Xylosyltransferase, coding gene, recombinant plasmid, strain and application of xylosyltransferase in preparation of xylosylated glucooligosaccharide

Abstract

The invention relates to the technical field of gene engineering, in particular to xylosyltransferase, a coding gene, a recombinant plasmid, a strain and application of the xylosyltransferase in preparation of xylosylated glucooligosaccharide. In the invention, the xylosyl transferase is selected from one of TiXXT1, TiXXT2 or TiXXT5; the amino acid sequence of the TiXXT1 is as shown in SEQ ID NO. 1; the amino acid sequence of the TiXXT2 is as shown in SEQ ID NO. 2; and the amino acid sequence of the TiXXT5 is as shown in SEQ ID NO. 3. According to the invention, xylosyltransferase coding genes TiXXT1, TiXXT2 and TiXXT5 are identified in a tamarind genome for the first time, and the unique continuous xylose transfer activity of the xylosyltransferase coding genes is confirmed through heterologous expression and enzymatic characterization; and a foundation is laid for low-cost synthesis of the functional high-substituted xylosylated glucan oligosaccharide.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to xylosyltransferase, encoding genes, recombinant plasmids, strains, and their applications in the preparation of xylosyl-modified glucosinolates. Background Technology

[0002] Xylglucan is a key hemicellulose component of the primary cell wall in plants, and its structural characteristics are closely related to its function. It typically consists of a backbone of β-1,4-glucose chains, with xylose side chains linked to the hydroxyl groups at position 6 of specific glucose residues via α-1,6-glycosidic bonds. This modification process is catalyzed by specific xylosyltransferases. These enzymes are responsible for transferring activated xylose donors (usually UDP-xylose) to the glucan backbone, thereby endowing xylglucan with a branched structure, which in turn affects its binding ability to cellulose microfibrils, cell wall plasticity, and overall mechanical properties (Scheller & Ulvskov, 2010; Zabotina, 2012). As one of the main cross-linked polysaccharides in the cell wall matrix, xylglucan plays an important role in regulating cell expansion, maintaining tissue toughness, and responding to external mechanical stimuli.

[0003] Tamarind seeds are rich in a unique type of xyloglucan, whose side chain pattern, branching frequency, and molecular weight distribution differ significantly from xyloglucans from common plant sources. This special structure endows them with excellent hydration, viscosity, and gelling properties, leading to their commercial applications as natural thickeners and stabilizers in the food industry, as drug delivery carriers in the pharmaceutical field, and as texture modifiers in cosmetics (Chinta et al., 2025; Durai et al., 2012). Although its end products have been widely developed and utilized, the enzyme playing a key role in its biosynthetic pathway—tamarind xylosyltransferase—has not yet been successfully cloned and systematically characterized, leaving the synthetic mechanism of this polysaccharide unclear (Narude et al., 2025; Sri et al., 2025; Zhang et al., 2024).

[0004] Currently, research on xylosyltransferases largely focuses on the model plant Arabidopsis thaliana. Xylosyltransferases in Arabidopsis are mainly encoded by the AtXXT gene family, several members of which have been successfully identified and functionally recombinantly expressed in heterologous systems such as Escherichia coli and yeast. Their in vitro activity, substrate preference, and protein structural characteristics have been preliminarily elucidated (Julian & Zabotina, 2022; Yamatoya et al., 2020). These studies provide important templates for understanding the synthetic basis of xyloglucan. However, homologous enzymes from the tropical economic crop tamarind may differ significantly from those in Arabidopsis in gene sequence, protein structure, substrate recognition mechanism, catalytic kinetic efficiency, and the regulatory mechanisms at the transcriptional and translational levels. This difference may stem from various factors such as evolutionary distance, adaptation to growth environment, and cell wall composition specificity (Cavalier et al., 2008; Zabotina, 2012). Therefore, directly applying the enzymological data of model plants to guide the synthesis or modification of tamarind glucan often fails to achieve the desired results, which greatly limits the high-yield and customized production of tamarind glucan with specific functional properties through synthetic biology.

