CN114958791B - Spermidine derivative glycosyltransferase LbUGT62 and encoding gene and application thereof - Google Patents
Spermidine derivative glycosyltransferase LbUGT62 and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses spermidine derivative glycosyltransferase LbUGT62, and a coding gene and application thereof. The amino acid sequence of the spermidine derivative glycosyltransferase LbUGT62 is shown as SEQ ID NO. 4. The glycosyltransferase of the spermidine derivative can catalyze the spermidine derivative to generate corresponding spermidine derivative glycoside, has important application value in the in-vitro biosynthesis of the Ningxia wolfberry caffeoyl spermidine substance, or has reference function and important application value for the in-vitro catalytic synthesis of spermidine derivative glycoside from other species.
Description
The present divisional application is patent application number 202110021501.8, invention name: a group of spermidine derivative glycosyltransferases, and encoding genes and application thereof, and application date: division of the invention patent application at 2021, 1 and 8.
Technical field:
the invention belongs to the field of plant genetic engineering and biotechnology, and in particular relates to a group of spermidine derivative glycosyltransferases, and a coding gene and application thereof.
The background technology is as follows:
ningxia wolfberry (Lycium barbarum L.) is a plant of Lycium of Solanaceae, and dried mature fruit of Lycium barbarum is used as a rare traditional Chinese medicine from ancient times, and has important medicinal history, and is also one of medicinal and edible varieties. The pharmacopoeia records that the medlar has the effects of nourishing liver, tonifying kidney, and nourishing essence and improving eyesight. Modern pharmacological researches show that Ningxia wolfberry has good effects of promoting immunity, reducing blood sugar and blood fat, resisting oxidation and radiation injury, promoting apoptosis of tumor cells, delaying aging and the like, and the effects are mainly derived from abundant secondary metabolic components such as polysaccharide, polyphenol, carotenoid and the like. In recent years, the Ningxia wolfberry also contains rich caffeoylspermine substances, the content of which exceeds 2.1 g/kg, and is inferior to carotenoid (2.2-2.3 g/kg), and the caffeoylspermine substances are one of the active ingredients emerging from Ningxia wolfberry. The caffeoylimide substance can improve short-time learning and memory capacity of transgenic drosophila, and can be developed into a medicine component for resisting Alzheimer disease; in addition, the fermentation production of the antitumor drug pingyangmycin also has a promoting effect. Although the substances are less common than secondary metabolites such as alkaloid, flavonoid, terpenoid and the like, the identification and activity evaluation in a plurality of species show that the substances also have the effects of treating parasitic trypanosomiasis, inhibiting vancomycin-resistant staphylococcus aureus, reducing blood pressure, easing pain and the like, and have great application prospects in the prevention or treatment of diseases.
The content of the caffeoylspermine substances in the Ningxia wolfberry is high, the structure variety is rich, and 15 caffeoylspermine substances with different structure forms are identified from the Ningxia wolfberry at present. The most obvious structural feature of these 15 compounds is that they all have glycosylation modifications. Glycosylation modification is completed by glycosyltransferase, and plays an important role in synthesis, transportation and storage of plant secondary metabolites. The dicaffeoylspermidine glycosides found in Lycium barbarum are also the first caffeoylspermidine glycosides found and identified in nature. Thus, ningxia wolfberry is a natural resource of rich caffeoylspermidine glycoside and also a source of caffeoylspermidine glycosyltransferase. The caffeoylspermidine glycoside in the Ningxia wolfberry has important application prospect and potential economic value in the aspect of treating diseases such as Alzheimer disease and the like. Therefore, the gene resource in Ningxia wolfberry can be utilized to develop in vitro synthesized caffeoylspermidine glycoside by means of biotechnology and the like, and the method has important industrial value.
The invention comprises the following steps:
the first object of the invention is to provide a group of spermidine derivative glycosyltransferases, which can catalyze spermidine derivatives to generate corresponding spermidine derivative glycosides, and has important application value in the in vitro biosynthesis of Ningxia wolfberry caffeoyl spermidine substances, or has reference function and important significance in the in vitro catalytic synthesis of spermidine derivative glycosides from other species sources.
