, the substrate is UDP-xylose; the buffer solution is Tris-HCl buffer solution with the pH value of 8.5;
preferably, the catalytic reaction conditions are 35 ℃ for 10min.
Group 3 examples, combinations of enzymes for arabinose modification of pentacyclic triterpene Compounds
This set of examples provides a set of enzymes for arabinose modification of pentacyclic triterpene compounds. All embodiments of this group share the following common features: the set of enzymes that modify arabinose for pentacyclic triterpene compounds comprises: the UDP-sugar epimerase PsUGE2 with the amino acid sequence shown in SEQ ID NO.1 or the gene sequence shown in SEQ ID NO.2, the sucrose synthase GuSuS 1-delta 9 with the amino acid sequence shown in SEQ ID NO.3 or the gene sequence shown in SEQ ID NO.4, the UDP-glucose dehydrogenase AtUGDH3 with the amino acid sequence shown in SEQ ID NO.5 or the gene sequence shown in SEQ ID NO.6, the UDP-glucuronic acid decarboxylase AtUXS3 with the amino acid sequence shown in SEQ ID NO.7 or the gene sequence shown in SEQ ID NO.8, the glycosyltransferase UGT99D1 with the amino acid sequence shown in SEQ ID NO.9 or the gene sequence shown in SEQ ID NO. 10.
In some embodiments, the pentacyclic triterpene compound is selected from the group consisting of: oleanolic acid and/or betulinic acid.
Group 4 example, method of the invention for arabinose modification of pentacyclic triterpene Compounds
The present set of examples provides a method for arabinose modification of pentacyclic triterpene compounds. All embodiments of this group share the following common features: the catalytic reaction is carried out by adopting UDP-glucose epimerase PsUGE2 with the amino acid sequence shown in SEQ ID NO.1 or gene sequence shown in SEQ ID NO.2, sucrose synthase GuSuS 1-delta 9 with the amino acid sequence shown in SEQ ID NO.3 or gene sequence shown in SEQ ID NO.4, UDP-glucose dehydrogenase AtUGDH3 with the amino acid sequence shown in SEQ ID NO.5 or gene sequence shown in SEQ ID NO.6, UDP-glucuronic acid decarboxylase AtUXS3 with the amino acid sequence shown in SEQ ID NO.7 or gene sequence shown in SEQ ID NO.8, glycosyltransferase UGT99D1 with the amino acid sequence shown in SEQ ID NO.9 or gene sequence shown in SEQ ID NO.10 and pentacyclic triterpene compound as a substrate.
In some embodiments, the catalytic reaction comprises the steps of:
(1) Sucrose synthase GuSuS 1-delta 9, UDP, substrate, glycosyl donor and NAD + Reacting the reaction system;
(2) After regulating the pH value, adding UDP-glucose dehydrogenase AtUGDH3 into the reaction system of the step (1) for reaction;
(3) Adding UDP-glucuronic acid decarboxylase AtUXS3 into the reaction system in the step (2) for reaction;
(4) Adding UDP-sugar epimerase PsUGE2 into the reaction system of the step (3) for reaction;
(5) Adding glycosyltransferase UGT99D1 into the reaction system of the step (4) for reaction.
In a further embodiment, in step (1), the reaction system comprises: the final concentration was 0.17. Mu.g/. Mu.L sucrose synthase GuSuS 1-. DELTA.9, 667. Mu.M UDP, 83. Mu.M substrate, 6667. Mu.M glycosyl donor, 1333. Mu.M NAD + The method comprises the steps of carrying out a first treatment on the surface of the The reaction condition is 50 ℃ for 30min;
preferably, in the step (2), the pH is adjusted to 8.5 by KOH, the concentration of UDP-glucose dehydrogenase AtUGDH3 in the reaction system is 0.17 mug/. Mu.L, and the reaction condition is 35 ℃ for 30min;
preferably, in the step (3), the final concentration of UDP-glucuronic acid decarboxylase AtUXS3 in the reaction system is 0.17 mug/. Mu.L, and the reaction condition is 35 ℃ for 30min;
preferably, in step (4), the final concentration of UDP-sugar epimerase PsUGE2 in the reaction system is 0.17. Mu.g/. Mu.L, and the reaction conditions are 25℃for 30min;
Preferably, in step (5), the final concentration of glycosyltransferase UGT99D1 in the reaction system is 0.17 μg/μl, and the reaction conditions are 35℃for 6h.
In specific embodiments, the glycosyl donor is sucrose; the substrate is pentacyclic triterpene compound;
the pentacyclic triterpene compound is selected from the group consisting of: oleanolic acid and/or betulinic acid.
Group 5 examples, anti-DLD-1 novel drugs
This group of examples provides a new class of anti-DLD-1 drugs. All embodiments of this group share the following common features: the active ingredients of the novel anti-DLD-1 drug comprise: 3-oxo-arabinose-betulinic acid.
In a preferred embodiment, 3-oxo-arabino-betulinic acid is used in the novel anti-DLD-1 drug at a concentration of 80 to 120. Mu.M, preferably 100. Mu.M.
In further embodiments, the novel anti-DLD-1 agent further comprises pharmaceutically acceptable excipients; specifically, the pharmaceutically acceptable excipients are selected from the group consisting of: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
Use of 3-oxo-arabino-betulinic acid in preparing anti-DLD-1 drugs in group 6 examples
This group of examples provides the use of 3-oxo-arabinose-betulinic acid in the preparation of anti-DLD-1 drugs. The invention provides the application of 3-oxygen-arabinose-betulinic acid in preparing the anti-DLD-1 medicine for the first time, and the inhibition rate of the 3-oxygen-arabinose-betulinic acid on the DLD-1 is obviously superior to that of betulinic acid.
Group 7 examples, preparation of novel anti-DLD-1 drugs
The present set of examples provides a method for preparing novel anti-DLD-1 agents. In all the examples of this group, the preparation method of the novel anti-DLD-1 drug has the following common characteristics: the method for carrying out arabinose modification on pentacyclic triterpene compounds provided in any one of the embodiments of group 4 is adopted to carry out arabinose modification on betulinic acid, so as to obtain the active ingredient of the novel anti-DLD-1 drug.
In a further embodiment, the method for preparing the novel anti-DLD-1 drug further comprises: the method comprises the steps of carrying out arabinose modification on betulinic acid to obtain 3-oxygen-arabinose-betulinic acid; 3-oxo-arabinose-betulinic acid is used as the active component of the new anti-DLD-1 medicine.
In a preferred embodiment, 3-oxo-arabino-betulinic acid is used in the novel anti-DLD-1 drug at a concentration of 80 to 120. Mu.M, preferably 100. Mu.M.
Group 8 example, preparation method of anti-MCF-7 drug of the invention
The present set of examples provides a method for preparing an anti-MCF-7 drug. In all the examples of this group, the preparation method of the anti-MCF-7 drug has the following common characteristics: the method for carrying out arabinose modification on pentacyclic triterpene compounds provided in any one of the embodiments of group 4 is adopted to carry out arabinose modification on oleanolic acid, so as to obtain the active ingredients of the anti-MCF-7 medicine.
In a further embodiment, the method for preparing the anti-MCF-7 drug further comprises: the method carries out arabinose modification on oleanolic acid to obtain 3-oxygen-arabinose-oleanolic acid; 3-oxo-arabinose-oleanolic acid as an active ingredient of the anti-MCF-7 drug.
In a preferred embodiment, 3-oxo-arabinose-oleanolic acid is used in the anti-MCF-7 drug in a concentration of 20-100. Mu.M, preferably 25. Mu.M.
Experimental example 1: acquisition of sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2, glycosyltransferase genes
A. Acquisition of the sucrose synthase GuSuS1- Δ9 Gene
The fragment of the sucrose synthase GuSuS 1-delta 9 gene is shown as SEQ ID NO.4, and the amino acid sequence expressed by the fragment is shown as SEQ ID NO.3
B. obtaining of UDP-glucose dehydrogenase AtUGDH3 Gene
The UDP-glucose dehydrogenase AtUGDH3 gene fragment is shown as SEQ ID NO.6, and the amino acid sequence expressed by the fragment is shown as SEQ ID NO.5
Acquisition of the UDP-glucuronic acid decarboxylase AtUXS3 Gene
The fragment of UDP-glucuronic acid decarboxylase AtUXS3 gene is shown as SEQ ID NO.8, and the amino acid sequence expressed by the fragment is shown as SEQ ID NO.7
Acquisition of UDP-sugar epimerase PsUGE2 Gene
The UDP-sugar epimerase PsUGE2 gene fragment is shown as SEQ ID NO.2, and the amino acid sequence expressed by the fragment is shown as SEQ ID NO.1
E. Acquisition of glycosyltransferase genes
Glycosyltransferase UGT99D1 gene fragment is shown as SEQ ID NO.10, and the amino acid sequence expressed by the fragment is shown as SEQ ID NO.9
Experimental example 2: construction of E.coli engineering bacteria expressing sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-glucose epimerase PsUGE2, glycosyltransferase UGT99D1
A. Construction of E.coli engineering bacteria expressing sucrose synthase GuSuS 1-delta 9
In order to construct E.coli BL21 (DE 3)/Pet 28a-GuSuS 1-delta 9 engineering bacteria expressing sucrose synthase GuSuS 1-delta 9, a chemically synthesized sucrose synthase GuSuS 1-delta 9 gene fragment is used as a template, an upstream primer and a downstream primer are designed, and enzyme cutting sites BamHI, xhoI and protecting bases are added:
sucrose synthase GuSuS 1-Delta9 gene fragment primer F:
GGATCC CACAGTCTCCGTGAGAGGCTCG(SEQ ID NO.11)
sucrose synthase GuSuS 1-Delta9 gene fragment primer R:
TGTGCCCCTAGCTGTTGAGGAGCTCGAG(SEQ ID NO.12)
the PCR reaction system is as follows: 1. Mu.L of template, 1. Mu.L of upstream and downstream Primer, 25. Mu.L of Primer star mix (Takara Shuzo Co., ltd.) 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 58 ℃ for 5s, extension at 72 ℃ for 2min, circulation for 30 times, 10min at 72 ℃ and preservation at 4 ℃.