[0005] Therefore, systematic research on tamarind xylosyltransferases—including gene cloning, recombinant protein expression and purification, in vitro enzyme activity detection, substrate-specific analysis, three-dimensional structural analysis, and in vivo functional verification—has significant scientific value and application potential. This will not only fill the gap in basic research on cell wall synthesis in this species and deepen our understanding of the diversity of hemicellulose biosynthetic pathways in non-model plants, but also lay the foundation for targeted modification of the cell wall structure of tamarind or other crops through genetic engineering and metabolic engineering. For example, by regulating the expression of this enzyme or altering its activity, the physicochemical properties of the cell wall may be optimized, thereby improving fruit texture and enhancing stress resistance. In in vitro synthesis systems, the use of efficiently expressed recombinant enzymes can achieve large-scale enzymatic preparation of structurally uniform and performance-controllable xyloglucan, thus promoting the wider application of high-performance, high-value-added xyloglucan products in food, medicine, and materials.

[0006] In conclusion, conducting research on the gene mining, recombinant expression, and application of tamarind xylosyltransferases in synthetic biology is a crucial link connecting basic plant science and industrial biotechnology, and is of great significance for the rational design and efficient utilization of plant cell wall resources. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide xylosyltransferase, encoding gene, recombinant plasmid, strain, and its application in the preparation of xylosyl-modified glucosinolates.

[0008] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide a xylosyltransferase selected from one of TiXXT1, TiXXT2, or TiXXT5; The amino acid sequence of TiXXT1 is shown in SEQ ID NO.1; The amino acid sequence of TiXXT2 is shown in SEQ ID NO.2; The amino acid sequence of TiXXT5 is shown in SEQ ID NO.3.

[0009] A second object of the present invention is to provide a gene encoding the above-mentioned xylosyltransferase, wherein the nucleotide sequence of TiXXT1 is shown in SEQ ID NO.4; The nucleotide sequence of TiXXT2 is shown in SEQ ID NO.5; The nucleotide sequence of TiXXT5 is shown in SEQ ID NO.6.

[0010] A third objective of this invention is to provide a recombinant plasmid containing the aforementioned genes.

[0011] A fourth object of the present invention is to provide a strain comprising the above-described recombinant plasmid.

[0012] The fifth object of this invention is to provide a method for preparing xylosyltransferase, comprising the following steps: (A1) Inoculate the above strains into the culture medium, culture them to the logarithmic growth phase, and then add an inducer to induce growth; (A2) After step (A1) is completed, collect the bacterial cells, lyse them, collect the supernatant, and purify it to obtain xylosyltransferase.

[0013] In one embodiment of the present invention, in step (A1), the inducer is IPTG, and the induction time is 16-24 hours.

[0014] In one embodiment of the present invention, in step (A2), the bacterial cells are collected by centrifugation, then washed and resuspended using PBS washing solution; After crushing, the clear liquid is collected by centrifugation; Purification was performed using nickel column affinity chromatography; After purification, the protein can be concentrated by ultrafiltration and centrifugation.

[0015] The sixth object of the present invention is to provide an application of a xylosyltransferase in the preparation of xylosyl-modified dextran, wherein the xylosyltransferase is the aforementioned xylosyltransferase.

[0016] The seventh object of the present invention is to provide a method for preparing xylosyl-modified dextran, comprising the following steps: Xyloylglycosylated glucan was prepared by mixing UDP-xylose, glucosinolate, MnCl2, Tris, NaCl and the above-mentioned xylosyltransferase.

[0017] In one embodiment of the present invention, the molar ratio of UDP-xylose, glucosinolate, MnCl2, Tris, NaCl and xylosyltransferase is 2 mM: 1 mM: 2.5 mM: 50 mM: 150 mM: 2~5 μM; During the reaction, the temperature was 37℃ and the time was 1~6 h.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention, through the first-ever identification and heterologous expression of xylosyltransferases TiXXT1, TiXXT2, and TiXXT5 with unique continuous xylose transfer activity in tamarind, constructs a highly efficient cell-free synthesis system, achieving the targeted and precise preparation of xylosylated glucosinolates. This technology uses glucosinolates with specific degrees of polymerization as acceptors, effectively overcoming the shortcomings of traditional natural extraction methods, such as high product structural heterogeneity, and the numerous impurities and susceptibility to endotoxin contamination in microbial fermentation methods. It significantly improves the controllability of the product's degree of polymerization and xylose substitution mode. While greatly simplifying downstream purification processes and ensuring high structural consistency of target fragments, it provides core active raw materials with controllable quality standards and clearly defined functional structures for the precision pharmaceutical and high-end skincare fields, and lays a solid foundation for the low-cost, large-scale synthesis of highly functional xylosylated glucosinolates. Attached Figure Description

[0019] Figure 1 Statistical graph for transcriptome functional annotation.