The glycosyltransferases LbUGT62, lbUGT64 and LbUGT73 of the spermidine derivatives are characterized in that the amino acid sequences are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
It is a second object of the present invention to provide genes encoding the above-mentioned group of spermidine derivative glycosyltransferases.
The coding genes are preferably LbUGT62, lbUGT64 and LbUGT73, and the nucleotide sequences of the coding genes are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
It is a third object of the present invention to provide the use of a group of spermidine derivative glycosyltransferases as described above in the synthesis of spermidine derivative glycosides.
Preferably, the use of spermidine derivative glycosyltransferase LbUGT62, lbUGT64 or LbUGT73 to catalyze the production of spermidine derivative glycosides from spermidine derivatives.
Further preferred, the spermidine derivative glycosyltransferase LbUGT62 catalyzes N 1 ，N 10 -dicaffeoylspermidine to two N 1 ，N 10 Use of-dicaffeoylspermidine glycoside, or spermidine derivative glycosyltransferase LbUGT62 in the catalysis of N 1 caffeoyl-N 10 -dicaffeoylspermidine to form an N 1 caffeoyl-N 10 -the use of dicaffeoylspermidine glycosides; the amino acid sequence of the spermidine derivative glycosyltransferase LbUGT62 is shown as SEQ ID NO. 4.
Further preferably, the spermidine derivative glycosyltransferase LbUGT64 catalyzes N 1 ，N 10 -dicaffeoylspermidine to two N 1 ，N 10 Use of a dicaffeoylspermidine glycoside, or spermidine derivative glycosyltransferase LbUGT64 in catalyzing N 1 caffeoyl-N 10 -dicaffeoylspermidine to two N 1 caffeoyl-N 10 -the use of dicaffeoylspermidine glycosides; the amino acid sequence of the spermidine derivative glycosyltransferase LbUGT64 is shown as SEQ ID NO. 5.
Further preferred, the spermidine derivative glycosyltransferase LbUGT73 catalyzes N 1 ，N 10 -dicaffeoylspermidine to two N 1 ，N 10 Use of-dicaffeoylspermidine glycoside, or spermidine derivative glycosyltransferase LbUGT73, for catalyzing N 1 caffeoyl-N 10 -dicaffeoylspermidine to three N 1 caffeoyl-N 10 -the use of dicaffeoylspermidine glycosides; the amino acid sequence of the spermidine derivative glycosyltransferase LbUGT73 is shown as SEQ ID NO. 6.
The invention obtains 3 spermidine derivative glycosyltransferases from the Ningxia wolfberry clone, can catalyze spermidine derivatives to generate corresponding spermidine derivative glycosides, has important application value in the in vitro biosynthesis of Ningxia wolfberry caffeoyl spermidine substances, or has reference function and important significance for the in vitro catalytic synthesis of spermidine derivative glycosides from other species sources.
Description of the drawings:
FIG. 1 shows the SDS-PAGE results of LbUGT62 (A), lbUGT64 (B) and LbUGT73 (C) protein purification. M: protein marker, T: total protein, S: after disruption, the supernatant protein, F: flow-through liquid, W1: first wash section, W2: a second wash section, E: an elution portion; the right arrow indicates the protein of interest.
FIG. 2 LbUGT62 catalyzes N 1 ，N 10 -dicaffeoylspermidine generates new compounds P1 and P2.
FIG. 3 LbUGT62 catalytic N production 1 ，N 10 Primary mass spectrum (left) and secondary mass spectrum (right) of dicaffeoylspermidine glycosides P1 and P2.
FIG. 4 LbUGT64 catalyzes N 1 ，N 10 -dicaffeoylspermidine generates new compounds P3 and P4.
