Oxidase derived from Securinega suffruticosa and its applications

Abstract

The present invention discloses an oxidase SEQ ID NO:2 derived from Securinega suffruticosa, which can catalyze the oxidative condensation reaction of L-ascorbic acid / dehydroascorbic acid and allosecurinine to prepare the Securinega alkaloid fluesuffine A and its analogs, and has research and development value.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to an oxidase FsBBE derived from Flueggea suffruticosa and its application in the preparation of the alkaloid fluesuffine A from Flueggea suffruticosa. Background Art

[0002] Flueggea suffruticosa (Pall.) Baill., scientifically named, is a plant of the genus Securinega in the Euphorbiaceae family. It is widely distributed in China. Except for the northwest where it has not been found, it is distributed in other provinces and regions. Flueggea suffruticosa is used in traditional Chinese medicine to treat various nerve-related diseases such as paralysis, neurasthenia, and lethargy. Flueggea suffruticosa contains rich alkaloids, which are considered to be the bioactive components of the Flueggea suffruticosa plant, including securinine and allosecurinine. Securinine is a drug used to treat sequelae of poliomyelitis. In recent years, some novel active compounds have been isolated from Flueggea suffruticosa, and these new compounds have various activities such as anti-tumor, anti-viral, antibacterial, and central nervous system activities. The research on the alkaloids in Flueggea suffruticosa can discover more bioactive components. The analysis of the biosynthesis pathway of Flueggea suffruticosa alkaloids can find some enzymes with new functions and new tools for biocatalysis, thus laying a foundation for synthesizing Flueggea suffruticosa alkaloids by synthetic biology methods, and therefore can make more full use of this traditional Chinese medicine plant resource. Summary of the Invention

[0003] We extracted, separated, and identified some alkaloid components in Flueggea suffruticosa, and separated a novel alkaloid from Flueggea suffruticosa, which has the molecular structure shown in the following formula 1, and named it fluesuffine A:

[0004]

[0005] The ABCD ring part in the molecular structure of this compound 1 is extremely similar to the structure of allosecurinine / securinine, as shown in the following formula.

[0006]

[0007] It is speculated that the ABCD ring part is very likely to be derived from allosecurinine or securinine, and the highly oxidized 6-carbon structure part in the FG ring is very likely to be derived from vitamin C or L-ascorbic acid (L-AA). Therefore, compound 1 is very likely to be a vitamin C-modified natural product. The biosynthesis pathway of compound 1 is inferred to be the following path.

[0008]

[0009] After multi-step enzymatic modification of L-tyrosine and L-lysine, they polymerize to form allosecurinine / securinine, and then allosecurinine / securinine and L-ascorbic acid (L-AA) polymerize to form Compound 1 under the catalytic action of one or more steps by a certain / some oxidase.

[0010] To verify the above speculation, we isolated an oxidase and its encoding gene from the Flueggea suffruticosa plant. Through sequence analysis and comparison, it was found that it should belong to the oxidoreductase of the BBE family, so it was named FsBBE. It can catalyze the oxidative condensation reaction of L-ascorbic acid or dehydroascorbic acid (DHA) with securinine to obtain Compound 1.

[0011] Therefore, the first aspect of the present invention provides an isolated polypeptide, and the polypeptide is selected from the following group:

[0012] (a) A polypeptide having the amino acid sequence of SEQ ID NO:2:

[0013] MNPLKHSSSTPLVFVLLTVCSCATSVTIPELFFQCLSNTTTTSTSIFNVLYTPRNTSYTSILESRIQNLRFNTTDTPKPLAIVTPLDASHIQATIICARKHNLQIRIRSGGHDYEGLSYVSPLPFVVLDLINLRNITVDVENRVAWVGCGATLGEFYYRIAEKTRTLAFPAGACPTVGVGGHFSGGGYGYLLRKFGLAADNILDASLVDVNGRILDRASMGEDLFWAIRGGGGNSFGVVIAWKVNLVPVPSTLTSFKVSKSLEQNTMIQLLNKWQYVANKLPDELSMFAVVSKKNSTISVKFYSLYVGGIDSLLPLMEERFPELGLKRADCNEMSWIESAVSFAGYASNTSLDVLLNHTNNYEIASGRFKGKSDFVKEPVPEAALEGLLKWLSDKDITNAAIYMVPLGGKMGEITETSIPFPHRAGNLYLLAYYVKWEGQGTEAAQKPLSWIRKGYKYMAPYVSKNPREAHLNDRDLDIGTNNISGNTSYEQASIWGTKYFKNNFDRLVRVKTSVDPSDFFRNEQSVPPLLS (SEQ ID NO:2);

[0014] (b) A polypeptide derived from (a) formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO:2 and having the function of the polypeptide in (a);

[0015] (c) A polypeptide derived from (a) having a homology of more than 95%, preferably more than 98%, more preferably more than 99% with the polypeptide sequence defined in (a) and having the function of the polypeptide in (a); or

[0016] (d) A derivative polypeptide containing the polypeptide sequence described in (a) or (b) or (c) in its sequence.

[0017] The second aspect of the present invention provides an isolated polynucleotide, and the polynucleotide is selected from:

[0018] (A) A polynucleotide encoding the polypeptide recited in claim 1;

[0019] (B) A polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:2;

[0020] (C) A polynucleotide having a nucleotide sequence as shown in SEQ ID NO:1;

[0021] (D) A polynucleotide having a nucleotide sequence with a homology of ≥95%, preferably ≥98%, more preferably ≥99% to the nucleotide sequence shown in SEQ ID NO:1;

[0022] (E) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A)-(D).

[0023] Preferably, the above polynucleotide is SEQ ID NO:1.

[0024] Another aspect of the present invention provides a vector containing the above polynucleotide, and a microorganism transformed with the vector.

[0025] The above microorganism can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Bacillus subtilis. Preferably it is Escherichia coli BL21(DE3).

[0026] The fourth aspect of the present invention provides the use of the above polypeptide, or the above microorganism, in the preparation of fluesuffine A and compound 2 (fluesuffine B) shown in Formula 1.

[0027] When preparing compound 1, for example, using L-ascorbic acid (L-AA) or dehydroascorbic acid (DHA) and allosecurinine as substrate raw materials, compound 1 is prepared by an oxidative condensation reaction catalyzed by the above polypeptide.

[0028] Specifically, the above substrate raw materials are selected from L-ascorbic acid and allosecurinine or dehydroascorbic acid and allosecurinine.