[0020] Figure 2 Phylogenetic tree and transmembrane topology analysis diagram of TiXXT and Arabidopsis AtXXT family.

[0021] Figure 3 The image shows the SDS-PAGE analysis of recombinant xylosyltransferase; where A: pET-30α(+)-GB1-tr95989; B: fusion protein GB1-tr95989; C: fusion protein GB1-tr40083; D: fusion protein GB1-tr3543.

[0022] Figure 4 A comparative graph showing the xylosyltransferase activities of tamarind candidate proteins; where A. absolute rate: in 1 mmol L -1 Using glucosamine G5 as a substrate, the amount of UDP generated was measured after reacting at 37℃ for 60 min (pmol min).-1 B. Relative activity: With the enzyme-free blank as 1, calculate the fold change in UDP generation rate for each sample.

[0023] Figure 5 The images show the HPLC-HPAEC-MALDI-TOF chromatograms of xylosylated products of glucosinolate (G6); where A. HPLC chromatogram (UV 192 nm); B. HPAEC pulsed amperometric detection chromatogram; C. MALDI-TOF MS: G6 standard; D. Local magnification of TiXXT1 mono / di / tri / tetraxylosylated ions; E. Local magnification of TiXXT2 mono / dixylosylated ions; F. Local magnification of TiXXT5 monoxylosylated ions. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0025] In the following embodiments, the 3' end of the fusion tag GB1 was introduced into the speI restriction site and then inserted into the 3' end of the NdeI restriction site in pET-30a(+) (the pET-30a(+) sequence did not have any deletions). The pET-30a(+)-GB1 plasmid was synthesized by a company. Unless otherwise specified, all reagents used were commercially available, and all detection methods and techniques used were conventional detection methods and techniques in the art.

[0026] Example 1 This embodiment provides the experimental materials and methods, as detailed below: (1) Materials: LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, for culturing Escherichia coli. Solid medium with 20 g / L agar powder.

[0027] PBS buffer solution (10×): Na₂HPO₄ 80 mM, NaCl 1.36 M, KH₂PO₄ 20 mM, KCl 26 mM, pH 7.4.

[0028] NPI-5: Tris-HCl 20 mM, NaCl 500 mM, imidazole 5 mM, pH 7.5.

[0029] NPI-20: Tris-HCl 20 mM, NaCl 500 mM, imidazole 20 mM, pH 7.5.

[0030] NPI-50: Tris-HCl 20 mM, NaCl 500 mM, imidazole 100 mM, pH 7.5.

[0031] NPI-250: Tris-HCl 20 mM, NaCl 500 mM, imidazole 100 mM, pH 7.5.

[0032] NPI-500: Tris-HCl 20 mM, NaCl 500 mM, imidazole 500 mM, pH 7.5.

[0033] (2) Instruments and equipment: Spectra Max i3x multi-functional microplate reader, Nanodrop 2000 ultra-micro nucleic acid and protein quantification system, S1000 PCR instrument, Power Basic electrophoresis system, ChemiDoc XRS+ gel imaging system, Thermo Scientific Dionex ICS-5000 / 6000 ion chromatography system, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometer.

[0034] (3) Transcriptome mining: Three biological replicates each of tamarind leaves, seed endosperm, and seeds were collected. Total RNA was extracted and sequenced using Illumina PE150 sequencing. Reference transcripts were obtained by Trinity de novo assembly. BLASTx(Swiss-Prot+TAIR10, e≤1×10⁻⁶) was performed using the Trinotate pipeline. -5 The candidate gene pool was constructed by annotating Pfam (GT-family-34, DxD motif) with GO:0016757 "glycosyltransferase activity" and TPM≥5.

[0035] Transcriptome analysis of tamarind leaves, seed endosperm, and seeds yielded a total of 241.03 Gb of clean data. After trinity splicing, the number of unigenes obtained was 68,640, the number of transcripts was 142,695, and the average N50 length was 2,777 bp. Figure 1 The transcriptome functional annotation statistics are presented. The functional annotation shows that 346 unigenes possess "glycosyltransferase activity," which will be used for subsequent homology comparison.