FIG. 5 LbUGT64 catalytic N production 1 ，N 10 Primary mass spectrum (left) and secondary mass spectrum (right) of dicaffeoylspermidine glycosides P3 and P4.
FIG. 6 LbUGT73 catalyzes N 1 ，N 10 -dicaffeoylspermidine generates new compounds P1 and P2.
FIG. 7 LbUGT73 catalytic N production 1 ，N 10 Primary mass spectrum (left) and secondary mass spectrum (right) of dicaffeoylspermidine glycosides P1 and P2.
FIG. 8 LbUGT62 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to form new compound P5.
FIG. 9 LbUGT62 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to give new compound P5 primary mass spectrum (a) and secondary mass spectrum (B).
FIG. 10 LbUGT64 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to give new compounds P6 and P7.
FIG. 11 LbUGT64 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to give new compounds P6 (left column) and P7 (right column) primary Mass Spectrum (MS) and secondary mass spectrum (MS 2).
FIG. 12 LbUGT73 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to form new compounds P5, P6 and P8.
FIG. 13 LbUGT73 catalyzes N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to give new compounds P5 (left column), P6 (middle column) and P8 (right column) primary Mass Spectrum (MS) and secondary mass spectrum (MS 2).
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Experimental methods not specifically noted in the following examples can be performed according to conventional methods.
Example 1: screening of spermidine derivative glycosyltransferase genes
Taking a PSPGbox sequence conserved by plant secondary metabolite glycosyltransferase as a bait sequence, and searching in a Ningxia wolfberry transcriptome database to obtain 70 Ningxia wolfberry glycosyltransferase sequences and 59 lycium ruthenicum homologous glycosyltransferase sequences. The lycium ruthenicum and the lycium ruthenicum belong to the lycium genus, and fruits of the lycium ruthenicum contain spermidine derivative substances, but the fruits do not contain glycoside, so that candidate genes of the invention are mainly screened based on expression in the lycium ruthenicum and not expression in the lycium ruthenicum. The genes obtained by final screening were named LbUGT62, lbUGT64 and LbUGT73, respectively.
Example 2: cloning spermidine derivative glycosyltransferase gene, constructing overexpression vector and sequencing analysis
RNA of Lycium barbarum fruit was extracted, and genomic DNA was removed and reverse transcription of RNA was performed using a Prime Script RT Reagent Kit With gDNA Eraser (Takara) kit, respectively. The reaction system for genomic DNA removal is as follows: 5X gDNAEraser Buffer 2.0.0. Mu.L, gDNAEras 1.0. Mu.L, total RNA 1. Mu.g, RNase Free H 2 O to 10. Mu.L; reacting for 2 minutes at 42 ℃; after the end, the system was placed on ice. The reaction system for reverse transcription is as follows: the reaction system for digestion of genomic DNA was 10. Mu.L, which included 5X PrimeScript Buffer 2 (for real time) 4.0. Mu.L, primeScript RT Enzyme Mix I1.0.0. Mu.L, RT Primer Mix 1.0. Mu.L, RNase Free dH 2 O4.0. Mu.L; after 15 minutes of reaction at 37 ℃,85 ℃ for 5 seconds; the obtained product is cDNA of Lycium barbarum fruit in Ningxia, and is stored in a refrigerator at-20deg.C.
Primer sequences of spermidine derivative glycosyltransferase gene clones were designed using Primer5.0 software based on full-length coding sequences in the transcriptome of Lycium barbarum fruit, wherein the forward Primer and reverse Primer sequences are shown in Table 1, respectively. The obtained cDNA of Lycium barbarum fruit was used as a template, and PCR amplification was performed using PrimeSTAR max (2X) (Takara) using the forward and reverse primer pairs shown in Table 1, and the PCR reaction system was: primeSTAR Max (2X) 25. Mu.L, cDNA 3. Mu.L, forward and reverse primers at 10. Mu.M concentration of 2. Mu.L each, ddH 2 O18. Mu.L. The PCR reaction conditions were: 98 ℃ for 2min;98℃for 30s,55-65℃for 30s (specific annealing temperature based on the primer synthesized) and 72℃for 1min;35 cycles; extending at 72℃for 10min.