The cloned sucrose synthase GuSuS 1-delta 9 gene is recovered by using a Thermo agarose gel DNA recovery kit for purification and recovery. 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 Xdigest buffer 5. Mu.L, and 30. Mu.L of the DNA fragment were used to make up 50. Mu.L of the cleavage system with double distilled water at 37℃for 2 hours. After cleavage, the cleavage product was recovered using a Thermo agarose gel DNA recovery kit.
The inserted fragment sucrose synthase gene was seamlessly joined to linearized vector pET28a using the Gibson Assembly method (using NEB official website https:// interactive. NEB. Com/recommended method). 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: template LB plates were single-colony, guSuS 1-. DELTA.9 upstream and downstream primers were each 1. Mu.L, 2 XTaq mix (Beijing Polymer Biotechnology Co., ltd.) 7.5. Mu.L, and 15. Mu.L was made up 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 1min, circulation for 25 times, 10min at 72 ℃ and preservation at 4 ℃. The colony PCR verifies that the transformant containing the correct target band is subjected to DNA sequencing (Jin Weizhi biotechnology Co., ltd.) to determine that the E.coli BL21 (DE 3)/pET 28a-GuSuS 1-delta 9 engineering bacteria are successfully constructed.
B. Construction of engineering bacterium of E.coli expressing UDP-glucose dehydrogenase AtUGDH3
In order to construct E.coli BL21 (DE 3)/Pet 28a-AtUGDH3 of escherichia coli engineering bacteria expressing UDP-glucose dehydrogenase AtUGDH3, chemically synthesizing UDP-glucose dehydrogenase AtUGDH3 gene fragments as templates, designing an upstream primer and a downstream primer, and adding enzyme cutting sites BamHI, xhoI and a protecting base:
UDP-glucose dehydrogenase AtUGDH3 gene fragment primer F:
GGATCCATGGTGAAAATTTGCTGCATTGGCG(SEQ ID NO.13)
UDP-glucose dehydrogenase AtUGDH3 gene fragment primer R:
CTGAAAGATATGCCGGCGGTGGCGCTCGAG(SEQ ID NO.14)
e.coli BL21 (DE 3)/pET 28a-UGT7399D1 engineering bacterium containing glycosyltransferase UGT7399D1 gene constructed by adopting same method of E.coli engineering bacterium for constructing sucrose synthase in experimental example A
C. Construction of engineering bacterium of E.coli expressing UDP-glucuronic acid decarboxylase AtUXS3
In order to construct an escherichia coli engineering bacterium E.coli BL21 (DE 3)/Pet 28a-AtUXS3 for expressing UDP-glucuronic acid decarboxylase AtUXS3, chemically synthesizing a UDP-glucuronic acid decarboxylase AtUXS3 gene fragment as a template, designing an upstream primer and a downstream primer, and adding enzyme cutting sites BamHI, xhoI and a protecting base:
UDP-glucuronic acid decarboxylase AtUXS3 gene fragment primer F:
GGATCCATGACCTTTAACGCGTATAGCGGCCT(SEQ ID NO.15)
UDP-glucuronic acid decarboxylase AtUXS3 gene fragment primer R:
TGCGCCTGAACGTGCCGCGCAACCTCGAG(SEQ ID NO.16)
E.coli BL21 (DE 3)/pET 28a-AtUXS3 engineering bacterium containing glycosyltransferase UGT7399D1 gene is constructed by adopting same method of constructing E.coli engineering bacterium of sucrose synthase in experimental example A
D. Construction of engineering bacterium of E.coli expressing UDP-sugar epimerase PsUGE2
In order to construct an engineering bacterium E.coli BL21 (DE 3)/Pet 28a-PsUGE2 of Escherichia coli expressing UDP-sugar epimerase PsUGE2, chemically synthesizing a UDP-sugar epimerase PsUGE2 gene fragment as a template, designing an upstream primer and a downstream primer, and adding enzyme cutting sites BamHI, xhoI and a protecting base:
UDP-sugar epimerase PsUGE2 gene fragment primer F:
GGATCCATGGCGAGCACGAGTCAGAAAATTC(SEQ ID NO.17)
the UDP-sugar epimerase PsUGE2 gene fragment primer R:
GTGGGGCTATAGCGGCAAACCGCTCGAG(SEQ ID NO.18)
e.coli BL21 (DE 3)/pET 28a-PsUGE2 engineering bacteria containing glycosyltransferase UGT7399D1 gene constructed by the same method as the E.coli engineering bacteria constructing sucrose synthase in experimental example A
E. Construction of engineering bacteria of E.coli expressing glycosyltransferase UGT99D1
In order to construct an engineering bacterium E.coli BL21 (DE 3)/Pet 28a-UGT99D1 for expressing glycosyltransferase UGT99D1, a glycosyltransferase UGT99D1 gene fragment is chemically synthesized as a template, an upstream primer and a downstream primer are designed, and enzyme cutting sites BamHI, xhoI and a protecting base are added:
Glycosyltransferase UGT99D1 gene fragment primer F:
GGATCCATGGGGAAACCAGCAGCAGGCG(SEQ ID NO.19)
glycosyltransferase UGT99D1 gene fragment primer R:
CGTCCAACGCTTCACCATCTAGCTCGAG(SEQ ID NO.20)
e.coli BL21 (DE 3)/pET 28a-UGT99D1 engineering bacterium containing glycosyltransferase UGT7399D1 gene constructed by the same method as the E.coli engineering bacterium for constructing sucrose synthase in experimental example A
Experimental example 3: fermentation and protein purification of escherichia coli genetically engineered bacteria
A. Fermentation of genetically engineered escherichia coli
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 190 rpm. The seed solution was transferred to 400mL of LB liquid medium containing 100mg/L kanamycin at 1% of the inoculum size, cultured at 37℃for 2-3 hours, and when the OD600 value was 0.6, isopropyl beta-D-thiogalactoside (IPTG, 0.1 mM) was added to induce gene expression at 16 ℃. After induction for 16h, cells were collected by centrifugation (12,160 g,5 min) and then following 40:1 volume ratio cells were resuspended in phosphate buffer (50 mM, pH 7.0). After being cracked by a high-pressure crushing device at 4 ℃, the mixture is centrifuged for 25 minutes at 21,610g at 4 ℃, and the supernatant is crude enzyme liquid containing target protein.
B. Purification of sucrose synthase GuSuS1- Δ9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2, glycosyltransferase UGT99D1
Protein purification was performed using the protein purification system AKTA purifier.
(1) Sample pretreatment: the crude enzyme solution obtained in the previous step is filtered by a 0.45 μm pore size filter and then stored at 4 ℃.
(2) Ni column pretreatment: before loading, the equilibrated Ni column was washed with not less than 10 column volumes of Binding Buffer (25 mM imidazole, 50mM PBS, pH 7.0) at a flow rate of 1 mL/min.
(3) Loading: the sample subjected to the sample loading pretreatment enters the Ni column through a constant flow pump at a flow rate of 0.5mL/min, and penetrating fluid is collected, so that the sample can be repeatedly loaded to improve the column hanging efficiency of the sample. Ni column after equilibrium loading: the nickel column was again equilibrated with a Binding Buffer of greater than 10 column volumes at a flow rate of 1mL/min to remove unbound protein from the column.
(4) Gradient elution: eluting the Ni column according to the different gradient ratio of the solution Buffer (1M imidazole) and the Binding Buffer, wherein the flow rate is 1mL/min, observing and monitoring an OD280 value, collecting eluent corresponding to a protein absorption peak, and determining the target protein after detection by SDS-PAGE.
(5) Concentrating protein with ultrafiltration tube, replacing buffer, detecting protein concentration with Nanodrop 2000c, and storing at 4deg.C.
Experimental example 4: UDP-sugar epimerase PsUGE2 catalyzes the production of UDP-arabinose from UDP-xylose
UDP-arabinose and UDP-xylose were absorbed at ultraviolet 260nm, so UPLC was used to detect UDP-arabinose production.
The total volume of the reaction was 100. Mu.L, consisting of 50. Mu.g of recombinase (UDP-sugar epimerase PsUGE 2), 1Mm UDP-Xyl and 50mM Tris-HCl buffer (pH 8.5). The reaction was placed in a 35℃water bath for 10min, and 400. Mu.L of methanol was added to terminate the reaction. The reaction product is detected by an Shimadzu UPLC system, and UPLC conditions are as follows:
mobile phase: the mobile phase is 100mM potassium phosphate, 8mM tetrabutylammonium bisulfate (pH 6.5), the model of a liquid chromatographic column for analysis is Shim-pack GIST-HP C18 (150×2.1mM,3 μm), the flow rate is 0.3mL/min, the detection wavelength is 260nm, and the temperature of a column incubator is 35 DEG C
The sample to be tested was centrifuged at 12000rpm for 1min to remove the precipitate, and the supernatant was filtered through a 0.22 μm pore size filter to a liquid phase vial to prepare a sample.