[0036] (4) Homology comparison to identify candidates: Using the unigene obtained in step (3) as the target, and the Arabidopsis XXT1-5 protein sequence as the query, a local BLASTp (e≤1×10) was performed. -20(identity ≥ 35%, coverage ≥ 70%) and construct a phylogenetic tree; select genes that cluster with AtXXT on the same branch and have typical type II membrane topology (Nin-Cout, TMHMM verification), and finally determine TiXXT as a candidate gene for tamarind xylosyltransferase (TXT). Figure 2 ).

[0037] Using 346 high-confidence glycosyltransferase unigenes as the target, local BLASTp was performed using the Arabidopsis xylosyltransferase AtXXT1-5 protein sequences as queries, resulting in six candidate tamarind xylosyltransferases (TiXXT): DN85573, DN40083, DN95989, DN3543, DN4254, and DN28158, with homology to AtXXT of 79.5%, 79.5%, 80.1%, 81.1%, 34.5%, and 40.1%, respectively. Further MEGA7 alignment and phylogenetic tree construction were performed. TMHMM prediction showed that TiXXT has an N-terminus in the cytoplasm and a C-terminus in the Golgi lumen, anchored to the Golgi membrane by a single transmembrane helix, exhibiting a type II membrane protein topology.

[0038] The candidate genes for tamarind xylosyltransferase were finally identified as: DN85573, DN40083, DN95989, DN3543, DN4254 and DN28158.

[0039] (5) Preparation and transformation of competent Escherichia coli cells: Preparation of competent states: picking E. coli Top 10 or E. coli A single colony of BL21(DE3) was incubated in 4 mL LB medium on a shaker for 12 h (37°C, 200 rpm). A 1% inoculum was then added to 50 mL LB medium and incubated on a shaker until OD (occurrence depth) was reached. 600 The bacterial cells were collected by centrifugation (4℃, 4500 rpm, 10 min) with a pH of 0.3–0.5. After washing the bacterial cells with 0.1 mol / L CaCl2 solution, the cells were resuspended in 1 mL of 0.1 mol / L CaCl2 solution containing 15% glycerol to obtain competent cells. The cells were aliquoted into 100 μL tubes and stored at -80℃ for later use.

[0040] Transformation experiment: The competent cells obtained above were mixed with 10 μL of ligation product and incubated on ice for 30 min. After heat shock at 42℃ for 90 s, 900 μL of LB medium was added, and the cells were cultured on a shaker for 60 min (37℃, 200 rpm). The cells were collected by centrifugation (4500 rpm, 5 min) and plated on antibiotic plates. Clones were obtained by incubation at 37℃. PCR amplification was performed to verify the product size.

[0041] (6) PCR amplification reaction system: PCR reaction system (25 μL): 12.5 μL 2× Phanta Max Master Mix, 1 μL primer-F, 1 μL primer-R, 1 μL DNA template and ddH2O.

[0042] PCR amplification program: 95℃ for 3 min; 98℃ for 10 s; Tm (primer annealing temperature) for 30 s; 72℃ for 30 s / kb, 30 cycles; 72℃ for 10 min. Amplification products were verified by agarose gel electrophoresis.

[0043] PCR product recovery: The target fragment amplified into a single band was recovered using the Axygen Clean Recovery Kit, and the DNA concentration after recovery was measured.

[0044] (7) Seamless cloning reaction system: The target fragment and linearized vector amplified by PCR were recovered using a gel extraction kit, and the concentration of the recovered DNA was determined using a Nanodrop 2000 ultra-micro nucleic acid and protein quantification instrument. Ligation was performed according to the Clon Express II one-step cloning kit, with a vector-to-target fragment molar ratio of 1:2. The 10 μL reaction system included 0.4 pmol of target fragment, 0.2 pmol of vector fragment, 2 μL of 5×CEⅡ Buffer, 1 μL of Exnase, and ddH2O.

[0045] (8) Construction of heterologous expression plasmids: By extracting tamarind ( Tamarindus indica Total RNA was used to obtain cDNA templates via reverse transcription (RT-PCR). Sequence design was performed based on six potential xylosyltransferase (TiXXT) candidate fragments screened by tamarind transcriptome sequencing analysis. To verify the functions of the six potential TiXXT proteins, taking the DN95989 gene as an example, a heterologous expression plasmid pET-30a(+)-DN95989 was first constructed (DN95989 was inserted between Nde I and Xho I in pET-30a(+)). Figure 3 A), and transformed into E. coli BL21(DE3) competent cells, and picked the correct single colony into LB medium. IPTG induced the expression of DN95989 protein; the results showed that the complete amino acid sequence of DN95989 in SDS-PAGE was in the form of inclusion bodies, so the transmembrane region of DN95989 protein was truncated and the fusion tag GB1 was added to reconstruct pET-30a(+)-GB1-tr95989 (with T. indicaUsing cDNA as a template, the target gene tr95989 was amplified using primers tr95989F and tr95989R; the same applies to other candidate genes). The DN95989 protein was then purified and expressed using IPTG-induced protein purification, and it was found to be a soluble protein. Figure 3 B).

