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CN-122012648-A - Purification method for efficiently producing fucose by using genetically engineered bacteria

CN122012648ACN 122012648 ACN122012648 ACN 122012648ACN-122012648-A

Abstract

The invention provides a purification method for efficiently producing fucose by using genetically engineered bacteria, which has the preservation number of CCTCC (China center for type culture collection) NO: M20252837, and is characterized by comprising the steps of S1, filtering fermentation liquor by using a ceramic membrane to obtain filtrate I, S2, adding a decolorizing agent into the filtrate I, stirring for decolorizing, filtering by using a filter membrane to obtain filtrate II, S3, ultrafiltering the filtrate II, washing the filtrate with pure water to obtain filtrate III, collecting all the filtrate, S4, sequentially carrying out cation resin and anion resin combined chromatography on the filtrate III to obtain a chromatographic liquid, S5, carrying out electrodialysis on the chromatographic liquid to remove salt until the conductivity is less than 100 mu S/cm, obtaining a desalting liquid, S6, concentrating the desalting liquid, adding absolute ethyl alcohol, heating and stirring first, cooling to obtain a suspension, S7, filtering the suspension, leaching the solid by using absolute ethyl alcohol, and carrying out vacuum drying to obtain fucose. The purification method provided by the invention has the advantages of high purity, high yield and short period, and is suitable for industrial production.

Inventors

  • DING FENGLING
  • Deng Junyun
  • ZHU LIHE
  • XIAO CONG
  • CAO XUEFENG
  • YE SIJIA

Assignees

  • 武汉糖智药业有限公司

Dates

Publication Date
20260512
Application Date
20251230

Claims (9)

  1. 1. A purification method for efficiently producing fucose by using genetically engineered bacteria is characterized in that the genetically engineered bacteria has a preservation number of CCTCC NO: M20252837, the purification object is fermentation broth obtained by inoculating CCTCC NO: M20252837 bacteria into a fermentation medium, and the purification steps are as follows: S1, filtering fermentation liquor by using a ceramic membrane to obtain filtrate I; S2, adding a decoloring agent into the filtrate I, stirring for decoloring, and filtering by a filter membrane to obtain filtrate II; S3, ultrafiltering the filtrate II, performing top washing by using pure water, and collecting all filtrate to obtain filtrate III; S4, sequentially carrying out cation resin and anion resin combined chromatography on the filtrate III to obtain a chromatographic liquid; S5, carrying out electrodialysis desalination on the chromatographic liquid to ensure that the conductivity of the chromatographic liquid is less than 100 mu S/cm, and obtaining a desalination liquid; s6, concentrating the desalted liquid until no obvious solvent is left, adding absolute ethyl alcohol, heating and stirring, and then cooling to stir to obtain a suspension; and S7, filtering the suspension, leaching the solid by using absolute ethyl alcohol, collecting the solid, and drying in vacuum to obtain the fucose.
  2. 2. The purification method according to claim 1, wherein the pore diameter of the ceramic membrane in step S1 is 50-200 nm, and the specific filtration method is as follows: the ceramic membrane is firstly circularly cleaned by 1M NaOH solution, then cleaned by pure water until the pH value is neutral, then poured into fermentation liquor for filtration, then top-cleaned by pure water, and finally the filtrate I is obtained by collecting all filtrate.
  3. 3. The purification method according to claim 1, wherein the decolorizing agent in the step S2 is activated carbon, and the mass-volume ratio of the activated carbon to the filtrate I is 1 (50-100); The aperture of the filtering membrane is 0.4-0.5 mu m.
  4. 4. The purification method according to claim 1, wherein the ultrafiltration membrane used in step S3 has a molecular weight cut-off of 8-12 kd and an ultrafiltration pressure of <0.2MPa.
  5. 5. The method according to claim 1, wherein the cationic resin in step S4 is a 001 x 4 type cationic resin and the anionic resin is a D301 type anionic resin.
  6. 6. The method according to claim 5, wherein the combined chromatography is specifically as follows: Activating 001×4 type cation resin with 1M HCl solution, washing with pure water to neutrality, activating D301 type anion resin with 1M NaOH solution, washing with pure water to neutrality, pouring filtrate III into 001×4 type cation resin chromatographic column, standing, eluting with pure water, eluting with 0.5M NaCl solution, collecting all chromatographic liquid, pouring chromatographic liquid into D301 type anion resin chromatographic column, standing, eluting with pure water, eluting with 0.5M NaCl solution, collecting all chromatographic liquid, and performing combined chromatography.
  7. 7. The purification method according to claim 1, wherein electrodialysis desalination in step S5 is specifically as follows: firstly, washing electrodialysis equipment until the conductivity is less than 10 mu s/cm, then adding NaCl solution with the concentration of 3% into a salt solution box, adding pure water into a filtrate box, adding chromatographic liquid into a raw material box, and finally starting electrodialysis; wherein, the volume ratio of NaCl solution, pure water and chromatographic solution is 1:1:1.
  8. 8. The purification method according to claim 1, wherein the volume ratio of the absolute ethanol to the concentrated solution in the step S6 is (3-5) 1; Heating and stirring conditions: the temperature is 40-50 ℃, the rotating speed is 150-250 rpm, the time is 1.5-2.5 hours; And cooling and stirring for 5-7 hours at the temperature of 0-4 ℃ and the rotating speed of 150-250 rpm.
  9. 9. The purification method according to claim 1, wherein the filtration in step S7 is performed using a buchner funnel; The vacuum drying condition is that the temperature is 45-55 ℃ and the time is 7-9 h.