[0029] When preparing compound 2, for example, using isoascorbic acid and allosecurinine as substrate raw materials, compound 2 is prepared by an oxidative condensation reaction catalyzed by the above polypeptide.

[0030] Specifically, the above substrate raw materials are selected from isoascorbic acid and allosecurinine.

[0031] The polypeptide SEQ ID NO:2 disclosed in the present invention can catalyze the oxidative condensation of allosecurinine with L-ascorbic acid (L-AA) or dehydroascorbic acid (DHA) to prepare a novel securinine alkaloid, fluesuffine A; and can also catalyze the oxidative condensation of allosecurinine with isoascorbic acid to prepare a novel compound, fluesuffine B. This lays a foundation for analyzing the biosynthesis of alkaloids in Securinega suffruticosa, promotes the development and utilization of new natural drugs, and has prospects for further research and development. Description of the Drawings

[0032] Figure 1 Photographs showing the cultivation of axenic seedlings of Securinega suffruticosa and the photograph of Securinega suffruticosa leaves fed with L-AA precursor.

[0033] Figure 2 Results of the content of compound 1 in Securinega suffruticosa fed with different L-AA precursors and the statistical chart of the isotope abundance of compound 1 fed with isotope-labeled L-ascorbic acid (1- 13 C). Figure A: Results of the content of compound 1 fed with different substrates, with the vertical axis being the relative content; Figure B: i: Isotope abundance of compound 1 fed with non-isotope-labeled L-ascorbic acid (1- 13 C); ii: Isotope abundance of compound 1 fed with isotope-labeled L-ascorbic acid (1- 13 C); iii: Isotope abundance of compound 1 obtained by the reaction of 3 with L-AA in D 2 O.

[0034] Figure 3 Statistical chart of the relative content of compound 1 in Securinega suffruticosa leaves induced by MeJA at different times. The vertical axis is the relative content of fresh weight, where water represents water, leaf represents leaf, root represents root, and stem represents stem.

[0035] Figure 4It is the result diagram of Q-TOF detection of proteins in Securinega suffruticosa leaves. The abscissa is the retention time. The left side shows the results of extracting EIC (390.1183). i: The results of total proteins in Securinega suffruticosa leaves with allosecurinine and L-AA activities; ii: The results of total proteins in Securinega suffruticosa leaves with allosecurinine and DHA activities; iii: The results of purified FsBBE protein with allosecurinine and L-AA activities; iv: The results of purified FsBBE protein with allosecurinine and DHA activities; v: The results of purified FsBBE protein with allosecurinine and L-AA activities; vi: The results of purified FsBBE protein with allosecurinine and isoascorbic acid activities; vii: Standard of compound 1. The right side is the result of the FsBBE mechanism study. i and ii are respectively the result diagrams of extracting EIC 216.1019 of the reaction of purified FsBBE protein with allosecurinine and the reaction of total proteins in tobacco leaves injected with the FsBBE gene with allosecurinine; iii, iv and v are respectively the result diagrams of extracting 390.1183 of the reaction of enamine intermediate 3 with L-AA, DHA and isoascorbic acid; vi and vii are respectively the standards of compound 2 and 1.

[0036] Figure 5 It shows the silver staining analysis photo of SDS-PAGE gel electrophoresis of the active fraction after the total proteins in Securinega suffruticosa leaves pass through the ion exchange column.

[0037] Figure 6 It shows the silver staining analysis photo of SDS-PAGE gel electrophoresis of the active fraction after passing through the molecular sieve. The marked target bands FS-F0-11-33-1 and FS-F0-11-34-1 in the figure are the target protein bands.

[0038] Figure 7 It shows the silver staining photo of SDS-PAGE gel electrophoresis of the candidate active protein purified from tobacco, and the FsBBE protein band is marked by the arrow.

[0039] Figure 8 It shows the absolute configuration diagrams of compound 1 and compound 2.

[0040] Figure 9 It shows the speculated synthesis mechanisms of compound 1 and compound 2. Specific implementation manners

[0041] To analyze and identify the alkaloid components in Securinega suffruticosa, we extracted the branches and leaves of Securinega suffruticosa with organic solvents such as ethanol and ethyl acetate. The total alkaloids obtained were subjected to silica gel column chromatography and separated by semi-preparative high performance liquid chromatography to obtain Compound 1. Using spectroscopic analysis, including high resolution electrospray ionization mass spectrometry (HRESIMS), 1 H and 13 C-NMR combined with two-dimensional nuclear magnetic resonance, infrared (IR), etc., the molecular structure was determined, and the absolute configuration was determined by X-ray diffraction.

[0042] Compound 1 contains the skeletal structure of allosecurinine or securinine and should be the oxidative condensation product of allosecurinine / securinine and L-ascorbic acid / dehydroascorbic acid. This oxidative condensation process is carried out under the catalysis of the oxidase FsBBE.

[0043] In order to enable the oxidase FsBBE to be applied to similar catalytic reactions, expressing this protein by microorganisms is the best method for preparing this enzyme.

[0044] Since the amino acid sequence of the oxidase FsBBE of the present invention is clear, those skilled in the art can easily obtain its coding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmid. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well-known to those skilled in the art.

[0045] The above-mentioned transformant host can be any microorganism suitable for expressing the enzyme SEQ ID NO:1, including bacteria and fungi. Preferred microorganisms are selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Bacillus subtilis. More preferably, it is Escherichia coli BL21(DE3).

[0046] When used as a biocatalyst for the production of alkaloids with similar structures such as fluesuffine A, the oxidase FsBBE of the present invention can be in the form of an enzyme or a cell. The forms of the enzyme include free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, enzyme immobilized on a carrier, etc.; the forms of the cell include viable cells and dead cells.

[0047] The separation and purification of the oxidase FsBBE of the present invention, including the preparation technology of immobilized enzyme, are also well-known to those skilled in the art.

[0048] The following further elaborates on the present invention with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0049] In this article, the addition amounts, contents and concentrations of various substances are involved. Unless otherwise specified, the percentage content mentioned refers to the mass percentage content.

[0050] In the examples of this article, if no specific description is made for the reaction temperature or operating temperature, the temperature generally refers to room temperature (15 - 30 °C).

[0051] Example 1 Separation and Identification of Compound 1

[0052] 1.1 Extraction and Separation of Securinine Alkaloids

[0053] The stems and leaves of Securinega suffruticosa were purchased from Nanyang, Henan in September 2017 and identified by Researcher Yanhong Yan of the Shanghai Chenshan Botanical Science Research Center, Chinese Academy of Sciences.