[0046] Protein purification and plasmid construction: T. indica Using cDNA as a template, the target gene tr95989 was amplified using primers tr95989F and tr95989R; the target gene tr40083 was amplified using primers tr40083F and tr40083R; the target gene tr3543 was amplified using primers tr3543F and tr3543R; the target gene tr85573 was amplified using primers tr85573F and tr85573R; the target gene tr4254 was amplified using primers tr4254F and tr4254R; and the target gene tr28158 was amplified using primers tr28158F and tr28158R. After verifying the correctness of the bands by agarose gel electrophoresis, the cDNA was purified and recovered. The target gene and pET-30a(+)-GB1 plasmid were digested with restriction enzymes Nde I and Xho I, respectively, to obtain linear vectors. After seamless cloning and ligation of the target gene and the linearized vector, the product was transformed into E. coli Top10 competent cells, and the following plasmids were selected for resistance: pET-30a(+)-GB1-tr95989, pET-30a(+)-GB1-tr40083, pET-30a(+)-GB1-tr3543, pET-30a(+)-GB1-tr85573, pET-30a(+)-GB1-tr4254, and pET-30a(+)-GB1-tr28158.

[0047] (9) Protein expression and purification: The correctly sequenced plasmids pET-30a(+)-GB1-tr95989, pET-30a(+)-GB1-tr40083, pET-30a(+)-GB1-tr3543, pET-30a(+)-GB1-tr85573, pET-30a(+)-GB1-tr4254, and pET-30a(+)-GB1-tr28158 were transformed into E. coli BL21(DE3) competent cells. A single colony of the correct sequence was picked and activated in 4 mL of LB medium for 12 h. The colonies were then inoculated at a 3% ratio into LB liquid medium and cultured at 37°C on a shaker until OD200 reached. 600Approximately 0.6, add a final concentration of 0.5 mmol / L IPTG, and induce induction at 20℃ for 20 h. Collect bacterial cells by centrifugation (4℃, 8000 rpm, 10 min), wash twice with PBS buffer and resuspend, then sonicate on ice (800 W, 5 s operation, 5 s pause). Separate the precipitate and supernatant by centrifugation (4℃, 8000 rpm, 10 min). Take the original bacterial culture, the sonicated supernatant, and the precipitate (PBS resuspended), treat with loading buffer, and observe protein expression by 12.5% ​​SDS-PAGE.

[0048] Take an appropriate amount of Ni 2+ - Pack the affinity chromatography column with NTA agarose and equilibrate with 5 volumes of NPI-5 buffer. 2+ Column. The supernatant after disruption was filtered through a 0.45 μm needle filter and loaded onto the column at a flow rate of 10 column volumes / h. Impurities were eluted with 5 column volumes of NPI-20 and NPI-100, followed by 10 column volumes of NPI-500, and the target protein was collected. The protein was concentrated by ultrafiltration centrifugation (10 kDa, 4℃, 5000 g).

[0049] The results showed that GB1-tr40083 and GB1-tr3543 are soluble proteins. Figure 3 C Figure 3 D), while GB1-tr85573 is still an inclusion body; in addition, GB1-tr4254 and GB1-tr28158 cannot be expressed, and will not be further explored and discussed later.

[0050] (10) Xylosyltransferase activity detection Xylosyltransferase reaction system: 2 mM UDP-xylose, 1 mM glucosyl oligosaccharide G5 (a pentasaccharide oligosaccharide, DP5, GGGGG, with 5 glucose molecules linked by β-1,4-glycosidic bonds), 2.5 mM MnCl2, 50 mM Tris, 150 mM NaCl, and 2.5 μM protein (GB1-tr95989, GB1-tr3543, and GB1-tr40083, respectively) were added. The total reaction volume was 25 μL, and the reaction was carried out at 37℃ for 1 h. Using the UDP-Glo™ Glycosyltransferase Assay (V4096) kit, 25 μL of the detection working solution was mixed with 25 μL of the product reaction system in the microplate, incubated at room temperature for 1 h, and then added to the microplate for bioluminescence detection.

