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CN-122012434-A - Xylosyltransferase, coding gene, recombinant plasmid, strain and application thereof in preparation of xylosylated glucose oligosaccharide

CN122012434ACN 122012434 ACN122012434 ACN 122012434ACN-122012434-A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a xylosyltransferase, a coding gene, a recombinant plasmid, a strain and application thereof in preparation of xylosylated glucose oligosaccharide. In the invention, the xylosyltransferase is selected from one of TiXXT, tiXXT2 or TiXXT, the amino acid sequence of TiXXT is shown as SEQ ID NO.1, the amino acid sequence of TiXXT is shown as SEQ ID NO.2, and the amino acid sequence of TiXXT is shown as SEQ ID NO. 3. The invention identifies the coding genes TiXXT, tiXXT, tiXXT5 of the xylosyltransferase in the tamarind genome for the first time, confirms that the xylosyltransferase has unique continuous xylose transfer activity through heterologous expression and enzymatic characterization, and lays a foundation for low-cost synthesis of functional high-substituted xylosylated xylooligosaccharide.

Inventors

  • AI LIANZHONG
  • XIONG ZHIQIANG
  • XIA YONGJUN
  • WANG GUANGQIANG
  • ZHANG HUI
  • SONG XIN
  • XIE FAN

Assignees

  • 上海理工大学

Dates

Publication Date
20260512
Application Date
20260115

Claims (10)

  1. 1. A xylosyltransferase, wherein the xylosyltransferase is selected from one of TiXXT1, tiXXT2, or TiXXT; the amino acid sequence of TiXXT is shown as SEQ ID NO. 1; The amino acid sequence of TiXXT is shown as SEQ ID NO. 2; The amino acid sequence of TiXXT is shown as SEQ ID NO. 3.
  2. 2. A gene encoding the xylosyltransferase of claim 1, wherein the nucleotide sequence of TiXXT1 is set forth in SEQ ID No. 4; the nucleotide sequence of TiXXT is shown as SEQ ID NO. 5; The nucleotide sequence of TiXXT is shown as SEQ ID NO. 6.
  3. 3. A recombinant plasmid comprising the gene of claim 2.
  4. 4. A strain comprising the recombinant plasmid of claim 3.
  5. 5. A method for preparing a xylosyltransferase, comprising the steps of: (A1) Inoculating the strain of claim 4 into a culture medium, culturing until the logarithmic phase, and adding an inducer for induction; (A2) After the step (A1) is finished, collecting thalli, crushing, collecting supernatant, and purifying to obtain the xylosyltransferase.
  6. 6. The method for preparing xylosyltransferase according to claim 5, wherein in the step (A1), the inducer is IPTG and the induction time is 16-24 h.
  7. 7. The method for producing a xylosyltransferase according to claim 5, wherein in the step (A2), the cells are collected by centrifugation and then washed and resuspended in PBS washing solution; Collecting clear liquid through centrifugation after crushing; Purifying by nickel column affinity chromatography; after purification, the protein was allowed to concentrate by ultrafiltration centrifugation.
  8. 8. Use of a xylosyltransferase for the preparation of a xylosylated glucan, wherein the xylosyltransferase is the xylosyltransferase of claim 1.
  9. 9. A method for preparing xylosylated glucooligosaccharide, which is characterized by comprising the following steps: And uniformly mixing UDP-xylose, glucooligosaccharide, mnCl 2 , tris, naCl and the xylosyltransferase according to claim 1, and reacting to obtain the xylosylated glucooligosaccharide.
  10. 10. The method for producing xylosylated glucan according to claim 9, wherein the molar ratio of UDP-xylose, glucan, mnCl 2 , tris, naCl and xylosyltransferase is 2 mM:1 mM:2.5 mM:50 mM:150 mM:2~5 μm; in the reaction process, the temperature is 37 ℃ and the time is 1-6 hours.