The result of the UPLC detection is shown in FIG. 3.
Experimental example 5: construction of UDP-arabinose circulation System and arabinoside Synthesis System
With oleanolic acid/betulinic acid as substrate, adding sucrose, uridine diphosphate and NAD + Adding sucrose synthase GuSuS 1-delta 9, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-glucose epimerase PsUGE2 and glycosyltransferase UGT99D1, and reacting for 6 hours to realize the modification of pentacyclic triterpene compound arabinose and synthesize 3-oxygen-arabinose-oleanolic acid/3-oxygen-arabinose-betulinic acid.
Specifically, 50. Mu.g GuSuS 1-. DELTA.9, 667. Mu.M UDP, 6667. Mu.M sucrose, 83. Mu.M OA/BA, 1333. Mu.M NAD + (nicotinamide adenine dinucleotide) was added to Tris-HCl reaction solution with a total volume of 300. Mu.L, placed in a water bath at 50℃for reaction for 30min, 1. Mu.L of KOH with a concentration of 2M was added to adjust pH to 8.5, 50. Mu.g of AtUGDH was added for reaction for 30min at 35℃and 50. Mu.g of AtUXS was added for reaction for 30min at 35℃and 50. Mu.g of PsUGE2 for reaction for 30min at 25℃and finally 50. Mu.g of UGT99D1 for reaction for 6h, 700. Mu.L of methanol was added to the reaction solution for termination of the reaction, centrifuged at 12000rpm for 1min, and the supernatant was filtered into a liquid phase vial with an organic filter membrane of 0.22. Mu.m, and analyzed with UPLC under the following conditions:
the mobile phase is acetonitrile (A) and 0.1% phosphoric acid (B), the proportion of the initial component B is 80%, the proportion of the component B is reduced to 65% in 3min, the proportion of the component B is reduced to 50% in 4min, the proportion of the component B is reduced to 35% in 8min, the proportion of the component B is reduced to 15% in 12min, the proportion of the component B is reduced to 5% in 15min, the proportion of the component B is increased to 80% in 16min, and the component B is kept for 4min for preparing for the next sample injection; the analytical liquid chromatography column was of the type Shim-pack GIST-HP C18 (150X 2.1mm,3 μm), the flow rate was 0.3mL/min, the detection wavelength was 203nm, and the column oven temperature was 35 ℃.
Experimental example 6: preparation and structural characterization of two pentacyclic triterpene arabinoside compounds
(1) Glycosyltransferase UGT99D1 is obtained according to the operation procedure of experimental example 3, 40mg UDP-arabinose, 1ml DMSO of 0.05M oleanolic acid/betulinic acid are added, samples are taken every 1h for reaction, UPLC detection is utilized to observe the conversion rate of oleanolic acid/betulinic acid, when the oleanolic acid/betulinic acid is about to be converted, the oleanolic acid/betulinic acid is added, a water bath shaking table of 35 ℃ is used for 200rpm, and the reaction is carried out for 10h.
(2) The reaction product was centrifuged at 5000rpm for 20 minutes, the pellet was resuspended in 20ml methanol and filtered through a 0.22 pore size organic filter 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: the 0.1% formic acid aqueous solution, the mobile phase solution is filtered by a 0.22 μm pore size filter membrane and is degassed by ultrasonic vibration. The flow rate was 5mL/min and the detection wavelength was 203nm. The liquid chromatographic column is Shimadzu C18 silica gel column, 20x250mm. The proportion of the initial component B is 80%, the proportion of the component B is reduced to 65% at 18min, the proportion of the component B is reduced to 50% at 24min, the proportion of the component B is reduced to 35% at 64min, the proportion of the component B is reduced to 15% at 72min, the proportion of the component B is reduced to 5% at 90min, and the proportion of the component B is increased to 80% at 96 min. Taking 5mL of the sample obtained in the step 1 to be detected, loading and separating in an injection sample injection mode, collecting liquid from each peak in the UV rays, and detecting by using an analytical high performance liquid chromatograph to judge the retention time corresponding to the glycosylated derivative and judge the purity. After all samples were collected, they were concentrated using a vacuum concentrator and dried to a powder using a freeze dryer and stored at 4 ℃.
(4) Two pentacyclic triterpene arabinoside compound powders were dissolved in deuterated methanol, and subjected to Bruker Assend 700M nuclear magnetic resonance spectrometer 1 H spectrum, 13 C spectrum, COZY and NOESY analysis to determine structural information.
Experimental example 7: construction of Saccharomyces cerevisiae cells for synthesizing 3-oxo-arabinose-oleanolic acid, 3-oxo-arabinose-betulinic acid
In the experimental example, genes of a amyrin alcohol synthase GgbAS, a reductase MtCPR, a P450 oxidase CYR716A12, a UDP-glucose dehydrogenase AtUGDH3, a UDP-glucuronic acid decarboxylase AtUXS3, a UDP-glucose epimerase PsUGE2 and a glycosyltransferase UGT99D1 with optimized yeast codons are introduced into a saccharomyces cerevisiae BY4741 to construct a saccharomyces cerevisiae cell BY4741-OA12-Ara for synthesizing 3-oxo-arabinose-oleanolic acid; yeast codon optimized lup synthase OeLUP, reductase AtCPR, P450 oxidase CYR716A12, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-sugar epimerase PsUGE2 and glycosyltransferase UGT99D1 genes are introduced into Saccharomyces cerevisiae BY4741 to construct Saccharomyces cerevisiae BY4741-BA12-Ara for synthesizing 3-oxygen-arabinose-betulinic acid. Realizes the intracellular synthesis of 3-oxo-arabinose-oleanolic acid and 3-oxo-arabinose-betulinic acid.
1. Test materials and methods
YPD medium: about 10g/L yeast extract, 20g/L glucose, 20g/L peptone, and 20g/L agar powder.
Defect medium: about 6.7g/L YNB (yeast nitrogen base without amino acids), 0.6g-1.3g/L other non-essential amino acid powder (excluding leucine, histidine, tryptophan, uracil), 20g/L glucose or galactose, etc., and the following raw materials are added according to different needs:
leucine powder: 60-100mg/L
Tryptophan powder: 20-40mg/L
Histidine powder: 20-40mg/L
Uracil powder: 20-40mg/L
The solid culture medium is prepared by adding 15-25g/L agar powder, regulating pH to 6.2-6.5, culturing with 100mL triangular flask, and culturing with constant temperature shaking table at 30deg.C and 200rpm for 5 days.
2. Synthesis of 3-oxo-arabinose-oleanolic acid from the head Using engineered Yeast 1
Saccharomyces cerevisiae BY4741 is used as a chassis host, the genes of the amyrin alcohol synthase GgbAS, the reductase MtCPR, the P450 oxidase CYR716A12, the UDP-glucose dehydrogenase AtUGDH3, the UDP-glucuronic acid decarboxylase AtUXS3, the UDP-glucose epimerase PsUGE2 and the glycosyltransferase UGT99D1 are introduced into the Saccharomyces cerevisiae BY4741 to construct a Saccharomyces cerevisiae cell BY4741-OA12-Ara (engineering yeast 1) for synthesizing 3-oxo-glucose-oleanolic acid, and the Saccharomyces cerevisiae cell BY4741-OA12-Ara is cultured for 7 days BY using a YPD culture medium, as shown in figure 11, so that the 3-oxo-arabino-oleanolic acid is synthesized from the head BY using the engineering yeast 1.
3. Synthesis of 3-oxo-arabinose-betulinic acid from the head using engineered yeast 2
Saccharomyces cerevisiae BY4741 is used as a chassis host, lupin synthase OeLUP, reductase MtCPR, P450 oxidase CYR716A12, UDP-glucose dehydrogenase AtUGDH3, UDP-glucuronic acid decarboxylase AtUXS3, UDP-glucose epimerase PsUGE2 and glycosyltransferase UGT99D1 genes are introduced into Saccharomyces cerevisiae BY4741, so that Saccharomyces cerevisiae BY4741-BA12-Ara (engineering yeast 2) for synthesizing 3-oxygen-glucose-betulinic acid is constructed, and cultured for 7 days BY using YPD culture medium, as shown in figure 12, and 3-oxygen-arabinose-betulinic acid is synthesized from the head BY using the engineering yeast 2.
Experimental example 8 pharmacological Activity of 3-oxo-arabinose-oleanolic acid, 3-oxo-arabinose-betulinic acid
The pharmacological activity test is carried out on the obtained 3-oxo-arabinose-oleanolic acid and 3-oxo-arabinose-betulinic acid in the experimental example, the oleanolic acid is taken as a 3-oxo-arabinose-oleanolic acid control, MCF-7 tumor cells are taken as a research object, and the concentrations of 100 mu M, 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M, 3.125 mu M, 1.5625 mu M, 0.7813 mu M and 0.3906 mu M are selected for the test; similarly, betulinic acid is used as a 3-oxygen-arabinose-betulinic acid control, DLD-1 tumor cells are used as study objects, and experiments are carried out by selecting the concentrations of 100 mu M, 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M, 3.125 mu M, 1.5625 mu M, 0.7813 mu M and 0.3906 mu M; the specific embodiment is as follows:
1. Experimental method and procedure
1.1 cell culture
The tumor cell lines were cultured at 37℃under 5% CO as shown in Table 3 2 Is cultured in an incubator of (a). Cells in the logarithmic growth phase were taken for plating at regular passages.