[0054] The total dry weight of the branches and leaves of Securinega suffruticosa was 1.8 kg. After being ground by a grinder, it was repeatedly ultrasonically extracted 4 times with 5 liters of ethanol. After the ethanol phase was distilled and concentrated, an ethanol extract was obtained. The ethanol extract was resuspended with 1 L of dilute hydrochloric acid aqueous solution with a pH of 6.0, and extracted three times with an equal volume of ethyl acetate. After the pH of the aqueous layer was adjusted to 8.0 with ammonia water, it was extracted three times with an equal volume of ethyl acetate again. The ethyl acetate phases were combined and distilled and concentrated to obtain 27.0 g of total alkaloids. The total alkaloids were separated by passing through a silica gel column with CH 2 Cl 2 :MeOH = 100:0, 30:1, 20:1, 15:1, 10:1, 7:1, 4:1, 2:1, 1:1, 0:1 as the mobile phase, detected by TLC, and similar fractions were combined to obtain 8 components: F1 - F8. Component F5 was further separated by semi - preparative high - performance liquid chromatography. Using a Luna C18 semi - preparative chromatographic column (250×10 mm, 5 μm), with the mobile phase: methanol: 0.1% formic acid water = 5:95 - 25:75, 0 - 10 min; 25:75 - 25:75, 10 - 15 min; 25:75 - 95:5, 15 - 25 min; 95:5, 25 - 30 min gradient elution, compound 1 (t R = 22.5 min) was separated. Compound 1 was dissolved in deuterated chloroform for NMR identification. Under the condition of ethyl acetate: methanol = 7:1 as the solvent, the single crystal of compound 1 was slowly evaporated at room temperature, and the single crystal was subjected to X - ray diffraction to determine its absolute configuration.

[0055] 1.2 Separation and Structure Identification of Compound 1

[0056] The separated compound 1 was a pale yellow solid, and HRESIMS (high - resolution electrospray ionization mass spectrometry) m / z [M + H] + 390.1183, indicating that its molecular formula was C 19 H 19 NO 8, the degree of unsaturation is 11. The IR spectrum shows hydroxyl groups (3452 cm -1 ), carbonyl groups (1745 cm -1 ), and double bonds (2943 cm -1 , 1441 cm -1 , and 1378 cm -1 ) absorption. 1 H and 13 C-NMR combined with two-dimensional NMR spectra show (see Table 1) that compound 1 has two aliphatic or amide carbonyl groups (δ C 171.4, 174.3), one sp 2 quaternary carbon δ C 163.7, three sp 2 methine groups (δ C / H 148.8 / 7.05, 122.4 / 6.57, 111.3 / 5.86), four lower-field sp 3 quaternary carbons (δ C 112.3, 101.5, 93.1, 80.4), four sp 3 methine groups (δ C / H 90.9 / 5.05, 74.4 / 4.52, 56.5 / 3.92, 37.0 / 2.11), one of which is a lower-field methine group, and five sp 3 methylene groups (Table 1.3.1). Comparing the above data with the reported NMR data of alkaloids isolated from Securinega suffruticosa shows that compound 1 contains a heterodimer with an allosecurinine or securinine skeleton structure. A detailed analysis of the two-dimensional NMR spectrum of compound 1 shows that the planar structure of compound 1 is formula 1. Compound 1 has 8 chiral carbon atoms, 4 of which are quaternary carbons. After many attempts, we were fortunate to obtain a single crystal capable of determining the absolute configuration quality of this compound. The single crystal diffraction results show that this compound has the 2R, 3R, 7S, 9S, 2’S, 3’R, 4’R, and 6’S configurations. We named compound 1 fluesuffine A.

[0057] Table 1. 1 H spectrum and 13 C NMR data of compound 1

[0058]

[0059]

[0060] The following analyzes and verifies the biosynthetic pathway of compound 1.

[0061] Example 2. Cultivation of axenic seedlings of Securinega suffruticosa

[0062] The seeds were placed in a 0.1% v / w 20 solution and soaked, shaken slowly at 90 rpm on a shaker for 5 min, and then rinsed 3 times with distilled water; then the seeds were surface-sterilized in 70% by mass ethanol for 1 min and immediately transferred to a 0.5% v / v sodium hypochlorite solution and shaken slowly at 90 rpm for 12 min; then the seeds were rinsed 3 times with sterile water in a laminar flow hood, placed in sterile water overnight, the sterile water was poured off in the laminar flow hood, the excess moisture on the surface of the seeds was blotted dry with sterilized filter paper, and then placed on the filter paper in a sterile moist sterile petri dish and left in a refrigerator at 4 °C overnight, and then placed in a constant temperature incubator at 24 °C in the dark for cultivation. About one week after the seeds were cultivated on the petri dish, the seeds began to germinate. At this time, they were cultivated at 24 °C with a 16 h / day light for another 3 days to grow into sterile seedlings. The roots of the sterile seedlings were cut off, and the sterile seedlings with the roots cut off were transferred to the PGMO5 medium and continued to be cultivated in a constant temperature incubator at 24 °C with a 16 h / day light for about 3 - 4 weeks, see Figure 1 。

[0063] PGMO5 medium: 2.2 g of MS medium, 10 g of sucrose, 4 g of phytogel, made up to 1 L with water, the pH was adjusted to 5.8 with NaOH solution, and sterilized at 121 °C for 20 min.

[0064] Example 3 Feeding experiments of L-AA precursors and isotope-labeled L-ascorbic acid (1- 13 C)

[0065] The feeding experiments of L-AA precursors were carried out using the leaves of sterile seedlings of Securinega suffruticosa that had grown for about 5 weeks. These precursors included D-glucose, D-mannose, L-galactose, L-galactono-1,4-lactone, L-ascorbic acid, etc. Immediately, three Securinega suffruticosa seedlings were taken out from the bottle (see Figure 1 ), the leaves of the sterile seedlings were cut off (the bottommost and topmost leaves were discarded), and the leaves were cut in the middle, and the leaves were immersed in an aqueous solution of the feeding precursor (concentration 5 mM, such as Figure 1As shown in the figure, three replicate experiments were conducted for each fed precursor compound. Water was used as the control group. During feeding, the sterile seedling leaves were completely immersed in the aqueous solution of the precursor compound. They were cultured in an incubator at 24°C with a 90 rpm shaker and 72 h of light for 72 h. After 72 h, the leaves were taken out, excess water was blotted with absorbent paper, and then weighed. They were placed in a 1.5 ml EP tube, frozen and ground in liquid nitrogen. For every 100 mg of wet weight sample, 1 ml of methanol containing 0.1% formic acid (HupA with a concentration of 50 nM was used as the internal standard) extraction solution was added, and ultrasonic extraction was carried out for 10 min. After centrifugation at 17000 g for 10 min, the supernatant was filtered through a 0.2 μM filter membrane and detected by Q-TOF. The relative content of compound 1 was calculated. The calculation method was: (1 / hupzine A * 50x10-6 * 1x10-3) * 389.36 / 100 (compound 1 and hupzine A represent the peak areas of EIC 390.1183 and 243.1497 extracted when identifying the content by mass spectrometry). The content results of compound 1 fed with different substrate compounds and the isotope abundance of compound 1 fed with isotope-labeled L-ascorbic acid (1- 13 C) The isotope abundance of compound 1 is shown in Figure 2 .