[0051] Definition of enzyme activity unit (U): In this study, one enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of UDP per minute under the optimal reaction conditions of the enzyme.

[0052] The purified fusion proteins GB1-tr95989, GB1-tr40083, and GB1-tr3543, along with a blank control (enzyme-free), were incubated with 1 mM glucosamine G5 and 2 mM UDP-xylose systems at 37°C for 60 min, respectively. UDP production was detected using the UDP-Glo™ method, and the UDP production rates were 68.4, 18.4, and 15.2 pmol / min, respectively. -1 ( Figure 4 A). With the enzyme-free blank as 1, the UDP generation rates of GB1-tr95989, GB1-tr40083, and GB1-tr3543 increased by 74.5 times, 42.5 times, and 32.1 times, respectively. Figure 4 B). In this invention, the genes DN95989, DN40083, and DN3543 (their corresponding truncated coding sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively) are formally named TiXXT1, TiXXT2, and TiXXT5.

[0053] (11) Application of xylosyltransferase in glucose oligosaccharide G6 Similar to (10) above, except that glucosamine G5 is replaced with glucosamine G6 (a hexasaccharide oligosaccharide, DP6, GGGGGG, with 6 glucose molecules linked by β-1,4-glycosidic bonds), and glucosamine G6 is used as the substrate. The product detection method is as follows: Liquid chromatography: Agilent 1260 Infinity II, Rezex ROA-H+ hydrogen column (300 × 7.8 mm, 8 µm), column temperature 55℃, mobile phase 2.5 mM sulfuric acid, flow rate 0.6 mL / min -1 The amount of xylose remaining was detected by UV 192 nm.

[0054] Ion chromatography: Thermo ICS-5000+, CarboPac PA20 column (3 × 150 mm), column temperature 50℃, pulsed amperometric detection; gradient: 0–20 min 100 mM NaOH → 50 mM NaOAc, linearly up to 100 mM NaOAc over 20 min, flow rate 0.5 mL / min -1 Inject 5 µL of sample to achieve oligosaccharide region separation.

[0055] Time-of-flight mass spectrometry: Bruker maXis II ESI-Q-TOF, negative ion mode, m / z 50–3000; samples were collected in PA20 and diluted 1:1 with acetonitrile, then directly injected at 3 µL / min. -1Source parameters: capillary -4.5 kV, dry gas 4 Lmin -1 200℃; sodium was added online using NaOH / NaOAc, and [MH] was observed. - A signal with a quality accuracy of ≤2 ppm was used to confirm the molecular weight of xylosylated glucosinolate G6.

[0056] The xylosyltransferases TiXXT1, TiXXT2, and TiXXT5 were applied to the catalysis of glucosamine oligosaccharides. HPLC-HPAEC-MALDI-TOF MS analysis showed that no free xylose was detected in the reaction system, indicating that UDP-xylose was quantitatively transferred to glucosamine oligosaccharide G6. CarboPac PA20 chromatography showed that G6 (tR 8.9 min) sequentially generated GGXGGG (9.3 min), GXXGGG (10.0 min), and GXXXGG (10.5 min). MALDI-TOF MS ([M+Na)) + The m / z values ​​were measured sequentially as 1145.371 (GGXGGG, Xyl1), 1277.414 (GXXGGG, Xyl2), 1409.457 (GXXXGG, Xyl3), and 1541.500 (GXXXXG, Xyl4), with a mass deviation ≤2 ppm. This confirms that TiXXT1 can catalyze the continuous introduction of one to four xylose groups into glucosinolate G6; TiXXT2 can introduce one to two xylose groups; and TiXXT5 introduces only one, corresponding to the formation of Xyl1–Xyl4, Xyl1–Xyl2, and Xyl1 xylylized glucosinolates. Figure 5 The above studies demonstrate that xylosyltransferases TiXXT1, TiXXT2, and TiXXT5 derived from tamarind can efficiently achieve targeted modification of glucosyl oligosaccharide substrates, thereby catalyzing the synthesis of a series of novel xylosylated glucosyl oligosaccharides containing 1 to 4 xylose residues. This discovery not only confirms the potential of this enzyme system in constructing diverse side-chain structures but also provides core technical support for the precise preparation of xylosylated glucosyl oligosaccharides with specific substitution patterns.