The PCR product and the expression plasmid pET32a were digested with double enzymes (using Takara restriction enzymes), and the reaction system was: 10×QuickCut Buffer 5. Mu.L, pET32a plasmid/PCR product not exceeding 1. Mu.g/200ng,QuickCut BamHI 1. Mu.L, quickCut XhoI 1. Mu.L, sterilized ddH 2 O was made up to 50. Mu.L. And (3) purifying and recycling the product obtained after the double enzyme digestion reaction by using a product recycling kit of a Meiy company. After purification, the concentration was determined by combining the gene fragment of interest with vector 3:1 (T4 ligase from Takara Co., ltd.) and the ligation reaction system was: 10×T DNA Ligase Buffer 1.mu.L, DNA 5.mu.L, vector 2.mu.L, T4 DNALigase 1.mu.L, sterilized ddH 2 O1. Mu.L. The ligation was carried out overnight at 4 ℃.
The ligation product was transformed into DH 5. Alpha. Clone competence, and the transformed bacterial liquid was spread on LB plates containing ampicillin, and cultured upside down in a constant temperature incubator at 37℃for 12-16 hours. The following day, colony PCR analysis (using T5 PCR enzyme from Optimago) was performed using T7 forward primer and reverse primer of gene, the reaction system was: 2×T5 Supermix 5. Mu.L, T7 forward primer (10. Mu.M) 0.5. Mu.L, gene reverse primer (10. Mu.M) 0.5. Mu.L, bacterial liquid template 1. Mu.L, sterilized ddH 2 O3. Mu.L. The PCR reaction procedure was: 98 ℃ for 2min;98℃for 10s,55-65 ℃ (specific annealing temperature is based on the synthetic primer) for 10s,72℃for 30s;30 cycles; extending at 72℃for 3min.
3 clones of each gene, which are verified to be positive by colony PCR, are selected, plasmids are extracted and sent to a biological company for sequencing and identification, the nucleotide sequence of a spermidine derivative glycosyltransferase gene LbUGT62 obtained by cloning is shown as SEQ ID NO.1, the nucleotide sequence of the spermidine derivative glycosyltransferase gene LbUGT62 contains 1398 bases, the coded protein is named as spermidine derivative glycosyltransferase LbUGT62, the total number of the amino acid residues is 465, and the specific amino acid sequence is shown as SEQ ID NO. 4. The nucleotide sequence of the spermidine derivative glycosyltransferase gene LbUGT64 is shown as SEQ ID NO.2, the spermidine derivative glycosyltransferase gene LbUGT contains 1392 bases, the coded protein is named as spermidine derivative glycosyltransferase gene LbUGT64, the total number of amino acid residues is 463, and the specific amino acid sequence is shown as SEQ ID NO. 5. The nucleotide sequence of the spermidine derivative glycosyltransferase gene LbUGT73 is shown as SEQ ID NO.3, the coded protein is named as spermidine derivative glycosyltransferase gene LbUGT73, the total number of the coded protein is 477 amino acid residues, and the specific amino acid sequence is shown as SEQ ID NO. 6.
TABLE 1 candidate Gene full Length CDS cloning primer sequence (underlined indicates the cleavage site)
Example 3: synthesis of spermidine derivative glycosyltransferase gene, construction of overexpression vector and sequencing analysis
Full-length sequences of spermidine derivative glycosyltransferase genes LbUGT62 (specifically shown as SEQ ID NO. 1), lbUGT64 (specifically shown as SEQ ID NO. 2) and LbUGT73 (specifically shown as SEQ ID NO. 3) are directly synthesized by adopting a total synthesis method, the spermidine derivative glycosyltransferase genes are constructed on an expression vector pET32a in a connection mode of example 2, the spermidine derivative glycosyltransferase genes are transformed into DH5 alpha, and the sequences are determined to be correct by sequencing again after plasmids are extracted. Thus, an expression vector containing spermidine derivative glycosyltransferase gene LbUGT62, lbUGT64, or LbUGT73 was obtained.