[0066] It can be seen from Figure 2 that after feeding Securinega suffruticosa leaves with L-galactose, L-galactono-1,4-lactone, and L-ascorbic acid, the yield of compound 1 increased significantly, indicating that these added compounds are all precursors for the biosynthesis of compound 1, which confirmed our inference about the biosynthesis pathway of compound 1.

[0067] Example 4 Treatment of Securinega suffruticosa seedlings with methyl jasmonate and detection of the content of compound 1 in the treated seedling leaves

[0068] Securinega suffruticosa seedlings that had grown for about one month and had about 7 leaves at this time (see Figure 1 ) were placed in each bottle with 8 plants, and sprayed with an aqueous solution of methyl jasmonate (MeJA) at a concentration of 50 μM for 24, 48, and 72 hours respectively. Water was used as the blank control group (sprayed every 12 h), and a total of 4 groups of experiments were conducted, with three replicates in each group. The method for determining the content of new compounds in the seedling leaves was the same as that in Example 3. The content of compound 1 in Securinega suffruticosa leaves is shown in Figure 3 .

[0069] It can be seen from Figure 3 that treating Securinega suffruticosa seedlings with methyl jasmonate MeJA can increase the biosynthesis amount of compound 1, and the longer the treatment time, the higher the yield of compound 1.

[0070] Example 5: MeJA Induction Experiment on Aseptic Seedlings of Securinega suffruticosa and Transcriptome Sequencing of Leaves Induced by MeJA at Different Times

[0071] The leaves of the seedlings at 0 h (water blank control group), 24 h, and 72 h after MeJA treatment in Example 4 were subjected to second-generation transcriptome sequencing, and the instrument used for sequencing was Illumina HiSeq TM The obtained data were filtered to obtain clean data, and Trinity was used to splice the obtained clean data, resulting in a total of 145,153 transcripts

[0072] The obtained transcripts were subjected to prediction of coding sequences CDS (coding sequence). The CDS prediction method includes the following steps: 1. Align Gene with the NR protein library and the Swissprot protein library in the order of priority. If a match is found, extract the ORF coding frame information of the transcript from the alignment result, and translate the coding region sequence into an amino acid sequence according to the standard codon table (in the order of 5'->3'); 2. For sequences that do not match the NR protein library or the Swissprot protein library, or sequences that match but no results are predicted, use the estscan (3.0.3) software to predict their open reading frame ORF (open reading frame) to obtain the nucleic acid sequence and amino acid sequence encoded by these genes

[0073] Example 6: Extraction and Activity Experiment of Total Protein from Leaves of Aseptic Seedlings of Securinega suffruticosa

[0074] The aseptic seedlings of Securinega suffruticosa that had grown for about one month were cut and weighed. After the leaves with a wet weight of 8.2 g were ground in liquid nitrogen, 41 ml of extraction buffer A (100 mM NaPi, pH 7.4, 5 mM sodium bisulfite, 5 mM dithiothreitol, 1 mM EDTA, 10% (v / v) glycerol, 1% (w / v) PVP, 4% (w / v) PVPP) was added and incubated on ice. It was gently shaken up and down several times every 15 minutes. After 1 h, it was centrifuged at 5000 g at 4 °C for 30 min, and the supernatant was desalted through a pD10 column and replaced with buffer B (10 mM Tris-HCl, pH 7.5). After the protein was extracted, the protein concentration was measured by the Bradford method, and the protein concentration was 3.5 mg / ml

[0075] 50 μL of the desalted protein was added with allosecurinine / securinine (0.5 μL, initial concentration of 100 mM) and L-ascorbic acid (L-AA) / dehydroascorbic acid (DHA) (0.5 μL, initial concentration of 100 mM). The control group was the heat-inactivated protein, and the others were the same. After reacting in a shaker at 30 °C for 2 h, an equal volume of methanol was added to terminate the reaction. After centrifuging at 17,000 g for 10 min and filtering through a 0.2 μM filter membrane, Q-TOF (quadrupole-time of flight mass spectrometry) detection was performed. The detection results are shown in Figure 4 .

[0076] From Figure 4 the left spectrum in it, it can be seen that the total protein from the leaves of Securinega suffruticosa can catalyze the oxidative condensation of allosecurinine and L-AA to obtain compound 1 (see i in the spectrum), and can catalyze the oxidative condensation of allosecurinine and DHA to obtain compound 1 (see ii in the spectrum).

[0077] Example 7 Fractionation and Protein Mass Spectrometry Verification of the Total Protein from the Leaves of Securinega suffruticosa

[0078] The extraction method of the total protein from Securinega suffruticosa was the same as that in Example 6. Approximately 175.0 mg of the desalted total protein was fractionated using a HitrapQ HP column (GE). Buffer B with a gradually increasing NaCl concentration was used for elution at a flow rate of 2.5 ml / min. The F0 protein fraction that did not bind to the column showed activity after activity testing (the activity testing method was the same as that for the total protein, and the data was not shown). Therefore, the protein fraction F0 that did not bind to the column was concentrated using a 10 kDa ultrafiltration tube, and buffer C (10 mM MES, pH 6.5) was gradually added to the ultrafiltration tube to replace the buffer.

