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CN-121975900-A - Application of beta-glucosidase BglD or mutant thereof in synthesis of ginsenoside Rg3 and synthesis method of ginsenoside Rg3

CN121975900ACN 121975900 ACN121975900 ACN 121975900ACN-121975900-A

Abstract

The invention provides application of beta-glucosidase BglD or a mutant thereof in synthesizing ginsenoside Rg3, wherein the beta-glucosidase BglD is derived from Pseudothermotoga sp, and the amino acid sequence is shown as SEQ ID NO: 2. The invention also provides a beta-glucosidase BglD mutant, which takes beta-glucosidase BglD as a parent, wherein the 214 th asparagine (N) is mutated into serine (S), the 216 th leucine (L) is mutated into phenylalanine (F) or/and the 341 th valine (V) is mutated into alanine (A). The synthesis method can carry out whole-cell feeding under the catalysis of single enzyme, does not need cell disruption or extra hydrogen donor, has the conversion rate of over 90 percent for high-concentration (40 g/L) substrates, can efficiently prepare target products by a one-pot method, has simple process and better cost benefit and industrial application prospect.

Inventors

  • LUO ZUYUAN
  • WEI WANTAO
  • HE ZE

Assignees

  • 成都盈源博泰生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260202
Priority Date
20251225

Claims (9)

  1. 1. The application of the beta-glucosidase BglD or a mutant thereof in the synthesis of ginsenoside Rg3, wherein the beta-glucosidase BglD is derived from Pseudothermotoga sp, and the amino acid sequence is shown as SEQ ID NO. 2.
  2. 2. The method of claim 1, wherein the synthetic substrate is ginsenoside Rb1 or/and Rd.
  3. 3. The method according to claim 1 or 2, wherein the ginsenoside Rg3 is 20 (S) -Rg3.
  4. 4. A beta-glucosidase BglD mutant is characterized in that beta-glucosidase BglD is taken as a parent, the 214 th asparagine (N) is mutated into serine (S), the 216 th leucine (L) is mutated into phenylalanine (F) or/and the 341 th valine (V) is mutated into alanine (A).
  5. 5. The mutant of β -glucosidase BglD as claimed in claim 4, wherein the mutant is characterized in that leucine (L) at position 216 is mutated to phenylalanine (F) and valine (V) at position 341 is mutated to alanine (A).
  6. 6. A polynucleotide encoding the mutant β -glucosidase according to claim 4 or 5.
  7. 7. An expression vector, characterized in that: the expression vector comprising the polynucleotide of claim 6.
  8. 8. A recombinant cell comprising the polynucleotide of claim 7 or the expression vector of claim 8.
  9. 9. A method for synthesizing ginsenoside Rg3 is characterized in that ginsenoside Rb1 or/and Rd is used as a substrate, and the ginsenoside Rg3 is prepared through catalytic reaction of beta-glucosidase BglD or a mutant thereof, wherein the beta-glucosidase BglD is derived from Pseudothermotoga sp, and the amino acid sequence is shown as SEQ ID NO: 2.

Description

Application of beta-glucosidase BglD or mutant thereof in synthesis of ginsenoside Rg3 and synthesis method of ginsenoside Rg3 Technical Field The invention relates to an application of beta-glucosidase BglD or a mutant thereof in synthesizing ginsenoside Rg3, and a method for synthesizing ginsenoside Rg 3. Background Ginsenoside Rg3 (English name: 20 (R) -GinsenosideRg 3), CAS number 38243-03-7, molecular formula C 42H72O13. White to pale yellow powder or crystal in appearance, and is extracted from dried root of Panax ginseng C.A. Meyer of Araliaceae. Common specifications comprise 20mg to 1g, the purity is not lower than 98%, and the product is mainly used for drug content measurement and has the activities of resisting tumor, inhibiting cancer cell metastasis and the like. The substance belongs to tetracyclic triterpene panaxadiol saponin monomer, and researches show that the substance has the effects of inhibiting vascular activity, resisting fatigue and enhancing immunity. The current recombinant lactobacillus bioconversion method is a method for realizing Rg3 directional synthesis, such as literature Su Min, and the like, the separation and identification of beta-glucosidase-producing saccharomycetes and the application thereof in ginsenoside Rg3 conversion, food science, 2018, vol.39, no.14, screening of beta-glucosidase-producing microorganisms and the application thereof in ginsenoside Rg3 conversion. Fermentation by kluyveromyces marxianus to produce beta-glucosidase to catalyze Rb1 and Rd to produce Rg3, fermentation for 3d is required and conversion rate is 248%, but conversion rate in this method= (unfermented sample ash mass-post-fermentation sample ash mass)/unfermented sample ash mass x 100. Application number 202510634060.7, the name of the invention is beta-glucosidase and a preparation method thereof, and application thereof in the production of ginsenoside Rg3, relates to the technical field of ginsenoside, and specifically discloses beta-glucosidase and a preparation method thereof, and application thereof in the production of ginsenoside Rg3, aiming at the problem of low conversion rate of ginsenoside Rg3 prepared by the existing microbial conversion method, wherein the conversion rate is 98% after reaction for 6 hours at 85 ℃, but the calculation mode of the conversion rate is not mentioned. CN 201610219176.5A method for preparing rare ginsenoside 20 (S) -Rg3 by using an enzymatic method, and degrading ginsenoside Rb1, rb2 and Rc to produce 20 (S) -Rg3 by using beta-glucosidase from Thermotogapetrophila and arabinofuranosidase from ThermotogathermarumDSM 5069. However, the enzyme preparation in the method is a compound enzyme preparation, and beta-glucosidase and arabinofuranosidase are added simultaneously for catalytic conversion. The invention discloses a beta-glucosidase and application thereof in preparing ginsenoside, and the application number is CN202311376189.X, the invention name is beta-glucosidase and application thereof in preparing ginsenoside. The beta-glucosidase or the recombinant microorganism expressing the beta-glucosidase can generate rare ginsenoside Rg3 and/or Rh1 through whole-cell catalysis, and further provides application of the beta-glucosidase in preparation of ginsenoside, wherein the amino acid sequence of the beta-glucosidase is RLF05189.1 on NCBI, but the method is not beneficial to subsequent separation and purification while the cost is increased due to the fact that catalytic conversion is carried out under the condition of adding a hydrogen donor. In summary, the currently reported bioconversion method generally has the problems of complicated operation and treatment steps, higher production cost, low conversion utilization rate of reaction substrates, namely raw materials and the like, so that the industrialized feasibility of the amplified production is limited. Disclosure of Invention The invention provides an application of beta-glucosidase BglD or a mutant thereof in synthesizing ginsenoside Rg3, and a method for synthesizing ginsenoside Rg 3. The invention provides application of beta-glucosidase BglD or a mutant thereof in synthesizing ginsenoside Rg3, wherein the beta-glucosidase BglD is derived from Pseudothermotoga sp, and the amino acid sequence is shown as SEQ ID NO: 2. Wherein the synthetic substrate is ginsenoside Rb1 or/and Rd. Wherein, the ginsenoside Rg3 is 20 (S) -Rg3. The invention also provides a beta-glucosidase BglD mutant, which takes beta-glucosidase BglD as a parent, wherein the 214 th asparagine (N) is mutated into serine (S), the 216 th leucine (L) is mutated into phenylalanine (F) or/and the 341 th valine (V) is mutated into alanine (A). Wherein, the mutant is formed by mutating the 216 th leucine (L) into phenylalanine (F) and mutating the 341 th valine (V) into alanine (A). The present invention provides a polynucleotide encoding a beta-glucosidase mutant as described. The invention also provides an expression vector