Example 4: target protein induced expression and purification
The expression vectors containing spermidine derivative glycosyltransferase genes LbUGT62, lbUGT64 or LbUGT73 obtained in example 3 were extracted and then transformed into BL21 (DE 3) expression competence, respectively. Positive monoclonal was picked and cultured in 5mL LB liquid medium containing Amp antibiotics at 37℃overnight at 200 rpm. The following day is 1:100 proportion of the strain is inoculated into 400mL of fresh LB liquid medium containing Amp antibiotics, the temperature is 37 ℃, and the strain is cultured at 200rpm until the strain reaches OD 600 To 0.6, the inducer IPTG was added at a final concentration of 0.1mM and the incubation was continued for 16-20 hours at 16 ℃. Collecting thallus by low temperature centrifugation, adding appropriate amount of sterilized ddH 2 O was washed once, centrifuged again, and the supernatant discarded.
The cells were resuspended in 10mL lysis buffer (pH 7.5, containing 20mM Tris-HCl, 0.5M NaCl and 10mM imidazole), repeatedly freeze-thawed 3 times in a water bath at 40℃and liquid nitrogen, PMSF was added to a final concentration of 1mM, and the resuspended cells were incubated in ice water and sonicated under the following conditions: ON 5s, off 5s,30min. Centrifuging at 4deg.C and 6000g for 15min after crushing, and collecting supernatant as total protein after induction.
The total protein after induction was purified using Ni-NTA affinity packing (Qiagen Inc.), the method being carried out with reference to the packing instructions. And desalting and concentrating the eluted target protein by adopting an ultrafiltration centrifuge tube. Finally, SDS-PAGE was used to examine the purification effect of the proteins, and the purification effects of LbUGT62, lbUGT64 and LbUGT73 were as shown in FIG. 1, respectively. Thereby obtaining purified spermidine derivative glycosyltransferases LbUGT62, lbUGT64 and LbUGT73.
Example 5: n (N) 1 ,N 10 Biosynthesis and identification of dicaffeoylspermidine glycosides
N using the purified spermidine derivative glycosyltransferase LbUGT62 obtained in example 4 1 ，N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 ，N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT62 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min,5% -40% B;12-17min,40% -99% B. After liquid phase elution analysis, lbUGT62 can catalyze N 1 ，N 10 Dicaffeoylspermidine gives two new compounds P1 and P2, as shown in figure 2.
The compounds P1 and P2 are respectively identified by mass spectrum, and the primary mass spectrum results of the compounds P1 and P2 are shown as [ M+H ]] + m/z= 632.28, as shown in FIG. 3 (left), shows that the molecular weight of the compound is 631, to N 1 ，N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (469), and the secondary mass spectrum produced mainly two fragments, m/z 470.23 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 3 (right), demonstrating that glucosyl group is linked to N 1 ，N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that both compounds P1 and P2 were N 1 ，N 10 -dicaffeoylspermidine glycoside, both of which are isomers of each other.
Example 6: n (N) 1 ,N 10 Biosynthesis and identification of dicaffeoylspermidine glycosides
By means ofN-treatment of the purified spermidine derivative glycosyltransferase LbUGT64 obtained in example 4 1 ，N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 ，N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT64 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min,5% -40% B;12-17min,40% -99% B. After liquid phase elution analysis, lbUGT64 can catalyze N 1 ，N 10 The dicaffeoylspermidine gives two new compounds P3 and P4, as shown in figure 4.
The compounds P3 and P4 are respectively identified by mass spectrum, and the primary mass spectrum results of the compounds P3 and P4 are shown as [ M+H ]] + m/z= 632.28, as shown in FIG. 5 (left), shows that the molecular weight of the compound is 631, to N 1 ，N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (469), and the secondary mass spectrum produced mainly two fragments, m/z 470.23 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 5 (right), demonstrating that glucosyl group is linked to N 1 ，N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that both compounds P3 and P4 were N 1 ，N 10 -dicaffeoylspermidine glycoside, both of which are isomers with respect to each other, and compounds P1 and P2 are also isomers.