[0079] The fraction F0 with the buffer replaced was then fractionated using a Hitrap SP HP column (GE) at a flow rate of 1.0 ml / min. Buffer C with a gradually increasing NaCl concentration was used for elution. Every 1.5 ml was collected as one fraction. A total of 40 fractions F0-F1-F0-F40 were collected. And the activity of each fraction was tested (the activity testing conditions were the same as those for F0, extracting EIC (390.0, [M+H] + ), and the peak areas obtained by extraction were used to judge the activity strength of each fraction). It was found that the fractions F0-F10 and F0-F11 had the best activity (the data was not shown). At the same time, silver staining analysis of SDS PAGE (12% gel) was performed on the active fractions F0-F9-F0-F17. It was found that there were still multiple protein bands in the fractions F0-F10 and F0-F11, and further fractionation was required (Figure 5 )。

[0080] After concentrating the fraction F0-11 with the best activity obtained by Hitrap SP HP column separation to less than 500 μL using a 10 kDa ultrafiltration tube, it was further separated and purified using a Superdex 200 Increase 5 / 150 GL molecular sieve column, eluted with buffer B at 0.3 ml / min, and 0.5 ml was collected as one fraction, with a total of 60 fractions collected (F0-11-1 - F0-11-60). Activity tests showed that fractions F0-11-33 and F0-11-34 had the best activity (the activity test conditions were the same as those for the F0 fraction, and the test result data are not shown), and the active fractions (F0-11-29 - F0-11-37) were analyzed by SDS PAGE (10% gel) silver staining ( Figure 6 )。

[0081] Corresponding to the activity test results, Figure 6 the bands marked in red are the target protein bands. Therefore, F0-11-33-1 ( Figure 6 the target band marked in Figure 6 )、F0-11-34-1 ( 2 the target band marked in 2 O were subjected to in-gel digestion and mass spectrometry identification. After cutting the gel into 1 mm × 1 mm gel particles and placing them in a 1.5 ml centrifuge tube, the following operations were performed on the samples simultaneously: 200 μL of ddH 3 O was used to rinse 3 times. Silver staining decolorization solution (100 mM NaS 2 O 4 and 30 mM potassium ferricyanide) was added and incubated at 37 °C for 30 mins for decolorization, and the operation was repeated several times until the color was completely removed. 200 μL of ddH 3 O was added and incubated for 5 min, and the operation was repeated once. 100 μL of 50% AcN was added and incubated for 5 min, then centrifuged, the supernatant was discarded, and another 100 μL of AcN was added and incubated for 5 min until the gel particles turned white. Centrifuged at 1000 g for 10 min, the supernatant was discarded, and it was left to dry at room temperature. 200 μL of 10 mM DTT (1 M DTT diluted with 25 mM NH 4 HCO 3 ) was added, gently shaken, and incubated at 56 °C for 1 h. 100 μL of 55 mM IAA was quickly added to the gel and incubated at room temperature in the dark for 30 mins. After rinsing once with 50 mM NH 4 HCO 3Extract twice, dry at 80 °C, redissolve the sample with 0.1% formic acid water, desalt on a C18 column, and perform mass spectrometry analysis and protein identification with 20 μL of 0.1% formic acid deionized water.

[0082] Perform a comparison analysis on the raw protein data obtained from mass spectrometry with the obtained Securinega suffruticosa protein database on Proteome Discoverer TM Software 2.3 to obtain a list of candidate proteins.

[0083] Obtain the full-length gene through 3' and 5' RACE of the candidate gene in Example 8

[0084] The candidate gene Cluster-10344.81450 (FsBBE) is very likely the target gene to be searched for. However, this gene has no full length in the transcriptome data, only 438 bp. Therefore, the RACE method is needed to obtain the full-length sequence of this gene. The primers required for 3' and 5' RACE are designed according to the existing sequence, as shown in the materials section. The RACE method is described in the clongtech kit. The full-length sequence of the obtained gene (FsBBE) is shown as SEQ ID NO:1, and the amino acid sequence of the encoded oxidase is SEQ ID NO:2.

[0085] Heterologous expression, protein purification and functional verification of the candidate gene in Example 9

[0086] Use Agrobacterium tumefaciens GV3101 containing the constructed pGambia 2301::81450-HIS Tag (using BamHI and SalI restriction sites) vector to grow in LB medium containing rifampicin and kanamycin for about 24 h, measure OD 600 = 1.0, centrifuge the bacterial cells at 4000 g, and resuspend them with buffer (10 mM MES, 10 mM MgCl 2 and 100 μM acetosyringone, pH 5.6) to OD 600After reaching 0.6, tobacco was injected. Four days after injection, tobacco leaves were collected, ground while frozen, and proteins were extracted. The extraction method was the same as that for extracting total proteins from Securinega suffruticosa: 1 g of fresh leaves were ground while frozen in liquid nitrogen and extracted on ice for 1 h using 5 mL of buffer A (100 mM NaPi, pH 7.4, 5 mM sodium bisulfite, 5 mM dithiothreitol, 1 mM EDTA, 10% (v / v) glycerol, 1% (w / v) PVP, 4% (w / v) PVPP) at 4°C. Centrifugation was performed at 5,000 x g for 30 min at 4°C. The supernatant was desalted using pD 10 columns (GE-Healthcare) and the buffer was replaced with buffer B (10 mM Tris-HCl, pH 7.5). Subsequently, we purified the recombinant protein with HIS-TAG of the Cluster-10344.81450 gene. The total protein was adsorbed by a nickel column and eluted with imidazole at different gradients. The fractions eluted with 100 mM imidazole were verified by SDS PAGE (see Figure 7 ). The fractions eluted with 100 mM imidazole were desalted using a pD 10 desalting column and the buffer was replaced with Tris-HCl pH 7.5 buffer, and then enzyme activity tests were performed. The test results are shown in Figure 4 .

[0087] From Figure 4 the left graph, it can be seen that after heterologously expressing the FsBBE protein in tobacco, the total proteins in tobacco leaves can also catalyze the oxidative condensation of allosecurinine with L-AA or DHA or isoascorbic acid to obtain compounds 1 or 2 (see iii, iv, v in the right graph).

[0088] Example 10 Verification of the Catalytic Mechanism of Oxidase FsBBE

[0089] The FsBBE protein purified from tobacco leaves was incubated with allosecurinine. The heat-inactivated protein was used as a control group. The total proteins from tobacco leaves expressing the Cluster-10344.81450 gene were incubated with allosecurinine. The total proteins from tobacco leaves injected with the empty pGambia2301 Agrobacterium tumefaciens were used as a control group. After reacting at 30°C for 3 h, an equal volume of methanol was added to terminate the reaction. The supernatant was taken after centrifugation at 17,000 g, and 1 μL was injected for Q-TOF detection. The detection results are shown in Figure 4 .