[0057] The specific sequences involved in this invention are as follows: The amino acid sequence of TiXXT1 is shown in SEQ ID NO.1, as follows: MLDRLLGPRRVRQIQRACRHGTVTFLCLFLTVVVLRGTIGAGKFGTPEQDLNEIRDLYSRGRRVEPHRVLEEVHPENTQSDQSNNYATFDISKILKDEGGDDEKRDPNMPYSLGPKISDWDEQRAEWLKNNPDYPNFIGPNKPRVLLVTGSSPKPCENPVGDHYLLKSIKNKIDYCRLHGIEIFYNMALLDAEMAGFWAKLPLIRKLLLSHPEIEFLWWMDSDAMFTDMAFEVPWERYKNHNFVMHGWNDMVYDEKNWIGLNTGSFLLRNCQWSLDILDAWAPMGPKGKIRDEAGKILTRELKNRPVFEADDQSAMVYLLATERAKWGEKVYLENHYYLHGYWGILVDRYEEMIENYHPGLGDHRWPLVTHFVGCKPCGKFGDYPVERCLRQMDRAYNFGDNQILQMYGFTHKNLGSRRVKRVRNESSNPLEVKDELGLLHPAFKAIRLPNSS* The amino acid sequence of TiXXT2 is shown in SEQ ID NO.2, as follows: MLDRSLSPRSVRQIQRACRQGTVTFLCLFLTVVVLRGTIGAGKFGTPEKDFNEIRDHLYSPGRRVEPHRVLVEAQPENTESDQSNNYATFDISKILKDEETDDEKRDPNAPYSLGPKISDWDVQRAEWLKNNPDYPNFIGPNKPRVLLVTGSSPKPCENPVGDHYLLKSIKNKIDYCRLHGIEIFYNMALLDAEMAGFWAKLPLIRKLLLSHPEIEFLWWMDSDAMFTDMAFEVPWERYKDHNFVMHGWNEMVYDQKNWIGLNTGSFLLRNSQWSLDILDAWAPMGPKGKIRDEAGKILTRELKDRPVFEADDQSAMVYLLAKEKEKWGEKVYLENAYYLHGYWGILVDRYEEMIENYHPGLGDHRWPLVTHFVGCKPCGKFGDYPVERCLRQMDRAYNFGDNQILQMYGFTHKSLLSRRVKRVRNDSSNPLEVKDEFGLLHPAFKAVRLPASS* The amino acid sequence of TiXXT5 is shown in SEQ ID NO.3, as follows: MGQENPTAQKRSSGGGLPTATAAAAANGNGRGLAFSGLPRGRQINKTFNNIKITILCGFVTILVLRGTIGVNFGSSDADAVNQHLIEETNRILAEIRSDSDPSDPDEPTETELN PNITFTLGPKISNWDEERKVWLDQNPEYPNYIKGKARILLLTGSPPKPCDNPIGDHYLLKSIKNKIDYCRLHGIEIVYNLAHLDKELAGYWAKLPMIRRLMMSHPEVEWIWWMDS DAFFTDMVFELPLSKYDKYNLVLHGYPDLLFEQKSWIAVNTGSFLFRNCQWSLDLLDAWAPMGPKGPIREEAGKILTANKGRPAFEADDQSALIYLLLSKKDQWMEKVFLENSY YLHGYWAGLVDRYEEMIEKYHPGLGDERWPPFVTHFVGCKPCGSYGDYPVERCLSSMERAFNFADNQVLKLYGFSHRGLLSPRIRRFRNETVTPLEFVDQFDIRRHHSESSGSKS* The nucleotide sequence of TiXXT1 is shown in SEQ ID NO.4 (5'-3'), as follows: The nucleotide sequence of TiXXT2 is shown in SEQ ID NO.5 (5'-3'), as follows: The nucleotide sequence of TiXXT5 is shown in SEQ ID NO.6 (5'-3'), as follows: The nucleotide sequence of GB1 is shown in SEQ ID NO.7 (5'-3'), as follows: tacaaactgatccttaacggtaaaactttgaaaggcgaaaccactaccgaagctgttgatgctgcgactgcagaaaaagttttcaaacagtacgctaacgacaacggtgttgacggtgaatggacttacgacgatgcgactaagaccttcaccgttatactgaa The nucleotide sequence of tr95989F is shown in SEQ ID NO.8 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGGCGCCGGGAAGTTTGGAACCC The nucleotide sequence of tr95989R is shown in SEQ ID NO.9 (5'-3'), as follows: gttagcagccggatctcactcgagAGAAGAATTCGGTAACCTAATAGCTTTGA The nucleotide sequence of tr40083F is shown in SEQ ID NO.10 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGCGCCGGAAAGTTTGGGACTC The nucleotide sequence of tr40083R is shown in SEQ ID NO.11 (5'-3'), as follows: gttagcagccggatctcactcgagAGAAGAAGCTGGCAACCTGA The nucleotide sequence of tr3543F is shown in SEQ ID NO.12 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGGCTTCGGAAGCTCTGATGCGG The nucleotide sequence of tr3543R is shown in SEQ ID NO.13 (5'-3'), as follows: gttagcagccggatctcactcgagGCTTTTTCGATCCGCTGCTC The nucleotide sequence of tr85573F is shown in SEQ ID NO.14 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGGCAAGTTCGGCACACCGGAAC The nucleotide sequence of tr85573R is shown in SEQ ID NO.15 (5'-3'), as follows: gttagcagccggatctcactcgagAGATGATGATAACCTTAACTGCTTTAAAAGC The nucleotide sequence of tr4254F is shown in SEQ ID NO.16 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGGCATGTCCCCTATCCCTAATG The nucleotide sequence of tr4254R is shown in SEQ ID NO.17 (5'-3'), as follows: gttagcagccggatctcactcgagTCAAGCAGGGTAATCGAAAGGCATAGGCCG The nucleotide sequence of tr28158F is shown in SEQ ID NO.18 (5'-3'), as follows: ggcGAGAACCTGTACTTTCAGGGCATGTTCGCATATTGGGCTAAGT The nucleotide sequence of tr28158R is shown in SEQ ID NO.19 (5'-3'), as follows: gttagcagccggatctcactcgagTCAAGCAGAAGCAGGATAATCAGAAGGTAT The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A xylosyltransferase, characterized in that, The xylosyltransferase is selected from one of TiXXT1, TiXXT2 or TiXXT5; The amino acid sequence of TiXXT1 is shown in SEQ ID NO.1; The amino acid sequence of TiXXT2 is shown in SEQ ID NO.2; The amino acid sequence of TiXXT5 is shown in SEQ ID NO.