1.2 cell plating
(1) Cell staining was performed with trypan blue and living cells were counted.
(2) The cell concentration was adjusted to the appropriate concentration.
TABLE 1 cell line Density (per well)
(3) 90. Mu.L of the cell suspension was added to each well of the culture plate as shown in Table 1 or Table 2, and the cell-free medium was added to the blank air.
(4) The plates were incubated overnight at 37℃in an incubator with 5% CO2 and 100% relative humidity.
1.3 preparation of Compound storage plates
Preparation of 400X compound storage plates: compounds were diluted with DMSO from the highest concentration gradient to the lowest concentration as shown in table 2 below. Is prepared at any time.
TABLE 2 400X schematic diagrams of Compound storage plates (concentration. Mu.M)
1.4 Preparation of 10X compound working solution and compound treatment cell
(1) Preparing a 10X compound working solution: 78. Mu.L of the cell culture solution was added to a 96-well plate having a V-shaped bottom, and 2. Mu.L of the compound was aspirated from a 400X compound storage plate and added to the cell culture solution in the 96-well plate. 2. Mu.L DMSO was added to the vehicle control and the blank. After adding the compound or DMSO, the mixture is blown and evenly mixed by a gun.
(2) Adding the medicine: mu.L of 10 XCompound working solution was added to the cell culture plates as shown in Table 1 or Table 2. To the vehicle control and the blank control, 10. Mu.L of DMSO-cell culture medium mixture was added. The final DMSO concentration was 0.25%.
(3) The 96-well cell plate was returned to the incubator for culturing, and the detection and reading were performed at 48h and 72h after the drug treatment.
1.5cell Titer-Glo luminescence method cell Activity assay
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit (Promega-G7573).
1. CellTiter-Glo buffer was thawed and left to stand to room temperature.
2. The CellTiter-Glo substrate was left to stand to room temperature.
3. CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.
4. The slow vortex shaking allowed for adequate dissolution.
5. The cell culture plates were removed and allowed to stand for 30 minutes to equilibrate to room temperature.
6. To each well 50. Mu.L (equal to half the volume of cell culture broth in each well) of CellTiter-Glo working fluid was added. The cell plates were wrapped with aluminum foil paper to protect from light.
7. The plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.
8. The plates were left at room temperature for 10 minutes to stabilize the luminescence signal.
9. The luminescence signal is detected at 2104EnVision reader.
1.6 data analysis
The Inhibition Rate (IR) of the test compound was calculated using the following formula: IR (%) = (1- (RLU compound-RLU placebo)/(RLU vehicle control-RLU placebo)) × 100%. Inhibition rates of compounds at different concentrations were calculated in Excel, then plotted using GraphPad Prism software and relevant parameters were calculated.
2. Test results
The pharmacological activity of the 3-oxygen-arabinose-oleanolic acid and the 3-oxygen-arabinose-betulinic acid are obviously increased. After 48h of reaction, as shown in FIG. 13, the inhibition rate of MCF-7 by oleanolic acid is about 33% at maximum, whereas after 48h of reaction, the inhibition rate of 25. Mu.M of 3-oxo-arabinose-oleanolic acid can reach 100%. After 72h of reaction, as shown in FIG. 14, the inhibition rate of DLD-1 by betulinic acid was about 49.5% at the highest, whereas after 72h of reaction, the inhibition rate of 3-oxo-arabinose-betulinic acid was 75.5%.
SEQUENCE LISTING
<110> university of Beijing technology
<120> UDP-sugar epimerase PsUGE2 and use thereof
<130> P210890/BLG
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 436
<212> PRT
<213> Artificial Sequence
<220>
<223> UDP-sugar epimerase PsUGE2
<400> 1
Met Ala Ser Thr Ser Gln Lys Ile Leu Val Thr Gly Gly Ala Gly Phe
1 5 10 15
Ile Gly Ala His Thr Val Val Gln Leu Leu Lys Asp Gly Phe His Val
20 25 30
Ser Ile Ile Asp Asn Phe Asp Asn Ser Ala Met Glu Ala Val Asp Arg
35 40 45
Val Arg Glu Ile Val Gly Pro Asn Leu Ser Gln Asn Leu Glu Phe Thr
50 55 60
Leu Gly Asp Leu Arg Asn Lys Asp Asp Leu Glu Lys Leu Phe Ser Lys
65 70 75 80
Thr Lys Phe Asp Gly Val Ile His Phe Ala Gly Leu Lys Ala Val Gly
85 90 95
Glu Ser Val Ala Asn Pro Arg Arg Tyr Phe Asp Asn Asn Leu Val Gly
100 105 110
Thr Ile Asn Leu Tyr Glu Val Met Ala Thr His Asn Cys Lys Asn Met
115 120 125
Val Phe Ser Ser Ser Ala Thr Val Tyr Gly Gln Pro Lys Lys Met Pro
130 135 140
Cys Val Glu Asp Phe Glu Leu Gln Ala Thr Asn Pro Tyr Gly Arg Thr
145 150 155 160
Lys Leu Phe Leu Glu Glu Ile Ala Arg Asp Ile Ser Lys Ala Glu Pro
165 170 175
Glu Trp Arg Ile Ile Leu Leu Arg Tyr Phe Asn Pro Val Gly Ala His
180 185 190
Glu Ser Gly Lys Leu Gly Glu Asp Pro Arg Gly Ile Pro Asn Asn Leu
195 200 205
Met Pro Tyr Ile Gln Gln Val Ala Val Gly Arg Leu Pro Glu Leu Asn
210 215 220
Val Tyr Gly His Asp Tyr Pro Thr Arg Asp Gly Ser Ala Val Arg Asp
225 230 235 240
Tyr Ile His Val Met Asp Leu Ala Asp Gly His Ile Ala Ala Leu Arg
245 250 255
Lys Leu Phe Thr Thr Glu Asn Ile Gly Cys Thr Ala Tyr Asn Leu Gly
260 265 270
Thr Gly Arg Gly Thr Ser Val Leu Asp Lys Leu Gly Glu Asp Pro Arg
275 280 285
Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Gln Gln Val Ala Val Gly
290 295 300
Arg Leu Pro Glu Leu Asn Val Tyr Gly His Asp Tyr Pro Thr Arg Asp
305 310 315 320
Gly Ser Ala Val Arg Asp Tyr Ile His Val Met Asp Leu Ala Asn Gly
325 330 335
His Ile Ala Ser Leu Arg Lys Leu Phe Ala Thr Glu Asp Ile Gly Cys
340 345 350
Thr Ala Tyr Asn Leu Gly Thr Gly Arg Gly Thr Ser Val Leu Glu Met
355 360 365
Val Asp Ala Phe Asn Lys Ala Ser Gly Lys Lys Ile Thr Leu Lys Leu
370 375 380
Cys Pro Arg Arg Pro Gly Asp Ala Ile Glu Val Tyr Ala Ser Thr Glu
385 390 395 400
Lys Ala Glu Arg Glu Leu Gly Trp Lys Ala Lys Tyr Gly Val Glu Glu
405 410 415
Met Cys Arg Asp Gln Trp Asn Trp Ala Lys Asn Asn Pro Trp Gly Tyr
420 425 430
Ser Gly Lys Pro
435
<210> 2
<211> 1308
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of UDP-sugar epimerase PsUGE2
<400> 2
atggcgagca cgagtcagaa aattctggtg accggcggcg cgggctttat tggcgcgcat 60
accgtggtgc agctgctgaa agatggcttt catgtgagca ttattgataa ctttgataac 120
agcgcgatgg aagcggtgga tcgcgtgcgc gaaattgtgg gcccgaacct gagtcagaac 180
ctggaattta ccctgggcga tctgcgcaac aaagatgatc tggaaaaact gtttagcaaa 240
accaaatttg atggcgtgat tcattttgcg ggcctgaaag cggtgggcga aagcgtggcg 300
aacccgcgcc gctattttga taacaacctg gtgggcacca ttaacctgta tgaagtgatg 360
gcgacccata actgcaaaaa catggtgttt agcagtagcg cgaccgttta tggtcagccg 420
aaaaaaatgc cgtgcgtgga agattttgaa ctgcaagcga ccaacccgta tggccgcacc 480
aaactgtttc tggaagaaat tgcgcgcgat attagcaaag cggaaccgga atggcgcatt 540
attctgctgc gctattttaa cccggtgggc gcgcatgaaa gcggcaaact gggcgaggac 600