[0090] The total protein of tobacco leaves expressing the Cluster-10344.81450 gene was incubated with allosecurinine. After reacting at 30 °C for 3 h, an equal volume of n-butanol was added for extraction three times. The n-butanol phases were combined and dried by rotary evaporation. After liquid separation, the separated fraction of compound 3 (enamine intermediate) was dried by rotary evaporation and dissolved in 40 μL of methanol, and the following experiments were carried out: 1 μL of the compound 3 solution was added to 50 μL of Tris-HCl buffer at pH 7.5 as the control group (compound 3); 1 μL of L-AA (100 mM in ddH 2 O) aqueous solution was added to 50 μL of Tris-HCl buffer at pH 7.5 as the control group (L-AA); 1 μL of the compound 3 solution and 1 μL of L-AA were added to 50 μL of Tris-HCl buffer at pH 7.5 as the experimental group (compound 3 + L-AA); DHA was similar to the above reaction and was carried out in D 2 O. Except that the solution was the buffer prepared with D 2 O, the rest were the same. After reacting at 30 °C for 3 h, it was filtered through a 0.22 μM filter membrane, and 1 μL was injected for Q-TOF detection. The detection results are shown in Figure 4 .

[0091] Example 11 Synthesis and Structural Characterization of Another New Compound Catalyzed by Oxidase FsBBE

[0092] When searching for L-AA analogues, only isoascorbic acid could be purchased. Therefore, the purified FsBBE protein and allosecurinine were used for activity testing with isoascorbic acid. The test results are shown in Figure 4 . Method for separation, purification and identification of new compounds and large-scale enzyme activity: After desalting the total protein of tobacco leaves expressing the Cluster-10344.81450 gene, allosecurinine with a final concentration of 1 mM and isoascorbic acid with a final concentration of 1 mM were added. After reacting at 30 °C for 3 h, an equal volume of n-butanol was added for extraction three times. The n-butanol phases were combined and dried by rotary evaporation. After liquid separation, the liquid separation method was the same as that for separating compound 1. Compounds 2 were obtained respectively. After being dissolved in deuterated chloroform and deuterated methanol, nuclear magnetic resonance identification was carried out. Single crystal attempts were made for both compounds in different solvents. Compound 2 was slowly volatilized at room temperature in a solvent of ethyl acetate:methanol = 4:1 to obtain crystals, and its absolute configuration was determined by X-ray crystal diffraction (see Figure 8 ). That is, the molecular structural formula of compound 2 is as follows:

[0093]

[0094] It differs from the molecular structure of Compound 1 in that the spatial configuration of the 6'-hydroxy group is opposite, and it is named fluesuffine B.

[0095] Example 12 Physiological Activity Tests of Two New Compounds

[0096] The SRB method (Skehan P. et al. 1990) was used to test the growth inhibitory activities of Compound 1 (fluesuffine A) and Compound 2 (fluesuffine B) against tumor cells.

[0097] The tumor cell lines were glioma cell SF-268, breast cancer cell MCF-7, liver cancer cell HepG-2, non-small cell lung cancer cell A549, and hepatic stellate cell LX-2.

[0098] Experimental method: Compound 1 (fluesuffine A) and Compound 2 (fluesuffine B) were separately dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions with a concentration of 10 mmol / L, and then diluted to the required concentrations with RPMI-1640 medium. The positive control drug was cisplatin. SF-268, MCF-7, HepG-2, A549, and LX-2 cells in the logarithmic growth phase were digested with trypsin, stained with trypan blue for counting. After the cell viability was detected by the trypan blue exclusion experiment to be greater than 95%, the cell concentration was adjusted to 3×10 4 cells / mL with fresh RPMI-1640 medium. The cells were seeded in 96-well plates, 180 μL of cell suspension was added to each well, and 3 blank wells were set for zero adjustment. They were cultured in an incubator at 37 °C and 5% CO 2 for 24 h. After the cells adhered, 20 μL of a compound solution with a certain concentration was added to each well, 20 μL of RPMI-1640 medium was added as the negative control, and cisplatin was used as the positive control. They were cultured in an incubator at 37 °C and 5% CO 2 for 72 h. Then, 50 μL of 50% cold trichloroacetic acid was added to fix the cells. After standing at 4 °C for 1 h, they were washed 5 times with distilled water and air-dried naturally. Then, 100 μL / well of SRB solution with a concentration of 4 mg / mL prepared with 1% glacial acetic acid was added, and stained at room temperature for 30 min. The supernatant was removed, and they were washed 5 times with 1% glacial acetic acid. Finally, 200 μL / well of 10 mmol / mL Tris solution was added to dissolve, and the absorbance value (A) at 570 nm was measured with an enzyme-linked immunosorbent assay reader. The inhibition rate of the drug on cell growth was calculated using the following formula: Cell growth inhibition rate (%) = (1 - A 样品组 / A 对照组 ) × 100%.

[0099] Experimental results: The IC 50 values of compound 1, compound 2 prepared by the present invention and the positive control drug cisplatin against tumor cell lines SF-268, MCF-7, HePG-2, A549 and LX-2 are shown in Table 2.

[0100] Table 2. Inhibitory activities of compound 1 and 2 on tumor cell growth ( n = 3)

[0101]

[0102] The results show that compound 1 and compound 2 have significant growth inhibitory activities against tumor cells. Therefore, the implementation of the present invention provides candidate compounds for the research and development of new anti-tumor drugs and provides a scientific basis for the development and utilization of traditional Chinese medicine plant resources.