3.

2. The gene encoding the xylosyltransferase of claim 1, characterized in that, The nucleotide sequence of TiXXT1 is shown in SEQ ID NO.4; The nucleotide sequence of TiXXT2 is shown in SEQ ID NO.5; The nucleotide sequence of TiXXT5 is shown in SEQ ID NO.

6.

3. A recombinant plasmid, characterized in that, It contains the gene described in claim 2.

4. A strain, characterized in that, It includes the recombinant plasmid as described in claim 3.

5. A method for preparing a xylosyltransferase, characterized in that, Includes the following steps: (A1) The strain described in claim 4 is inoculated into a culture medium and cultured to the logarithmic growth phase, and then an inducer is added for induction. (A2) After step (A1) is completed, collect the bacterial cells, lyse them, collect the supernatant, and purify it to obtain xylosyltransferase.

6. The method for preparing a xylosyltransferase according to claim 5, characterized in that, In step (A1), the inducer is IPTG, and the induction time is 16~24h.

7. The method for preparing a xylosyltransferase according to claim 5, characterized in that, In step (A2), the bacterial cells are collected by centrifugation, then washed and resuspended using PBS washing buffer; After crushing, the clear liquid is collected by centrifugation; Purification was performed using nickel column affinity chromatography; After purification, the protein can be concentrated by ultrafiltration and centrifugation.

8. The application of a xylosyltransferase in the preparation of xylosyl-modified glucosinolates, characterized in that, The xylosyltransferase is the xylosyltransferase described in claim 1.

9. A method for preparing xylosylated glucosinolates, characterized in that, Includes the following steps: The xylose, glucosinolate, MnCl2, Tris, NaCl and the xylosyltransferase described in claim 1 are mixed and reacted to obtain xylosylated glucosinolate.

10. The method for preparing xylosyl-modified glucosinolates according to claim 9, characterized in that, The molar ratio of UDP-xylose, glucosinolates, MnCl2, Tris, NaCl, and xylosyltransferase was 2 mM: 1 mM: 2.5 mM: 50 mM: 150 mM: 2~5 μM; During the reaction, the temperature was 37℃ and the time was 1~6 h.

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