Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/30—Artificial sweetening agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
  • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01017—Glucuronosyltransferase (2.4.1.17)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/84—Pichia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the invention relates to a preparation method of rebaudioside J, in particular to a biological preparation method of rebaudioside J.
• Sweeteners are a class of food additives that are widely used in the production of foods such as beverages and confectionery. They can be added during the production of foods or appropriately diluted as a substitute for sucrose during home baking. Sweeteners include natural sweeteners and artificial sweeteners, the former including sucrose, high fructose corn syrup, honey, and the like, the latter including aspartame, saccharin, and the like.
• Stevia is a natural sweetener extracted from plant stevia and is currently widely used in foods and beverages. The extract of Stevia has a variety of steviosides including rebaudioside, and the different batch components of naturally extracted stevioside vary greatly, requiring subsequent purification.
• the ratio of stevioside in stevia leaves is less than 0.5%. It is extremely difficult to obtain high-purity Rebaudio J by traditional extraction methods, which limits the in-depth study of Rebaudi J and hinders Its commercial application.
• the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for preparing rebaudioside J by enzymatic method, which can produce high purity rebaudioside at a low cost and in a short cycle. J product.
• the present invention adopts the following technical solutions:
• the glycosyl donor is a rhamnose donor.
• the rhamnose donor is UDP-rhamnose.
• the UDP-glycosyltransferase is UGT-B from rice (Oryza sativa).
• amino acid sequence of UGT-B from rice has at least 60% identity to sequence 2 shown in the sequence listing.
• amino acid sequence of UGT-B from rice has at least 70% identity to sequence 2 shown in the sequence listing.
• amino acid sequence of UGT-B from rice has at least 80% identity to sequence 2 shown in the sequence listing.
• amino acid sequence of UGT-B from rice has at least 90% identity with the sequence 2 shown in the sequence listing.
• amino acid sequence of the UGT-B from rice is completely identical to the sequence 2 shown in the sequence listing.
• the reaction can be carried out in an aqueous phase system at a temperature of 4 to 50 ° C and a pH of 5.0 to 9.0.
• the reaction is carried out in an aqueous phase system at a temperature of 35-45 ° C and a pH of 7.5-8.5. More preferably, the reaction is carried out at a temperature of 40 ° C and the reaction is carried out at pH 8.0.
• reaction is carried out in a phosphate buffer solution.
• the reaction system contains a recombinant cell of UDP-glycosyltransferase and a cell permeabilizing agent, and the reaction is carried out in the presence of a cell permeabilizing agent.
• the cell penetrating agent is toluene, and the volume ratio of toluene in the reaction system is 1-3%. Further, the volume ratio of toluene was 2%.
• Rebaudio J product that meets the requirements for use can be obtained by purification treatment.
• a specific purification method is post-treatment including resin separation, according to which a rebaudioside J product having a purity of up to 95% can be obtained.
• the recombinant cell is a microbial cell.
• the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.
• the present invention has the following advantages compared with the prior art:
• the method for preparing rebaudioside J by the enzymatic method provided by the invention has important application value. Since the substrate produced by the enzymatic method, Rebaudio A, can be obtained in large quantities, the production of Rebaudio J is not limited by the raw materials, which greatly reduces the production cost. In addition, the stevioside content in plants is low, and there are many different structures of stevioside, it is difficult to extract a relatively pure product, and the technique of the present invention is compared with the technique of extracting Rebaudioside J from stevia leaves. The enzymatic synthesis method can provide higher purity products, and will certainly promote the research and application of the new stevioside rebaudioside J.
• the synthetic route of Rebaudioside J mainly provided by the present invention is as follows:
• the UGT-B used in the present invention may be present in the form of an enzyme lyophilized powder or present in recombinant cells.
• UGT-B is obtained as follows:
• UGT-B recombinant Escherichia coli (or other microbial) expression strain is obtained by molecular cloning technology and genetic engineering technology, and then recombinant Escherichia coli is fermented to prepare recombinant cells containing UGT-B, or The above recombinant cells were prepared to obtain a lyophilized powder of UGT-B.
• the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and the expression of the target protein was induced by IPTG to obtain a recombinant Escherichia coli expression strain of UGT-B.
• the recombinant Escherichia coli expression strain containing UGT-B was inoculated into 4 ml of liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 ° C, and the overnight culture was transferred to 50 ml at 1% inoculum.
• the liquid LB medium was shake cultured (200 rpm) at 37 ° C until the OD600 value reached 0.6-0.8, and the final concentration of 0.4 mM IPTG was added to shake culture at 20 ° C overnight.
• the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended in 5 ml of 2 mmol/L phosphate buffer (pH 7.0) to obtain the recombinant cells, and the cells were further ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged ( 8,000 rpm, 10 min), the supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder.
• phosphate buffer pH 7.0
• sequence 3 and sequence 4 the gene was synthesized into UGT-B gene fragment, and NdeI and BamHI restriction sites were added to both ends, and ligated into pUC57 vector (produced by Suzhou Jinweizhi Biotechnology Co., Ltd.).
• the UGT gene fragment was digested with restriction endonucleases NdeI and BamHI, and the purified fragment was recovered.
• the fragment was ligated into the pET30a corresponding restriction site by T4 ligase, and transformed into BL21 (DE3) strain.
• the UGT strain was inoculated into 4 ml of liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 ° C, and the overnight culture was transferred to 50 ml of liquid LB medium at a 1% inoculum and shake cultured at 37 ° C (200 rpm).
• the OD 600 value was 0.6-0.8, and the final concentration of 0.4 mM IPTG was added to shake culture at 20 ° C overnight.
• the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended in 5 ml of 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells containing UGT-B for catalysis.
• the recombinant cells of UGT-B prepared in Example 3 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of UGT-B.
• the UGT-B lyophilized powder prepared according to the method of Example 2 was used to catalyze the synthesis of Rebaudioside J.
• Example 4 Synthesis of Rebaudioside J under the catalysis of UDP-glycosyltransferase recombinant cells using Rebaudioside A as substrate

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• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
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• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
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• Proteomics, Peptides & Aminoacids (AREA)
• Nutrition Science (AREA)
• Food Science & Technology (AREA)
• Polymers & Plastics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 2016
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