[0103] The biosynthesis of compound 1 and compound 2 also lays a foundation for analyzing the alkaloid synthesis pathway in Securinega suffruticosa, thus promoting the development and utilization of new natural drugs. Sequence Listing <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Oxidase Derived from Securinega suffruticosa and Its Application <130> SHPI2010290 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1599 <212> DNA <213> Flueggea suffruticosa (Pall.) Baill. <220> <221> CDS <222> (1)..(1596) <400> 1 atg aat cca tta aag cac tct tca tca acg cct tta gtg ttt gtg ttg 48 Met Asn Pro Leu Lys His Ser Ser Ser Thr Pro Leu Val Phe Val Leu 1 5 10 15 ctg act gta tgt tca tgt gca act tca gta aca att cct gag ctg ttc 96 Leu Thr Val Cys Ser Cys Ala Thr Ser Val Thr Ile Pro Glu Leu Phe 20 25 30 ttt caa tgc ctg tcc aat aca acc aca acc agt act agt att ttc aat 144 Phe Gln Cys Leu Ser Asn Thr Thr Thr Thr Ser Thr Ser Ile Phe Asn 35 40 45 gtc cta tac aca cca aga aac acg tcc tac act tcc atc tta gaa tca 192 Val Leu Tyr Thr Pro Arg Asn Thr Ser Tyr Thr Ser Ile Leu Glu Ser 50 55 60 cgc att caa aac ctc agg ttc aat aca act gac acg ccg aaa cct ctg 240 Arg Ile Gln Asn Leu Arg Phe Asn Thr Thr Asp Thr Pro Lys Pro Leu 65 70 75 80 gcc ata gtc aca ccg ctg gat gca tct cac att caa gcc acc atc ata 288 Ala Ile Val Thr Pro Leu Asp Ala Ser His Ile Gln Ala Thr Ile Ile 85 90 95 tgt gcc cgg aaa cac aac ctc caa atc aga atc cga agc ggt ggc cac 336 Cys Ala Arg Lys His Asn Leu Gln Ile Arg Ile Arg Ser Gly Gly His 100 105 110 gac tat gag ggg ttg tct tat gtc tcg ccc ctc cct ttt gtt gtg ctt 384 Asp Tyr Glu Gly Leu Ser Tyr Val Ser Pro Leu Pro Phe Val Val Leu 115 120 125 gat cta atc aat ctt cga aac atc acc gtt gat gta gaa aat aga gtc 432 Asp Leu Ile Asn Leu Arg Asn Ile Thr Val Asp Val Glu Asn Arg Val 130 135 140 gca tgg gtc ggg tgc gga gca aca tta gga gaa ttc tac tat aga att 480 Ala Trp Val Gly Cys Gly Ala Thr Leu Gly Glu Phe Tyr Tyr Arg Ile 145 150 155 160 gca gag aaa act agg acc ctg gca ttc cct gca ggt gct tgt cct act 528 Ala Glu Lys Thr Arg Thr Leu Ala Phe Pro Ala Gly Ala Cys Pro Thr 165 170 175 gta gga gtt ggt ggg cat ttc agt gga ggc gga tac ggg tat ttg ttg 576 Val Gly Val Gly Gly His Phe Ser Gly Gly Gly Tyr Gly Tyr Leu Leu 180 185 190 cgt aaa ttt ggc ctc gca gca gat aac atc ctt gat gca agt tta gtt 624 Arg Lys Phe Gly Leu Ala Ala Asp Asn Ile Leu Asp Ala Ser Leu Val 195 200 205 gat gtg aat ggt aga att ctc gat aga gct tcc atg ggg gaa gat ttg 672 Asp Val Asn Gly Arg Ile Leu Asp Arg Ala Ser Met Gly Glu Asp Leu 210 215 220 ttt tgg gca att aga ggt ggt gga gga aat agt ttc gga gta gtt att 720 Phe Trp Ala Ile Arg Gly Gly Gly Gly Asn Ser Phe Gly Val Val Ile 225 230 235 240 gct tgg aag gtt aat ttg gtt cca gtc cct tcc aca ttg act tct ttc 768 Ala Trp Lys Val Asn Leu Val Pro Val Pro Ser Thr Leu Thr Ser Phe 245 250 255 aaa gtc tca aaa agt ttg gaa cag aat acg atg att cag ctt ctc aac 816 Lys Val Ser Lys Ser Leu Glu Gln Asn Thr Met Ile Gln Leu Leu Asn 260 265 270 aag tgg caa tat gtt gca aat aaa ctt cct gat gaa tta agc atg ttt 864 Lys Trp Gln Tyr Val Ala Asn Lys Leu Pro Asp Glu Leu Ser Met Phe 275 280 285 gct gta gtt tct aaa aaa aac tca aca ata tct gtt aag ttt tat tcc 912 Ala Val Val Ser Lys Lys Asn Ser Thr Ile Ser Val Lys Phe Tyr Ser 290 295 300 ttg tat gta ggt gga att gat agc ctc ctt cca tta atg gaa gaa agg 960 Leu Tyr Val Gly Gly Ile Asp Ser Leu Leu Pro Leu Met Glu Glu Arg 305 310 315 320 ttt cct gag ctt ggt tta aaa aga gcg gat tgc aat gag atg agc tgg 1008 Phe Pro Glu Leu Gly Leu Lys Arg Ala Asp Cys Asn Glu Met Ser Trp 325 330 335 ata gag tca gca gta tct ttc gcc ggg tac gca agt aat aca tca ttg 1056 Ile Glu Ser Ala Val Ser Phe Ala Gly Tyr Ala Ser Asn Thr Ser Leu 340 345 350 gat gtt ctc ctc aat cac act aat aat tac gaa att gct agt gga aga 1104 Asp Val Leu Leu Asn His Thr Asn Asn Tyr Glu Ile Ala Ser Gly Arg 355 360 365 ttc aaa ggc aaa tcg gac ttt gtc aaa gag ccc gtg cca gaa gct gca 1152 Phe Lys Gly Lys Ser Asp Phe Val Lys Glu Pro Val Pro Glu Ala Ala 370 375 380 tta gaa ggc tta ttg aaa tgg ctt tca gac aaa gac ata acg aat gca 1200 Leu Glu Gly Leu Leu Lys Trp Leu Ser Asp Lys Asp Ile Thr Asn Ala 385 390 395 400 gcg atc tat atg gtt cca tta gga gga aaa atg ggt gag ata aca gaa 1248 Ala Ile Tyr Met Val Pro Leu Gly Gly Lys Met Gly Glu Ile Thr Glu 405 410 415 aca agc att cca ttc cca cat aga gca ggg aat cta tac ttg ttg gcg 1296 Thr Ser Ile Pro Phe Pro His Arg Ala Gly Asn Leu Tyr Leu Leu Ala 420 425 430 tat tat gtt aaa tgg gag ggg caa gga aca gaa gca gct caa aag ccc 1344 Tyr Tyr Val Lys Trp Glu Gly Gln Gly Thr Glu Ala Ala Gln Lys Pro 435 440 445 cta agt tgg atc aga aag ggt tac aaa tac atg gct ccc tat gtc tcc 1392 Leu Ser Trp