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CN-117720593-B - Sphingosine gum oligosaccharide and preparation method and application thereof

CN117720593BCN 117720593 BCN117720593 BCN 117720593BCN-117720593-B

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a sphingosine gum oligosaccharide and a preparation method and application thereof. The invention provides a sphingosine gum oligosaccharide, the main chain structure of which is beta-L-Rhap- (1-3) -beta-D-Glcp- (1-4) -beta-D-GlcpA, the sphingosine gum (welan gum, gellan gum and Di gum) is degraded by sphingosine degrading enzyme SpnR, and then the sphingosine gum oligosaccharide is purified by liquid chromatography. The preparation method of the sphingosine gum oligosaccharide has mild reaction conditions and simple and convenient separation. The sphingosine gum oligosaccharide prepared by the method has good plant growth promoting capability and intestinal tract probiotics, can be used in the fields of agriculture, food, health care products and the like, and expands the application of the sphingosine gum and the sphingosine gum oligosaccharide.

Inventors

  • LI SHA
  • LI KECHENG
  • GAO PENG
  • HUANG JINSONG
  • XU HONG
  • QIU YIBIN
  • HUANG WEIWEI

Assignees

  • 南京工业大学

Dates

Publication Date
20260512
Application Date
20231211

Claims (7)

  1. 1. The sphingosine gum oligosaccharide is characterized in that the main chain structure of the sphingosine gum oligosaccharide is beta-L-Rhap- (1- & gt 3) -beta-D-Glcp- (1- & gt 4) -beta-D-GlcpA; wherein the sphingosine gum oligosaccharide is welan gum enzymolysis oligosaccharide, gellan gum enzymolysis oligosaccharide or diutan gum enzymolysis oligosaccharide; The welan gum enzymatic hydrolysis oligosaccharide, or gellan gum enzymatic hydrolysis oligosaccharide, or diutan gum enzymatic hydrolysis oligosaccharide is obtained by degrading welan gum, or gellan gum, or diutan gum by using sphingosine degrading enzyme SpnR, wherein sphingosine degrading enzyme SpnR is derived from Sphingomonas sp.HT-1, the preservation number is CCTCC NO: M2012062, and the nucleotide sequence is shown as SEQ ID NO: 1.
  2. 2. The sphingan oligosaccharide according to claim 1, wherein the molecular weight of the sphingan oligosaccharide is 560-725 g/mol and the degree of polymerization is 3-4.
  3. 3. The method for preparing the sphingosine gum oligosaccharide according to any one of claims 1 or 2, wherein the sphingosine gum is prepared by enzymolysis of a sphingosine degrading enzyme SpnR; The sphingosine gel is welan gel, gellan gum or Di gum, the sphingosine degrading enzyme SpnR is derived from Sphingomonas sp.HT-1, the preservation number is CCTCC NO: M2012062, and the nucleotide sequence is shown as SEQ ID NO: 1.
  4. 4. The method according to claim 3, wherein the sphingosine degrading enzyme SpnR is obtained by the steps of: (I) Amplifying the SpnR gene of sphingosine degrading enzyme SpnR, and introducing the amplified gene into a pET-28a plasmid to obtain a recombinant plasmid pET-28a-SpnR; (II) transforming the recombinant plasmid obtained in the step (I) into competent cells of escherichia coli BL21 (DE 3), and screening positive transformants on LB solid medium plates containing kanamycin to obtain recombinant strains; (III) centrifugally collecting thalli after induced expression of the recombinant strain obtained in the step (II), washing with normal saline, re-suspending in PBS buffer solution with pH of 7-7.5, finally ultrasonically crushing thalli, and centrifugally taking supernatant to obtain crude enzyme liquid of sphingosine degrading enzyme SpnR.
  5. 5. The preparation method of claim 3, wherein the addition amount of the sphingosine degrading enzyme SpnR is 5-50U/g of sphingosine gel, and the enzymolysis reaction condition is that the enzymolysis is carried out for 1-5 h at 25-40 ℃ and the pH is 6.0-8.0.
  6. 6. A method of preparation according to claim 3, comprising the steps of: (1) After fermentation and purification of Sphingomonas sp, sphingosine gum is obtained; (2) Dissolving the sphingosine gel obtained in the step (1) in water, and uniformly stirring to prepare a sphingosine gel solution; (3) Adding sphingosine degrading enzyme SpnR into the sphingosine gum solution prepared in the step (2) for enzymolysis reaction to obtain a sphingosine gum enzymolysis liquid; (4) Inactivating the sphingosine gum enzymatic hydrolysate obtained in the step (3), centrifuging to obtain a sphingosine gum enzymatic hydrolysate supernatant, filtering, and purifying by using a liquid chromatography to obtain the sphingosine gum oligosaccharide.
  7. 7. Use of a sphingosine gum oligosaccharide according to any of claims 1 or 2 in tomato growth promotion, wherein the sphingosine gum oligosaccharide is a welan gum enzymatic oligosaccharide.