Description

Xylosyltransferase, coding gene, recombinant plasmid, strain and application thereof in preparation of xylosylated glucose oligosaccharide Technical Field The invention relates to the technical field of genetic engineering, in particular to a xylosyltransferase, a coding gene, a recombinant plasmid, a strain and application thereof in preparation of xylosylated glucose oligosaccharide. Background Xyloglucan is a key hemicellulose component of the primary cell wall of plants, and its structural features are closely related to function. It is usually composed of a backbone of beta-1, 4-glucose chains and linked to xylose side chains via alpha-1, 6-glycosidic linkages at the hydroxyl group 6 of a specific glucose residue, the modification being catalyzed by a specific xylosyltransferase. Such enzymes are responsible for transferring the activated xylose donor (typically UDP-xylose) to the glucan backbone, thus conferring a xyloglucan branching structure, which in turn affects its binding capacity to cellulose microfibrils, the plasticity of the cell wall and the overall mechanical properties (Scheller & Ulvskov, 2010; zabotina, 2012). As one of the main cross-linked polysaccharides of the cell wall matrix, xyloglucan plays an important role in regulating cell expansion, maintaining tissue toughness, responding to external mechanical stimulus and the like. The tamarind seed is rich in xyloglucan with unique structure, and the side chain mode, the branch frequency and the molecular weight distribution of the xyloglucan are obviously different from those of xyloglucan from common plant sources. The special structure ensures that the modified starch has good hydration, viscosity and gel property, thus being used as a natural thickener and a stabilizer in the food industry, being used as a drug slow release carrier in the pharmaceutical field, being used as a texture modifier in cosmetics and the like, and being commercially applied (Chinta et al., 2025; durai et al., 2012). Although its end products have been widely developed and utilized, the enzyme, tamarind xylosyltransferase, which plays a key role in its biosynthetic pathway has not been successfully cloned and systematically characterized until now, which has led to an unclear mechanism of synthesis of the polysaccharide (Narude et al., 2025; sri et al., 2025; zhang et al., 2024). Currently, many studies on the xylitol transferase enzyme are focused on the model plant arabidopsis thaliana. The xylosyltransferase in arabidopsis is mainly encoded by AtXXT gene family, a plurality of members of which have been successfully identified and functionally recombinant expressed in heterologous systems such as escherichia coli, yeast and the like, and the in vitro activity, substrate preference and protein structural characteristics of which have been primarily resolved (Julian & Zabotina, 2022; yamatoya et al 2020). These studies provide an important template for understanding the basis of xyloglucan synthesis. However, homologous enzymes of tamarind, a tropical cash crop, may differ significantly from arabidopsis thaliana in terms of gene sequence, protein structure, substrate recognition mechanism, catalytic kinetic efficiency, and manner of regulation of transcription and translation levels. Such differences may result from factors such as species evolutionary distance, growth environmental adaptation, and cell wall composition specificity (CAVALIER ET al., 2008; zabotina, 2012). Thus, direct application of the enzymatic data of model plants to direct the synthesis or modification of tamarind xyloglucan often makes it difficult to achieve the desired effect, which greatly limits the high-yield, customized production of tamarind xyloglucan with specific functional properties by synthetic biological means. Therefore, the research of tamarind xylosyltransferase, including gene cloning, protein recombinant expression and purification, in-vitro enzyme activity detection, substrate specificity analysis, three-dimensional structure analysis and in-vivo functional verification, is developed systematically, and has important scientific value and application potential. The method can fill the blank of the species on the basic research of cell wall synthesis, deepen understanding of the diversity of non-model plant hemicellulose biosynthesis pathways, and lay a foundation for the subsequent directional modification of the cell wall structure of tamarind or other crops by means of genetic engineering, metabolic engineering and the like. For example, the physical and chemical properties of the cell wall can be optimized by regulating the expression or changing the activity of the enzyme, so that the physical and chemical properties of the cell wall can be improved, the stress resistance can be enhanced, and in an in-vitro synthesis system, the large-scale enzyme preparation of the xyloglucan with uniform structure and controllable performance can be realized by utilizing the h