Example 7: n (N) 1 ,N 10 Biosynthesis of dicaffeoylspermidine glycoside
N-treatment with the purified spermidine derivative glycosyltransferase LbUGT73 obtained in example 4 1 ，N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 ，N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT73 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min, 5-40%B, a step of preparing a composite material; 12-17min,40% -99% B. After liquid phase elution analysis, lbUGT73 can catalyze N 1 ，N 10 Dicaffeoylspermidine gives two new compounds P1 and P2, as shown in figure 6.
The compounds P1 and P2 are respectively identified by mass spectrum, and the primary mass spectrum results of the compounds P1 and P2 are shown as [ M+H ]] + m/z= 632.28, as shown in FIG. 7 (left), shows that the molecular weight of the compound is 631, to N 1 ，N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (469), and the secondary mass spectrum produced mainly two fragments, m/z 470.23 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 7 (right), demonstrating that glucosyl group is linked to N 1 ，N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that both compounds P1 and P2 were N 1 ，N 10 -dicaffeoylspermidine glycoside, both of which are isomers of each other, identical to the product catalyzed by the spermidine derivative glycosyltransferase LbUGT 62.
Example 8: n (N) 1 caffeoyl-N 10 Biosynthesis of dihydrocaffeoyl spermidine glycoside
N using the purified spermidine derivative glycosyltransferase LbUGT62 obtained in example 4 1 caffeoyl-N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 caffeoyl-N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT62 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min,5% -40% B;12-17min,40% -99% B. After liquid phase elution analysis, lbUGT62 can catalyze N 1 caffeoyl-N 10 -dicaffeoylspermidine gives a new compound P5, as shown in FIG. 8, wherein P5 is compared with the standard 4' -O-beta-D-glucopyranosyl-N 1 -caffeoyl-N 10 Dihydrocaffeoyl spermidine (Lycium barbarum D) has the same retention time.
The compound P5 is identified by mass spectrum, and the primary mass spectrum results of the compound P5 are allShown as [ M+H ]] + m/z= 634.30, as shown in FIG. 9 (A), shows that the molecular weight of the compound is 633, to N 1 caffeoyl-N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (471), and the secondary mass spectrum produced mainly two fragments, m/z 472.24 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 9 (B), demonstrating that the glucosyl group is linked to N 1 caffeoyl-N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that Compound P5 was N 1 caffeoyl-N 10 The mass spectrum cleavage mode and the retention time of the dicaffeoylspermidine glycoside are consistent with those of the Ningxia wolfberry spermidine D through comparison analysis with a standard substance, so that the P5 is determined to be the Ningxia wolfberry spermidine D, and the specific structure is shown in figure 8.
Example 9: n (N) 1 caffeoyl-N 10 Biosynthesis of dihydrocaffeoyl spermidine glycoside
N-treatment with the purified spermidine derivative glycosyltransferase LbUGT64 obtained in example 4 1 caffeoyl-N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 caffeoyl-N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT64 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min,5% -40% B;12-17min,40% -99% B. After liquid phase elution analysis, lbUGT64 can catalyze N 1 caffeoyl-N 10 The dicaffeoylspermidine gives two new compounds P6 and P7, as shown in figure 10.
The compounds P6 and P7 are respectively identified by mass spectrum, and the primary mass spectrum results of the compounds P6 and P7 are shown as [ M+H ]] + m/z= 634.30, as shown in FIG. 11, shows that the molecular weight of the compound is 633, to N 1 caffeoyl-N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (471), and the secondary mass spectrum produced mainly two fragments, m/z 472.24 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 11, demonstrating that the glucosyl group is attached toN 1 caffeoyl-N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that both compounds P6 and P7 were N 1 caffeoyl-N 10 -dicaffeoylspermidine glycoside, both of which are isomers, and P5 (lycium barbarum spermidine D) are also isomers. Wherein P6 is the main product.