ccgcgcggca tcccgaacaa tctgatgccg tatattcagc aagtggcggt gggccgttta 660
ccggaattaa acgtttatgg ccatgattat ccgacgcgtg atggcagcgc ggtgcgcgat 720
tacatccatg tgatggactt agcggatggc cacattgcgg cgctgcgcaa actgttcacc 780
accgaaaaca ttggctgcac cgcgtataac ctgggcaccg gccgcggcac gagcgtgctg 840
gataagttag gtgaagatcc gcgcggcatt ccgaacaact taatgccata cattcagcaa 900
gtggcggtgg gccgcctgcc ggaactgaac gtttatggcc atgattatcc gacccgcgat 960
ggcagcgcgg ttcgcgatta tattcatgtg atggatctgg cgaacggcca tattgcgagc 1020
ctgcgtaaac tgttcgcgac ggaagatatt ggctgcacgg cgtataatct gggcaccggc 1080
cgcggcacga gcgtgctgga aatggtggat gcgtttaaca aagcgagcgg caaaaaaatt 1140
accctgaaac tgtgcccgcg ccgcccgggc gatgcgattg aagtgtatgc gagcaccgaa 1200
aaagcggaac gcgaactggg ctggaaagcg aaatatggcg tggaagaaat gtgccgcgat 1260
cagtggaact gggcgaaaaa caacccgtgg ggctatagcg gcaaaccg 1308
<210> 3
<211> 797
<212> PRT
<213> Artificial Sequence
<220>
<223> sucrose synthase GuSuS1- Δ9
<400> 3
His Ser Leu Arg Glu Arg Leu Asp Glu Thr Leu Thr Ala Asn Arg Asn
1 5 10 15
Glu Ile Leu Ala Leu Leu Ser Arg Ile Glu Ala Lys Gly Lys Gly Ile
20 25 30
Leu Gln His His Gln Val Ile Ala Glu Phe Glu Glu Ile Pro Glu Glu
35 40 45
Asn Arg His Lys Leu Met Asp Gly Ala Phe Gly Glu Val Leu Arg Ser
50 55 60
Thr Gln Glu Ala Ile Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg
65 70 75 80
Pro Arg Pro Gly Val Trp Glu Tyr Leu Arg Val Asn Val His Ala Leu
85 90 95
Val Val Glu Glu Leu Gln Pro Ala Glu Phe Leu Arg Phe Lys Glu Glu
100 105 110
Leu Val Asp Gly Ser Ser Asn Gly Asn Phe Val Leu Glu Leu Asp Phe
115 120 125
Glu Pro Phe Thr Ala Ser Phe Pro Arg Pro Thr Leu Asn Lys Ser Ile
130 135 140
Gly Asn Gly Val Gln Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe
145 150 155 160
His Asp Lys Glu Ser Leu His Pro Leu Leu Glu Phe Leu Arg Leu His
165 170 175
Ser Tyr Lys Gly Lys Thr Leu Met Leu Asn Asp Arg Ile Gln Thr Pro
180 185 190
Asp Ser Leu Gln His Val Leu Arg Lys Ala Glu Glu Tyr Leu Gly Thr
195 200 205
Leu Ser Pro Glu Thr Pro Tyr Ser Val Phe Glu His Lys Phe Gln Glu
210 215 220
Ile Gly Leu Glu Arg Gly Trp Gly Asp Thr Ala Glu Arg Val Leu Glu
225 230 235 240
Ser Ile Gln Leu Leu Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr
245 250 255
Leu Glu Thr Phe Leu Gly Arg Ile Pro Met Val Phe Asn Val Val Ile
260 265 270
Leu Ser Pro His Gly Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro
275 280 285
Asp Thr Gly Gly Gln Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu
290 295 300
Glu Asn Glu Met Leu His Arg Ile Lys Gln Gln Gly Leu Asp Ile Val
305 310 315 320
Pro Arg Ile Leu Ile Ile Thr Arg Leu Leu Pro Asp Ala Val Gly Thr
325 330 335
Thr Cys Gly Gln Arg Leu Glu Lys Val Phe Gly Thr Glu His Cys His
340 345 350
Ile Leu Arg Val Pro Phe Arg Asn Glu Lys Gly Met Val Arg Lys Trp
355 360 365
Ile Ser Arg Phe Glu Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp
370 375 380
Val Ala His Glu Leu Ala Lys Glu Leu Gln Gly Lys Pro Asp Leu Ile
385 390 395 400
Val Gly Asn Tyr Ser Asp Gly Asn Ile Val Ala Ser Leu Leu Ala His
405 410 415
Lys Leu Gly Val Thr Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr
420 425 430
Lys Tyr Pro Glu Ser Asp Ile Tyr Trp Lys Lys Phe Glu Glu Lys Tyr
435 440 445
His Phe Ser Cys Gln Phe Thr Ala Asp Leu Phe Ala Met Asn His Thr
450 455 460
Asp Phe Ile Ile Thr Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Asp
465 470 475 480
Thr Val Gly Gln Tyr Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu
485 490 495
Tyr Arg Val Val His Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile
500 505 510
Val Ser Pro Gly Ala Asp Gln Thr Ile Tyr Phe Pro Tyr Thr Asp Thr
515 520 525
Ser Arg Arg Leu Thr Ser Phe His Pro Glu Ile Glu Glu Leu Leu Tyr
530 535 540
Ser Ser Val Glu Asn Glu Glu His Ile Cys Val Leu Lys Asp Arg Asn
545 550 555 560
Lys Pro Ile Ile Phe Thr Met Ala Arg Leu Asp Arg Val Lys Asn Ile
565 570 575
Thr Gly Leu Val Glu Trp Tyr Gly Lys Asn Ala Lys Leu Arg Glu Leu
580 585 590
Val Asn Leu Val Val Val Ala Gly Asp Arg Arg Lys Glu Ser Lys Asp
595 600 605
Leu Glu Glu Lys Ala Glu Met Lys Lys Met Tyr Gly Leu Ile Glu Thr
610 615 620
Tyr Lys Leu Asn Gly Gln Phe Arg Trp Ile Ser Ser Gln Met Asn Arg
625 630 635 640
Val Arg Asn Gly Glu Leu Tyr Arg Val Ile Cys Asp Thr Lys Gly Ala
645 650 655
Phe Val Gln Pro Ala Val Tyr Glu Ala Phe Gly Leu Thr Val Val Glu
660 665 670
Ala Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Cys Asn Gly Gly Pro
675 680 685
Ala Glu Ile Ile Val His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr
690 695 700
His Gly Ala Ala Ala Ala Asp Leu Leu Val Glu Phe Phe Glu Lys Cys
705 710 715 720
Lys Ala Asp Pro Ser His Trp Asp Asn Ile Ser His Gly Gly Leu Gln
725 730 735
Arg Ile Glu Glu Lys Tyr Thr Trp Gln Ile Tyr Ser Glu Arg Leu Leu
740 745 750
Thr Leu Thr Gly Val Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp
755 760 765
Arg Arg Glu Ser Arg Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr
770 775 780
Arg Lys Leu Ala Glu Ser Val Pro Leu Ala Val Glu Glu
785 790 795
<210> 4
<211> 2391
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of sucrose synthase GuSuS1- Δ9
<400> 4
cacagtctcc gtgagaggct cgatgaaacc ttgactgcta atagaaatga aattttggcc 60
cttctctcaa ggatcgaagc caagggcaag gggatcctgc aacaccacca ggtcattgct 120
gagtttgagg aaattcctga ggagaataga cataagctga tggatggggc atttggagaa 180
gtcttgagat ccacacagga agccatagtt ttaccaccat gggttgctct ggctgttcgt 240
ccaaggcctg gtgtttggga gtacctgaga gtgaatgtgc acgctcttgt tgtcgaagag 300
ttgcaacctg ctgagtttct ccgcttcaag gaggaacttg ttgatggaag ttctaatggc 360
aactttgtgc ttgagttgga ctttgaacca tttactgcat ccttcccccg cccaactctc 420
aacaagtcaa ttggaaatgg tgtgcaattc ctcaaccgtc acctttctgc aaaactcttc 480
catgacaagg agagcttgca tccacttctg gaattcctca gacttcacag ctacaaggga 540
aagacattga tgttgaatga cagaattcaa accccggatt ctcttcaaca tgttctgagg 600
aaagctgaag agtatcttgg aacactttct cctgagacac cctactcagt atttgagcac 660
aagttccagg agatcggttt ggagagaggg tggggtgaca ccgcggagcg tgtcctcgag 720
tccatccaac tcctcttgga tcttcttgag gctcctgacc cttgcaccct tgagactttc 780
cttggaagga tccccatggt ctttaatgtt gtgatccttt cgccccacgg ttactttgcc 840
caagataatg tcttgggata ccctgatacc ggtggccagg ttgtttacat cttggatcaa 900
gttcgcgcct tggagaatga gatgctccat cgcattaagc aacaaggctt ggatatcgtc 960
cctcgcattc tcattatcac ccgtcttctc cccgatgcag taggaactac ctgtggccaa 1020
cgactcgaga aggtctttgg aaccgagcat tgccacattc ttcgagttcc cttcagaaac 1080
gagaagggaa tggttcgcaa gtggatctca agattcgaag tctggccata cctagaaact 1140
tacactgagg atgttgccca tgaacttgcc aaagagttgc aaggcaagcc agatctgatt 1200
gttggaaact acagtgatgg aaacattgtt gcctctttgt tggcacataa attaggtgtc 1260
actcagtgta ccattgctca tgcacttgag aagaccaagt accctgaatc tgacatttac 1320
tggaaaaaat tcgaagagaa atatcacttc tcttgccaat tcacagctga tctctttgct 1380
atgaaccaca cagacttcat catcaccagt accttccaag agattgctgg aagcaaggac 1440
actgttggac agtatgagag tcacactgcc ttcacccttc ctggactcta ccgtgtcgtg 1500
cacggtattg atgtctttga tccaaaattc aacattgtat ctcccggagc tgatcagacc 1560