Description

Purification method for efficiently producing fucose by using genetically engineered bacteria Technical Field The invention belongs to the technical field of fermentation product purification, and particularly relates to a purification method for efficiently producing fucose by using genetically engineered bacteria. Background The application of the fucose is wide in the fields of (1) application to a culture medium of a biological medicine antibody manufacturing enterprise, application of the fucose to regulation and control of the content of the N-glycosylated core fucose of the antibody, (2) application to raw materials of a small molecular raw material medicine enterprise, and subsequent synthesis of the fucose serving as a raw material medicine core material, and (3) application to food, cosmetics and auxiliary material additives. However, the defects and deficiencies of the prior art lead to limited L-fucose application (1) the existing traditional extraction and chemical production methods are not renewable in plant source resources, and a large amount of environmentally unfriendly hazardous wastes are generated by acid-base and organic reagent treatment. (2) The traditional extraction and separation process is complex, the product purity is reduced, and the high-quality product has high cost. The existing domestic product is a low-purity and yellowish L-fucose product, and the application scene and the added value of the product are far different from those of the high-purity L-fucose. Based on this, in addition to the need to develop other routes to obtain fucose in high yields, purification processes for the product are also extremely urgent. Disclosure of Invention In view of the above, the invention uses the preservation number of CCTCC NO: M20252837, the preservation time of 2025, 12 months and 10 # which are constructed in advance, the preservation unit of China Center for Type Culture Collection (CCTCC), the classification name of which is ESCHERICHIA COLIFUC-WT22, and provides a high-efficiency purification technology on the basis of obtaining Gao Yanzao sugar fermentation quantity so as to obtain high-purity and high-yield fucose. In order to achieve the above purpose, the invention adopts the following technical scheme: A purification method for efficiently producing fucose by using genetically engineered bacteria, wherein the preservation number of the genetically engineered bacteria is CCTCC NO: M20252837, the purification object is fermentation liquor obtained by inoculating CCTCC NO: M20252837 bacteria into a fermentation medium, and the fermentation medium comprises 5g/L of yeast extract powder, 4g/L of diammonium hydrogen phosphate, 13.5g/L of anhydrous potassium dihydrogen phosphate, 1.4g/L of magnesium sulfate heptahydrate, 1ml/L of microelement solution, 2g/L of calcium chloride, 12.2mg/L of VB, 1g/L of citric acid, 5g/L of glucose, 10g/L of glycerin, 5g/L of ammonium sulfate and PPE defoamer; The microelement solution comprises 5g/L NaCl, 1g/L MnCl 2·4H2O 4g/L,ZnSO4·7H2 O, 0.5g/L sodium molybdate dihydrate, 4.75g/L FeCl 3·6H2 O, 0.575g/L boric acid, 0.4g/L CuSO 4·5H2 O and 2.038ml/L concentrated sulfuric acid; The purification steps are as follows: S1, filtering fermentation liquor by using a ceramic membrane to obtain filtrate I; S2, adding a decoloring agent into the filtrate I, stirring for decoloring, and filtering by a filter membrane to obtain filtrate II; S3, ultrafiltering the filtrate II, performing top washing by using pure water, and collecting all filtrate to obtain filtrate III; S4, sequentially carrying out cation resin and anion resin combined chromatography on the filtrate III to obtain a chromatographic liquid; S5, carrying out electrodialysis desalination on the chromatographic liquid to ensure that the conductivity of the chromatographic liquid is less than 100 mu S/cm, and obtaining a desalination liquid; s6, concentrating the desalted liquid until no obvious solvent is left, adding absolute ethyl alcohol, heating and stirring, and then cooling to stir to obtain a suspension; and S7, filtering the suspension, leaching the solid by using absolute ethyl alcohol, collecting the solid, and drying in vacuum to obtain the fucose. Further, in the step S1, the pore diameter of the ceramic membrane is 50-200 nm, and the specific filtering method is as follows: the ceramic membrane is firstly circularly cleaned by 1M NaOH solution, then cleaned by pure water until the pH value is neutral, then poured into fermentation liquor for filtration, then top-cleaned by pure water, and finally the filtrate I is obtained by collecting all filtrate. Further, in the step S2, the decoloring agent is activated carbon, and the mass-volume ratio of the activated carbon to the filtrate I is 1 (50-100); The aperture of the filtering membrane is 0.4-0.5 mu m. Further, the ultrafiltration membrane used in the step S3 has a molecular weight cutoff of 8-12 KD and an ultrafiltration pressure o