Example 10: n (N) 1 caffeoyl-N 10 Biosynthesis of dihydrocaffeoyl spermidine glycoside
N-treatment with the purified spermidine derivative glycosyltransferase LbUGT73 obtained in example 4 1 caffeoyl-N 10 Biosynthesis of dicaffeoylspermidine glycoside, per 100 μl of reaction system comprising: 200 mu M N 1 caffeoyl-N 10 -dicaffeoylspermidine as substrate, 1mM UDP-glucose, 50mM Tris-HCl buffer (ph 7.5) and 5 μl of purified LbUGT64 protein. After 1 hour of reaction at 35℃the reaction was terminated with 100. Mu.L of methanol. The reaction system was analyzed by LCMS, elution procedure was: 0-2min,5% b (acetonitrile) +95% a (0.5% formic acid water); 2-12min,5% -40% B;12-17min,40% -99% B. After liquid phase elution analysis, lbUGT73 can catalyze N 1 caffeoyl-N 10 Dicaffeoylspermidine gives three new compounds P5, P6 and P8, as shown in fig. 12, where P5 has the same retention time as spermidine D and P8 has the same retention time as spermidine a.
The compounds P5, P6 and P8 are respectively identified by mass spectrum, and the primary mass spectrum results of the three are shown as [ M+H ]] + m/z= 634.29 or 634.30, as shown in FIG. 13, shows that the molecular weight of the compound is 633, to N 1 caffeoyl-N 10 Molecular weight of one more glucosyl group (162) than dicaffeoylspermidine (471), and the secondary mass spectrum produced mainly two fragments, m/z 472.24 (fragments after cleavage of glucosyl group) and 220.10, respectively, as shown in FIG. 13, demonstrating that glucosyl group is attached to N 1 caffeoyl-N 10 -dicaffeoylspermidine on the hydroxyl group. Thus, it was confirmed that compounds P5, P6 and P8 were all N 1 caffeoyl-N 10 -dicaffeoylspermidine glycoside, and the three are isomers with each other, by retention times with the standard Ningxia wolfberry spermidine A and DComparing mass spectrum results to determine that P5 is Ningxia wolfberry spermidine D which is the same as a catalytic product of LbUGT 62; p8 is Ningxia wolfberry spermidine A. Wherein P5 is the main product.
Claims (6)
1. The spermidine derivative glycosyltransferase is characterized in that the amino acid sequence of the spermidine derivative glycosyltransferase is shown as SEQ ID NO. 4.
2. A gene encoding the spermidine derivative glycosyltransferase of claim 1.
3. The gene according to claim 2, wherein the nucleotide sequence of the gene encoding glycosyltransferase of spermidine derivative is shown in SEQ ID NO. 1.
4. Use of the spermidine derivative glycosyltransferase of claim 1 in the synthesis of a glycoside of a spermidine derivative.
5. The use according to claim 4, wherein the spermidine derivative glycosyltransferase is used for catalyzing the formation of spermidine derivative glycosides from spermidine derivatives.
6. Use according to claim 5, characterized in that the spermidine derivative glycosyltransferase LbUGT62 catalyzes N 1 ，N 10 -dicaffeoylspermidine to two N 1 ，N 10 Use of-dicaffeoylspermidine glycoside, or spermidine derivative glycosyltransferase LbUGT62 in the catalysis of N 1 caffeoyl-N 10 -dihydrocaffeoylspermidine to form an N 1 caffeoyl-N 10 -use of dihydrocaffeoyl spermidine glycosides; the amino acid sequence of the spermidine derivative glycosyltransferase LbUGT62 is shown in SEQ ID NO. 4.
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| 糖基转移酶在改善天然产物成药性方面的应用;金月;吴旭日;陈依军;;中国药科大学学报(第05期);第31-37页 * |
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