atctacttcc cctacaccga caccagccgc aggctgacat ccttccaccc cgaaatcgaa 1620
gagcttcttt acagctcagt ggagaatgaa gagcacatat gtgtattgaa ggaccgcaac 1680
aagccaatta tcttcaccat ggcgaggttg gaccgtgtga agaacatcac tggacttgtc 1740
gagtggtacg gcaagaacgc caagctccgt gagctggtga accttgtggt tgttgccgga 1800
gacaggagga aggagtccaa ggacttggaa gagaaggccg agatgaagaa gatgtacggc 1860
ctgattgaga cctacaagct gaatggccaa ttcaggtgga tctcctctca gatgaaccgg 1920
gtgaggaacg gggagctgta ccgtgtcatc tgcgacacaa agggagcttt cgtgcagcct 1980
gctgtctatg aggcctttgg attgacagtt gttgaggcca tgacttgtgg gttgccaaca 2040
tttgcaacat gcaatggtgg ccctgctgag atcattgttc atggcaagtc tggtttccac 2100
attgaccctt accacggcgc ggccgccgcc gatctccttg ttgaattctt tgagaagtgc 2160
aaggctgacc catctcactg ggacaacatc tcccatggtg gtctccaacg tattgaagag 2220
aagtatacat ggcaaattta ctctgagagg cttctcactc tcactggtgt ctatggcttc 2280
tggaagcatg tgtctaacct tgaccgccgc gagagccgcc gttatcttga gatgttctat 2340
gctctcaagt accgcaaatt ggctgagtct gtgcccctag ctgttgagga g 2391
<210> 5
<211> 480
<212> PRT
<213> Artificial Sequence
<220>
<223> UDP-glucose dehydrogenase AtUGDH3
<400> 5
Met Val Lys Ile Cys Cys Ile Gly Ala Gly Tyr Val Gly Gly Pro Thr
1 5 10 15
Met Ala Val Ile Ala Leu Lys Cys Pro Ser Val Glu Val Ala Val Val
20 25 30
Asp Ile Ser Val Pro Arg Ile Asn Ala Trp Asn Ser Asp Gln Leu Pro
35 40 45
Ile Tyr Glu Pro Gly Leu Asp Asp Val Val Lys Gln Cys Arg Gly Lys
50 55 60
Asn Leu Phe Phe Ser Thr Asp Val Glu Lys His Val Arg Glu Ala Asp
65 70 75 80
Ile Val Phe Val Ser Val Asn Thr Pro Thr Lys Thr Arg Gly Leu Gly
85 90 95
Ala Gly Lys Ala Ala Asp Leu Thr Tyr Trp Glu Ser Ala Ala Arg Met
100 105 110
Ile Ala Asp Val Ser Val Ser Asp Lys Ile Val Val Glu Lys Ser Thr
115 120 125
Val Pro Val Lys Thr Ala Glu Ala Ile Glu Lys Ile Leu Thr His Asn
130 135 140
Ser Lys Gly Ile Lys Phe Gln Ile Leu Ser Asn Pro Glu Phe Leu Ala
145 150 155 160
Glu Gly Thr Ala Ile Glu Asp Leu Phe Met Pro Asp Arg Val Leu Ile
165 170 175
Gly Gly Arg Glu Thr Thr Glu Gly Phe Ala Ala Val Lys Ala Leu Lys
180 185 190
Asp Ile Tyr Ala Gln Trp Val Pro Glu Glu Arg Ile Leu Thr Thr Asn
195 200 205
Leu Trp Ser Ala Glu Leu Ser Lys Leu Ala Ala Asn Ala Phe Leu Ala
210 215 220
Gln Arg Ile Ser Ser Val Asn Ala Met Ser Ala Leu Cys Glu Ala Thr
225 230 235 240
Gly Ala Asn Val Ser Glu Val Ser Tyr Ala Val Gly Lys Asp Ser Arg
245 250 255
Ile Gly Pro Lys Phe Leu Asn Ser Ser Val Gly Phe Gly Gly Ser Cys
260 265 270
Phe Gln Lys Asp Ile Leu Asn Leu Val Tyr Ile Cys Glu Cys Asn Gly
275 280 285
Leu Pro Glu Val Ala Glu Tyr Trp Lys Gln Val Ile Lys Ile Asn Asp
290 295 300
Tyr Gln Lys Thr Arg Phe Val Asn Arg Ile Val Ser Ser Met Phe Asn
305 310 315 320
Thr Val Ser Asn Lys Lys Ile Ala Val Leu Gly Phe Ala Phe Lys Lys
325 330 335
Asp Thr Gly Asp Thr Arg Glu Thr Pro Ala Ile Asp Val Cys Lys Gly
340 345 350
Leu Leu Gly Asp Lys Ala Arg Leu Ser Ile Tyr Asp Pro Gln Val Thr
355 360 365
Glu Glu Gln Ile Gln Arg Asp Leu Thr Met Asn Lys Phe Asp Trp Asp
370 375 380
His Pro Leu His Leu Gln Pro Met Ser Pro Thr Thr Val Lys Gln Val
385 390 395 400
Ser Val Ala Trp Asp Ala Tyr Thr Ala Thr Lys Asp Ala His Gly Ile
405 410 415
Cys Ile Leu Thr Glu Trp Asp Glu Phe Lys Lys Leu Asp Phe Gln Arg
420 425 430
Ile Phe Glu Asn Met Gln Lys Pro Ala Phe Val Phe Asp Gly Arg Asn
435 440 445
Val Val Asp Ala Asp Lys Leu Arg Glu Ile Gly Phe Ile Val Tyr Ser
450 455 460
Ile Gly Lys Pro Leu Asp Gln Trp Leu Lys Asp Met Pro Ala Leu Ala
465 470 475 480
<210> 6
<211> 1440
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of UDP-glucose dehydrogenase AtUGDH3
<400> 6
atggtgaaaa tttgctgcat tggcgcgggc tatgtgggcg gcccgaccat ggcggtgatt 60
gcgctgaaat gcccggatgt ggaagtggcg gtggtggata ttagcgtgcc gcgcattaac 120
gcgtggaaca gcgataccct gccgatttat gaaccgggcc tggatgatgt ggtgaaacag 180
tgccgcggca aaaacctgtt ttttagcacc gatgtggaaa aacatgtgcg cgaagcggat 240
attgtgtttg tgagcgtgaa caccccgacc aaaacccgcg gcctgggcgc gggcaaagcg 300
gcggatctga cctattggga aagcgcggcg cgcatgattg cggatgtgag cgtgagcgat 360
aaaattgtgg tggaaaaaag caccgtgccg gtgaaaaccg cggaagcgat tgaaaaaatt 420
ctgacccata acagcaaagg cattaaattt cagattctga gcaacccgga atttctggcg 480
gaaggcaccg cgattaaaga tctgtttaac ccggatcgcg tgctgattgg cggccgtgaa 540
accccggaag gctttaaagc ggtgcagacc ctgaaaaacg tgtatgcgca ttgggtgccg 600
gaaggtcaga ttattaccac caacctgtgg agcgcggaac tgagcaaact ggcggcgaac 660
gcgtttctgg cgcagcgcat tagcagcgtg aacgcgatga gcgcgctgtg cgaagcgacc 720
ggcgcggatg tgacccaagt gagctatgcg gtgggcaccg atagccgcat tggcccgaaa 780
tttctgaaca gcagcgtggg ctttggcggc agctgctttc agaaagatat tctgaacctg 840
gtgtatattt gcgaatgcaa cggcctgccg gaagtggcgg aatattggaa acaagtgatt 900
aaaattaacg attatcagaa aagccgcttt gtgaaccgcg tggtgagcag catgtttaac 960
agcgtgagca acaaaaaaat tgcggtgctg ggctttgcgt ttaaaaaaga taccggcgat 1020
acgcgcgaaa ccccggcgat tgatgtgtgc aaaggcctgc tggaagataa agcgcgcctg 1080
agcatttatg atccgcaagt gaccgaagat cagattcagc gcgatctgag catgaacaaa 1140
tttgattggg atcatccgct gcatctgcag ccgatgagcc cgaccaccgt gaaacaagtg 1200
accgtgacct gggatgcgta tgaagcgacc aaagatgcgc atggcatttg cattatgacc 1260
gaatgggatg aatttaaaaa cctggatttt cagaaaattt ttgataacat gcagaaaccg 1320
gcgtttgtgt ttgatggccg caacattatg aacctgcaga aactgcgcga aattggcttt 1380
attgtgtata gcattggcaa accgctggat gattggctga aagatatgcc ggcggtggcg 1440
<210> 7
<211> 357
<212> PRT
<213> Artificial Sequence
<220>
<223> UDP-glucuronic acid decarboxylase AtUXS3
<400> 7
Met Thr Phe Asn Ala Tyr Ser Gly Leu Arg Ser Leu Ser Gln Ala Met
1 5 10 15
Ala Ala Thr Ser Glu Lys Gln Asn Thr Thr Lys Pro Pro Pro Ser Pro
20 25 30
Ser Pro Leu Arg Asn Ser Lys Phe Cys Gln Pro Asn Met Arg Ile Leu
35 40 45
Ile Ser Gly Gly Ala Gly Phe Ile Gly Ser His Leu Val Asp Lys Leu
50 55 60
Met Glu Asn Glu Lys Asn Glu Val Val Val Ala Asp Asn Tyr Phe Thr
65 70 75 80
Gly Ser Lys Glu Asn Leu Lys Lys Trp Ile Gly His Pro Arg Phe Glu
85 90 95
Leu Ile Arg His Asp Val Thr Glu Pro Leu Leu Ile Glu Val Asp Arg
100 105 110
Ile Tyr His Leu Ala Cys Pro Ala Ser Pro Ile Phe Tyr Lys Tyr Asn
115 120 125
Pro Val Lys Thr Ile Lys Thr Asn Val Ile Gly Thr Leu Asn Met Leu
130 135 140
Gly Leu Ala Lys Arg Val Gly Ala Arg Ile Leu Leu Thr Ser Thr Ser
145 150 155 160
Glu Val Tyr Gly Asp Pro Leu Ile His Pro Gln Pro Glu Ser Tyr Trp
165 170 175
Gly Asn Val Asn Pro Ile Gly Val Arg Ser Cys Tyr Asp Glu Gly Lys
180 185 190
Arg Val Ala Glu Thr Leu Met Phe Asp Tyr His Arg Gln His Gly Ile
195 200 205
Glu Ile Arg Ile Ala Arg Ile Phe Asn Thr Tyr Gly Pro Arg Met Asn
210 215 220
Ile Asp Asp Gly Arg Val Val Ser Asn Phe Ile Ala Gln Ala Leu Arg
225 230 235 240
Gly Glu Ala Leu Thr Val Gln Lys Pro Gly Thr Gln Thr Arg Ser Phe
245 250 255
Cys Tyr Val Ser Asp Met Val Asp Gly Leu Ile Arg Leu Met Glu Gly
260 265 270
Asn Asp Thr Gly Pro Ile Asn Ile Gly Asn Pro Gly Glu Phe Thr Met
275 280 285
Val Glu Leu Ala Glu Thr Val Lys Glu Leu Ile Asn Pro Ser Ile Glu
290 295 300
Ile Lys Met Val Glu Asn Thr Pro Asp Asp Pro Arg Gln Arg Lys Pro
305 310 315 320