Ile Arg Lys Gly Tyr Lys Tyr Met Ala Pro Tyr Val Ser 450 455 460 aaa aat cca aga gaa gca cat ctc aac gac aga gat ctt gat att ggt 1440 Lys Asn Pro Arg Glu Ala His Leu Asn Asp Arg Asp Leu Asp Ile Gly 465 470 475 480 act aac aat atc tca gga aat acc agt tac gaa cag gct agt att tgg 1488 Thr Asn Asn Ile Ser Gly Asn Thr Ser Tyr Glu Gln Ala Ser Ile Trp 485 490 495 gga acc aag tat ttt aaa aat aat ttt gac agg ttg gtt cgg gtg aag 1536 Gly Thr Lys Tyr Phe Lys Asn Asn Phe Asp Arg Leu Val Arg Val Lys 500 505 510 act agt gtt gat cct tca gat ttt ttc aga aat gaa caa agc gtc cct 1584 Thr Ser Val Asp Pro Ser Asp Phe Phe Arg Asn Glu Gln Ser Val Pro 515 520 525 cct ctg tta tct tga 1599 Pro Leu Leu Ser 530 <210> 2 <211> 532 <212> PRT <213> Flueggea suffruticosa (Pall.) Baill. <400> 2 Met Asn Pro Leu Lys His Ser Ser Ser Thr Pro Leu Val Phe Val Leu 1 5 10 15 Leu Thr Val Cys Ser Cys Ala Thr Ser Val Thr Ile Pro Glu Leu Phe 20 25 30 Phe Gln Cys Leu Ser Asn Thr Thr Thr Thr Ser Thr Ser Ile Phe Asn 35 40 45 Val Leu Tyr Thr Pro Arg Asn Thr Ser Tyr Thr Ser Ile Leu Glu Ser 50 55 60 Arg Ile Gln Asn Leu Arg Phe Asn Thr Thr Asp Thr Pro Lys Pro Leu 65 70 75 80 Ala Ile Val Thr Pro Leu Asp Ala Ser His Ile Gln Ala Thr Ile Ile 85 90 95 Cys Ala Arg Lys His Asn Leu Gln Ile Arg Ile Arg Ser Gly Gly His 100 105 110 Asp Tyr Glu Gly Leu Ser Tyr Val Ser Pro Leu Pro Phe Val Val Leu 115 120 125 Asp Leu Ile Asn Leu Arg Asn Ile Thr Val Asp Val Glu Asn Arg Val 130 135 140 Ala Trp Val Gly Cys Gly Ala Thr Leu Gly Glu Phe Tyr Tyr Arg Ile 145 150 155 160 Ala Glu Lys Thr Arg Thr Leu Ala Phe Pro Ala Gly Ala Cys Pro Thr 165 170 175 Val Gly Val Gly Gly His Phe Ser Gly Gly Gly Tyr Gly Tyr Leu Leu 180 185 190 Arg Lys Phe Gly Leu Ala Ala Asp Asn Ile Leu Asp Ala Ser Leu Val 195 200 205 Asp Val Asn Gly Arg Ile Leu Asp Arg Ala Ser Met Gly Glu Asp Leu 210 215 220 Phe Trp Ala Ile Arg Gly Gly Gly Gly Asn Ser Phe Gly Val Val Ile 225 230 235 240 Ala Trp Lys Val Asn Leu Val Pro Val Pro Ser Thr Leu Thr Ser Phe 245 250 255 Lys Val Ser Lys Ser Leu Glu Gln Asn Thr Met Ile Gln Leu Leu Asn 260 265 270 Lys Trp Gln Tyr Val Ala Asn Lys Leu Pro Asp Glu Leu Ser Met Phe 275 280 285 Ala Val Val Ser Lys Lys Asn Ser Thr Ile Ser Val Lys Phe Tyr Ser 290 295 300 Leu Tyr Val Gly Gly Ile Asp Ser Leu Leu Pro Leu Met Glu Glu Arg 305 310 315 320 Phe Pro Glu Leu Gly Leu Lys Arg Ala Asp Cys Asn Glu Met Ser Trp 325 330 335 Ile Glu Ser Ala Val Ser Phe Ala Gly Tyr Ala Ser Asn Thr Ser Leu 340 345 350 Asp Val Leu Leu Asn His Thr Asn Asn Tyr Glu Ile Ala Ser Gly Arg 355 360 365 Phe Lys Gly Lys Ser Asp Phe Val Lys Glu Pro Val Pro Glu Ala Ala 370 375 380 Leu Glu Gly Leu Leu Lys Trp Leu Ser Asp Lys Asp Ile Thr Asn Ala 385 390 395 400 Ala Ile Tyr Met Val Pro Leu Gly Gly Lys Met Gly Glu Ile Thr Glu 405 410 415 Thr Ser Ile Pro Phe Pro His Arg Ala Gly Asn Leu Tyr Leu Leu Ala 420 425 430 Tyr Tyr Val Lys Trp Glu Gly Gln Gly Thr Glu Ala Ala Gln Lys Pro 435 440 445 Leu Ser Trp Ile Arg Lys Gly Tyr Lys Tyr Met Ala Pro Tyr Val Ser 450 455 460 Lys Asn Pro Arg Glu Ala His Leu Asn Asp Arg Asp Leu Asp Ile Gly 465 470 475 480 Thr Asn Asn Ile Ser Gly Asn Thr Ser Tyr Glu Gln Ala Ser Ile Trp 485 490 495 Gly Thr Lys Tyr Phe Lys Asn Asn Phe Asp Arg Leu Val Arg Val Lys 500 505 510 Thr Ser Val Asp Pro Ser Asp Phe Phe Arg Asn Glu Gln Ser Val Pro 515 520 525 Pro Leu Leu Ser 530

Claims

1. An isolated polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. An isolated polynucleotide, which encodes the polypeptide according to claim 1, and its nucleotide sequence is shown in SEQ ID NO:

1.

3. A vector comprising the polynucleotide according to claim 2.

4. A microorganism transformed with the vector according to claim 3.

5. The microorganism according to claim 4, characterized in that it is Escherichia coli BL21(DE3).

6. Use of the polypeptide according to claim 1 or the microorganism according to claim 5 in the preparation of fluesuffine A, an alkaloid of Securinega suffruticosa shown in Formula 1:

7. The use according to claim 6, characterized in that using L-ascorbic acid or dehydroascorbic acid and allosecurinine as substrate raw materials, and preparing fluesuffine A, an alkaloid of Securinega suffruticosa, through an oxidative condensation reaction catalyzed by the polypeptide according to claim 1.

8. Use of the polypeptide according to claim 1 or the microorganism according to claim 5 in the preparation of the compound shown in Formula 2:

9. The use according to claim 8, characterized in that using isoascorbic acid and allosecurinine as substrate raw materials, and preparing it through an oxidative condensation reaction catalyzed by the polypeptide according to claim 1.

Images