Description

Sphingosine gum oligosaccharide and preparation method and application thereof Technical Field The invention belongs to the field of bioengineering, and particularly relates to a sphingosine gum oligosaccharide and a preparation method and application thereof. Background With the continuous intensive research on natural polysaccharides, the preparation of degradation products of natural polysaccharides and the research on biological activity have also attracted more and more attention. In recent years, researchers find that some oligosaccharides obtained by degrading natural polysaccharide have better biological activity and application prospect, including xanthan gum oligosaccharides with oxidation resistance and bacteriostasis, alginate oligosaccharides with antibacterial, anti-tumor and immunoregulatory functions, carrageenan oligosaccharides with anti-inflammatory and blood coagulation functions, and the like. Sphingosine gum refers to a viscous high molecular polymer secreted by Sphingomonas extracellular in the growth metabolism process, and comprises gellan gum, welan gum, diutan gum, rhamnoides gum and the like. They have the same tetrasaccharide repeating unit backbone structure of D-glucose, D-glucuronic acid, D-glucose and L-rhamnose, except for a slight difference in the number and position of side chain groups. These same and different structural features combine to make this class of microbial polysaccharides a structurally and functionally rich resource. The polysaccharide has good rheological property, natural and nonhazardous property, short production period and stable product quality, and can be used as a cement concrete additive, an oil displacement preparation, a food additive, a chemical raw material and the like in various fields of industrial and agricultural production. The sphingosine gum can be degraded by a chemical method and an enzymatic method to obtain the sphingosine gum oligosaccharide. The enzymatic hydrolysis method can selectively break the glycosidic bond, can realize controllable and directional degradation, has the advantages of mild reaction conditions, high reaction speed and less pollutant generation, and can realize the rapid, environment-friendly and specific preparation of the sphingosine gum oligosaccharide. The obtained sphingosine gum oligosaccharide, which is a relatively novel microbial source hetero oligosaccharide, has application potential in the fields of agriculture, food, health care products, medical sanitation and the like, and greatly expands the downstream application market of the sphingosine gum and derivatives thereof. Disclosure of Invention The invention aims to solve the technical problem of providing the sphingosine gum oligosaccharide aiming at the defects of the prior art. The invention also solves the technical problem of providing a preparation method of the sphingosine gum oligosaccharide. The technical problem to be solved finally is to provide the application of the sphingosine gum oligosaccharide. In order to solve the technical problems, the invention adopts the following technical scheme: The sphingosine gum oligosaccharide has a main chain structure of beta-L-Rhap- (1- & gt 3) -beta-D-Glcp- (1- & gt 4) -beta-D-GlcpA. Wherein the molecular weight of the sphingosine gum oligosaccharide is 560-725 g/mol, and the polymerization degree range is 3-4. The preparation method of the sphingosine gum oligosaccharide is also within the scope of the protection of the invention, and the sphingosine gum oligosaccharide is prepared by carrying out enzymolysis on sphingosine gum by using a sphingosine degrading enzyme SpnR. Wherein the sphingosine gum comprises any one or a combination of a plurality of welan gum, gellan gum and diutan gum. Specifically, the sphingosine gum is a heteropolysaccharide having a repeating unit of Rha-Glc-GlcA-Glc tetraose. Wherein the sphingosine degrading enzyme SpnR is derived from Sphingomonas sp.HT-1, the preservation number is CCTCCNO: M2012062, and the nucleotide sequence is shown as SEQ ID NO: 1. Specifically, the Sphingomonas sp.HT-1, the preservation number of which is CCTCC NO: M2012062, and the detailed information of the strain are disclosed in patent CN 102618468A. (Note: alcaligenes sp.HT-1 was now classified into Sphingomonas according to Liu Hui, wei Lulu, zhu Longfa et al, 2023, provisions on the study of Sphingomonas by microbiology Notification; so the Latin brand name was modified from Alcaligenes sp. To Sphingomonas sp.) Wherein, the sphingosine degrading enzyme SpnR is obtained according to the following steps: (I) Amplifying the SpnR gene of sphingosine degrading enzyme SpnR, and introducing the amplified gene into a pET-28a plasmid to obtain a recombinant plasmid pET-28a-SpnR; (II) transforming the recombinant plasmid obtained in the step (I) into competent cells of escherichia coli BL21 (DE 3), and screening positive transformants on LB solid medium plates containing kanamycin to obtain recom