Asp Ile Ser Lys Ala Lys Glu Val Leu Gly Trp Glu Pro Lys Val Lys
325 330 335
Leu Arg Glu Gly Leu Pro Leu Met Glu Glu Asp Phe Arg Leu Arg Leu
340 345 350
Asn Val Pro Arg Asn
355
<210> 8
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of UDP-glucuronic acid decarboxylase AtUXS3
<400> 8
ggatccatga cctttaacgc gtatagcggc ctgcgcagcc tgagccaagc gatggcggcg 60
acgagcgaaa aacagaacac caccaaaccg ccaccgagcc cgagcccgct gcgcaacagc 120
aaattttgtc agccgaacat gcgcattctg attagcggcg gcgcgggctt tattggcagc 180
catctggtgg ataaactgat ggaaaacgaa aaaaacgaag tggttgtggc ggataactat 240
tttaccggca gcaaagaaaa cctgaaaaaa tggattggcc atccgcgctt tgaactgatt 300
cgccatgatg tgaccgaacc gctgctgatt gaagtggatc gcatttatca tctggcgtgc 360
ccggcgagcc cgatttttta taaatataac ccggtgaaaa ccattaaaac caacgtgatt 420
ggcaccctga acatgctggg cctggcgaaa cgcgtgggcg cgcgcattct gctgacgagc 480
acgagcgaag tgtatggcga tccgctgatt catccgcagc cggaaagcta ttggggcaac 540
gtgaacccga ttggcgtgcg cagctgctat gatgaaggca aacgcgtggc ggaaaccctg 600
atgtttgatt atcatcgtca gcatggcatt gaaattcgca ttgcgcgcat ttttaacacc 660
tatggcccgc gcatgaacat tgatgatggc cgcgtggtga gcaactttat tgcgcaagcg 720
ctgcgcggcg aagcgctgac cgtgcagaaa ccgggcacgc agacccgcag cttttgctat 780
gtgagcgata tggtggatgg cctgattcgc ctgatggaag gcaacgatac cggcccgatt 840
aacattggca acccgggcga atttaccatg gtggaactgg cggaaaccgt gaaagaactg 900
attaacccga gcattgaaat taaaatggtg gaaaacaccc cggatgatcc gcgtcagcgc 960
aaaccggata ttagcaaagc gaaagaagtg ctgggctggg aaccgaaagt gaaactgcgc 1020
gaaggcctgc cgctgatgga agaagatttt cgcctgcgcc tgaacgtgcc gcgcaacctc 1080
gag 1083
<210> 9
<211> 489
<212> PRT
<213> Artificial Sequence
<220>
<223> glycosyltransferase UGT99D1
<400> 9
Met Gly Lys Pro Ala Ala Gly Glu Glu Leu Ala Ala Ala Gly Tyr Glu
1 5 10 15
Gly Lys Gln Gln Gln Arg Ala His Phe Val Phe Ile Pro Leu Met Ala
20 25 30
Gln Gly His Val Ile Pro Ser Leu Asp Thr Ala Leu Leu Leu Ala Ser
35 40 45
Gln Gly Ala Val Cys Ser Met Val Ala Thr Pro Trp Thr Ala Ala Arg
50 55 60
Ile Arg Pro Cys Ile Glu Gln Cys Gly Leu Glu Val Arg Leu Leu Glu
65 70 75 80
Phe Pro Leu Glu Tyr Val Ser Asp Gly Ala Asp Asn Leu Asp Asn Ile
85 90 95
Pro Pro Glu Arg Val Val Gly Tyr Phe Gln Ala Val Ala Leu Leu Gln
100 105 110
Ala Pro Ile Gln Glu Arg Leu Gly Lys Leu Glu Pro Arg Val Ser Cys
115 120 125
Ile Val Ser Asp Phe Ser His Pro Trp Thr Ala Pro Val Ala Ala Ala
130 135 140
Leu Gly Val Pro Arg Ile Ser Phe Phe Pro Met Cys Ala Phe Cys Ala
145 150 155 160
Leu Ser Glu His Asn Val His Glu Tyr Asn Lys Cys Leu Cys Ser Pro
165 170 175
Gly Ser Glu Glu Val Val Ala Val Pro Leu Leu Asp Ala Thr Leu Leu
180 185 190
Glu Met Arg Arg Val Glu Ala Pro Cys Phe Phe Arg His Ala Ser Met
195 200 205
Gly Thr Leu Gly Glu Asp Ile Gln Arg Ala His Ser Gln Gly Ala Gly
210 215 220
Met Ile Phe Asn Ser Phe Leu Glu Leu Glu Pro Asp Tyr Val Arg Gly
225 230 235 240
Leu Ser Ser Ala Trp Gly Gly Lys Lys Val Trp Thr Val Gly Pro Val
245 250 255
Ser Leu His His Gln Leu Ala Ala Ala Thr Thr Cys Arg Gly Glu Glu
260 265 270
Ala Ser Met Asp Asp Asp Cys Leu Gln Trp Leu Asp Gly Lys Glu Pro
275 280 285
Gly Ser Val Val Tyr Val Ser Phe Gly Thr Ile Ala Pro Lys Met Glu
290 295 300
Pro Glu Met Leu Leu Glu Leu Ala Arg Ala Leu Glu Thr Ser Gly His
305 310 315 320
Pro Phe Ile Trp Ala Leu Ser Lys Ala Asp His Pro Phe Ala Glu Thr
325 330 335
Ser Gln Glu Leu Gln Glu Leu Glu Ala Arg Val Ala Ala Ser Gly Gly
340 345 350
Gly Arg Ile Val Arg Gly Trp Val Pro Gln Leu Leu Ile Leu Ser His
355 360 365
Ala Ala Val Gly Cys Leu Phe Thr His Ser Gly Trp Asn Ser Val Met
370 375 380
Glu Ala Ile Thr Ala Gly Lys Pro Ala Val Thr Trp Pro Arg Leu Ile
385 390 395 400
Gly Ser Asp His Phe Val Asn Glu Lys Phe Thr Val Glu Val Leu Arg
405 410 415
Ile Gly Val Ser Val Arg Pro Glu Asp Pro His Ile Gln Pro Val Glu
420 425 430
Val Arg Arg Glu Ala Ile Gln Ala Ala Leu Thr Ala Val Leu Glu Gly
435 440 445
Gly Asp Glu Gly Gln Glu Arg Arg Asn Arg Val Arg Asp Leu Ser Leu
450 455 460
Lys Ala Lys Ala Ala Met Gln Pro Gly Gly Ser Ser His Ala Asn Leu
465 470 475 480
Ser Asp Leu Val Gln Arg Phe Thr Ile
485
<210> 10
<211> 1470
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of glycosyltransferase UGT99D1
<400> 10
atggggaaac cagcagcagg cgaagaatta gcagcagctg ggtacgaggg gaagcagcag 60
cagcgtgcgc actttgtgtt catcccgctg atggcccagg gccacgtgat ccctagcctg 120
gacaccgcgc tgctgctcgc cagccagggc gccgtgtgca gcatggtggc caccccgtgg 180
acggccgcga ggatccggcc gtgcatcgag cagtgtggtc tggaggtccg tctgctggag 240
ttcccgctgg agtacgtgtc tgacggcgcc gacaacctgg acaacatccc gccggagcgg 300
gtggtgggct acttccaggc ggtggcgctc ctgcaggcgc ccatccagga gcgcctgggc 360
aagctggagc cgcgcgtgag ctgcatcgtg tcggacttct cccacccgtg gacggctccc 420
gtcgcggcgg ctctcggggt gccacgcatc agcttcttcc ccatgtgtgc cttctgcgcc 480
ctctccgagc acaacgtcca cgagtacaac aaatgccttt gctctccggg gtcggaggag 540
gtggtggcgg tgcctctgct ggacgcgacg ctgctggaga tgcggagggt ggaagccccg 600
tgtttcttcc ggcacgcgtc catggggacg ctgggcgagg acatccagcg agcgcattcc 660
cagggcgccg gcatgatctt caactccttc ctggagctgg agccggacta cgtccggggc 720
ttgagctccg cttggggtgg caagaaggtg tggactgtcg gcccggtatc cctccaccac 780
cagctcgccg ccgccaccac ctgtagaggc gaagaagctt ccatggacga cgactgcctc 840
cagtggctgg acggcaagga gcctggctcc gtcgtgtacg tcagcttcgg gaccatcgct 900
cccaagatgg agcccgagat gctgctggag ctggcgcggg cgctggagac gtcgggccac 960
cccttcatct gggccctcag caaggccgac catccgttcg cggagacgtc gcaggagctg 1020
caggagctgg aggcgcgcgt cgccgccagt ggcggtggcc ggatcgtgag aggatgggtg 1080
ccccagctgc tcatcctctc gcacgccgcc gtcggctgct tgttcacgca cagcgggtgg 1140
aactccgtca tggaggccat cacggctggg aagcccgcgg tgacgtggcc tcgtctcata 1200
gggtcggacc acttcgtgaa cgagaagttc accgtggagg tgctacggat cggtgtcagc 1260
gtccggccgg aggaccctca catccagccc gtggaggtgc gccgggaggc catccaggcg 1320
gcgctcaccg ccgtcctgga aggaggagac gagggccagg agaggcgaaa ccgcgtccgc 1380
gatctctctc tcaaggcaaa ggcggccatg cagcctggtg gatcgtcgca cgccaacctc 1440
tccgacctcg tccaacgctt caccatctag 1470
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> sucrose synthase GuSuS1- Δ9 Gene fragment primer F
<400> 11
ggatcccaca gtctccgtga gaggctcg 28
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> sucrose synthase GuSuS1- Δ9 Gene fragment primer R
<400> 12
tgtgccccta gctgttgagg agctcgag 28
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-glucose dehydrogenase AtUGDH3 Gene fragment primer F
<400> 13
ggatccatgg tgaaaatttg ctgcattggc g 31
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-glucose dehydrogenase AtUGDH3 Gene fragment primer R
<400> 14
ctgaaagata tgccggcggt ggcgctcgag 30
<210> 15
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-glucuronic acid decarboxylase AtUXS3 Gene fragment primer F
<400> 15
ggatccatga cctttaacgc gtatagcggc ct 32
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-glucuronic acid decarboxylase AtUXS3 gene fragment primer R
<400> 16
tgcgcctgaa cgtgccgcgc aacctcgag 29
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-sugar epimerase PsUGE2 Gene fragment primer F
<400> 17
ggatccatgg cgagcacgag tcagaaaatt c 31
<210> 18
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> UDP-sugar epimerase PsUGE2 Gene fragment primer R
<400> 18
gtggggctat agcggcaaac cgctcgag 28
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> glycosyltransferase UGT99D1 Gene fragment primer F
<400> 19
ggatccatgg ggaaaccagc agcaggcg 28
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> glycosyltransferase UGT99D1 Gene fragment primer R
<400> 20
cgtccaacgc ttcaccatct agctcgag 28
Claims (18)
1. The UDP-sugar epimerase PsUGE2 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
2. The UDP-sugar epimerase PsUGE2 according to claim 1, wherein the gene sequence thereof is shown in SEQ ID NO. 2.
3. A method for synthesizing UDP-arabinose is characterized in that UDP-sugar epimerase PsUGE2 with an amino acid sequence shown as SEQ ID NO.1 or a gene sequence shown as SEQ ID NO.2 is adopted to catalyze a reaction on a substrate.
4. A method for synthesizing UDP-arabinose according to claim 3, wherein said catalytic reaction system comprises: 1Mm substrate, 0.5. Mu.g/. Mu.LUDP-sugar epimerase PsUGE2, the remainder being buffer.
5. The method for synthesizing UDP-arabinose according to claim 4, wherein the substrate is UDP-xylose; the buffer is Tris-HCl buffer with pH of 8.5.
6. A process for synthesizing UDP-arabinose according to claim 4, wherein the catalytic reaction conditions are 35℃for 10min.
7. A set of enzymes for arabinose modification of pentacyclic triterpene compounds, comprising: the UDP-sugar epimerase PsUGE2 with the amino acid sequence shown in SEQ ID NO.1 or the gene sequence shown in SEQ ID NO.2, the sucrose synthase GuSuS 1-delta 9 with the amino acid sequence shown in SEQ ID NO.3 or the gene sequence shown in SEQ ID NO.4, the UDP-glucose dehydrogenase AtUGDH3 with the amino acid sequence shown in SEQ ID NO.5 or the gene sequence shown in SEQ ID NO.6, the UDP-glucuronic acid decarboxylase AtUXS3 with the amino acid sequence shown in SEQ ID NO.7 or the gene sequence shown in SEQ ID NO.8, the glycosyltransferase UGT99D1 with the amino acid sequence shown in SEQ ID NO.9 or the gene sequence shown in SEQ ID NO. 10.
8. The set of enzymes for arabinose modification of pentacyclic triterpene compounds according to claim 7, wherein said pentacyclic triterpene compounds are selected from the group consisting of: oleanolic acid and/or betulinic acid.
9. A method for carrying out arabinose modification on pentacyclic triterpene compounds is characterized in that UDP-glucose epimerase PsUGE2 with an amino acid sequence shown as SEQ ID NO.1 or a gene sequence shown as SEQ ID NO.2, sucrose synthase GuSuS 1-delta 9 with an amino acid sequence shown as SEQ ID NO.3 or a gene sequence shown as SEQ ID NO.4, UDP-glucose dehydrogenase AtUGDH3 with an amino acid sequence shown as SEQ ID NO.5 or a gene sequence shown as SEQ ID NO.6, UDP-glucuronic acid decarboxylase AtUXS3 with an amino acid sequence shown as SEQ ID NO.7 or a gene sequence shown as SEQ ID NO.8, and glycosyltransferase UGT99D1 with an amino acid sequence shown as SEQ ID NO.9 or a gene sequence shown as SEQ ID NO.10 are adopted, and the pentacyclic triterpene compounds are used as substrates for catalytic reaction.
10. A method of arabinose modification of pentacyclic triterpene compounds according to claim 9, wherein said catalytic reaction comprises the steps of:
(1) Sucrose synthase GuSuS 1-delta 9, UDP, substrate, glycosyl donor and NAD + Reacting the reaction system;
(2) After regulating the pH value, adding UDP-glucose dehydrogenase AtUGDH3 into the reaction system of the step (1) for reaction;
(3) Adding UDP-glucuronic acid decarboxylase AtUXS3 into the reaction system in the step (2) for reaction;
(4) Adding UDP-sugar epimerase PsUGE2 into the reaction system of the step (3) for reaction;
(5) Adding glycosyltransferase UGT99D1 into the reaction system of the step (4) for reaction.
11. The method for arabinose modification of pentacyclic triterpene compounds according to claim 10, wherein in the step (1), said reaction system comprises: 0.17. Mu.g/. Mu.L sucrose synthase GuSuS 1-. DELTA.9, 667. Mu.MUDP, 83. Mu.M substrate, 6667. Mu.M glycosyl donor, 1333. Mu.MNAD + The method comprises the steps of carrying out a first treatment on the surface of the The reaction conditions are 50 ℃ for 30min.
12. The method for performing arabinose modification on pentacyclic triterpene compound according to claim 10, wherein in the step (2), the pH is adjusted to 8.5 by KOH, the concentration of UDP-glucose dehydrogenase AtUGDH3 in the reaction system is 0.17. Mu.g/. Mu.L, and the reaction condition is 35℃for 30 minutes.
13. The method for performing arabinose modification on pentacyclic triterpene compound according to claim 10, wherein in the step (3), the concentration of UDP-glucuronic acid decarboxylase atuos 3 in the reaction system is 0.17 μg/μl, and the reaction condition is 35 ℃ for 30min.
14. The method for performing arabinose modification on pentacyclic triterpene compound according to claim 10, wherein in the step (4), the concentration of the UDP-sugar epimerase PsUGE2 in the reaction system is 0.17. Mu.g/. Mu.L, and the reaction condition is 25℃for 30 minutes.
15. The method for performing arabinose modification on pentacyclic triterpene compound according to claim 10, wherein in step (5), the concentration of glycosyltransferase UGT99D1 in the reaction system is 0.17 μg/μl, and the reaction condition is 35 ℃ for 6 hours.
16. The method for arabinose modification of pentacyclic triterpene compounds according to claim 10, wherein said glycosyl donor is sucrose; the substrate is pentacyclic triterpene compound;
the pentacyclic triterpene compound is selected from the group consisting of: oleanolic acid and/or betulinic acid.
17. The preparation method of the novel anti-DLD-1 drug is characterized in that the method for carrying out arabinose modification on the pentacyclic triterpene compound according to any one of claims 9 to 16 is adopted to carry out arabinose modification on betulinic acid so as to obtain the active ingredient of the novel anti-DLD-1 drug.
18. The preparation method of the anti-MCF-7 medicine is characterized in that the method for carrying out arabinose modification on pentacyclic triterpene compounds according to any one of claims 9 to 16 is adopted to carry out arabinose modification on oleanolic acid so as to obtain the active ingredient of the anti-MCF-7 medicine.
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CN110055232A (en) * | 2019-01-10 | 2019-07-26 | 北京理工大学 | Two Radix Glycyrrhizae sucrose synthases and its application in synthesis enoxolone glycosylated derivative |
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