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WO2019161634A1 - Procédé de préparation de rébaudioside a, enzyme pour la préparation de rébaudioside a, et application - Google Patents

Procédé de préparation de rébaudioside a, enzyme pour la préparation de rébaudioside a, et application Download PDF

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Publication number
WO2019161634A1
WO2019161634A1 PCT/CN2018/096211 CN2018096211W WO2019161634A1 WO 2019161634 A1 WO2019161634 A1 WO 2019161634A1 CN 2018096211 W CN2018096211 W CN 2018096211W WO 2019161634 A1 WO2019161634 A1 WO 2019161634A1
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Prior art keywords
glucosyltransferase
rebaudioside
stevioside
reaction
raw material
Prior art date
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Ceased
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PCT/CN2018/096211
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English (en)
Chinese (zh)
Inventor
傅荣昭
李振伟
刘文山
刘玉凤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BONTAC BIO-ENGINEERING (SHENZHEN) Co Ltd
Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd
Original Assignee
BONTAC BIO-ENGINEERING (SHENZHEN) Co Ltd
Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd
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Priority to CN201880001964.4A priority Critical patent/CN109196110A/zh
Priority to PCT/CN2018/096211 priority patent/WO2019161634A1/fr
Publication of WO2019161634A1 publication Critical patent/WO2019161634A1/fr
Anticipated expiration legal-status Critical
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    • 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

Definitions

  • the invention relates to the field of biomedical technology, in particular to a preparation method of rebaudioside A, an enzyme for preparing lysine A and application thereof.
  • Steviol glycoside also known as stevioside, is a low-calorie, high-intensity sweetener with a sweetness 150-300 times that of sucrose and about 1/300 of that of sucrose. Studies have shown that it has no toxic side effects, is safe to eat, and studies have shown that stevioside can be used to prevent high blood pressure, diabetes, obesity, heart disease and dental caries, and is an ideal sweetener for replacing sucrose.
  • Stevioside Stevioside
  • RA Lai Di Di A
  • the present invention provides a preparation method of Lai Di Di A, an enzyme for preparing Lai Di Di A and an application thereof; the preparation method has the advantages of simple process, high yield, low cost and green safety.
  • the present invention provides a method of preparing lysine A, comprising:
  • the reaction solution is prepared by using the stevioside raw material as a substrate, and the pH of the reaction solution is adjusted to 6.0-8.0 at a constant temperature of 20-45. After the stirring reaction is carried out at ° C, the rebaudioside A is collected; the stevioside raw material includes stevioside, and the amino acid sequence of the glucosyltransferase includes the amino acid sequence shown in SEQ ID NO: 1.
  • the glucosyltransferase of the present invention is derived from Gardenia jasminoides.
  • the uridine diphosphate glucose (UDPG) may also be referred to as uridine-5'-diphosphate glucose or uridine diphosphate glucose.
  • the stevioside (St), or stevioside has a molecular formula of C 38 H 60 O 18 and has a chemical structure as shown in Formula I.
  • the Lydia A (RA), or stevioside A, A-side stevioside has the formula C 44 H 70 O 23 , and the chemical structure is as shown in Formula II:
  • the stevioside raw material comprises stevioside (St), and the stevioside and the uridine diphosphate glucose are catalyzed by a glucosyltransferase to obtain rebaudioside A (RA), and the diphosphate Uridine glucose forms Uridine triphosphate (UDP) in the reaction.
  • the uridine diphosphate may also be referred to as uridine-5'-diphosphate.
  • the uridine diphosphate glucose in the reaction liquid is obtained by directly adding in the reaction liquid or by adding a mixed raw material to the reaction liquid, the mixed raw material including uridine diphosphate (UDP) And one or both of the uridine diphosphate glucose, Sucrose and Sucrose Synthase (SuS).
  • the mixed raw material includes uridine diphosphate, uridine diphosphate glucose, sucrose, and sucrose synthase, or the mixed raw material includes uridine diphosphate glucose, sucrose, and sucrose synthase, or the mixed raw material includes uridine Diphosphate, sucrose and sucrose synthase.
  • the sucrose synthase (SuS) also known as sucrose synthase, is a glycosyltransferase widely present in plants.
  • the process route of the preparation method of the rebaudioside A may also be as shown in the formula (2):
  • the uridine diphosphate, the sucrose and the sucrose synthase are reacted in the reaction solution to obtain the uridine diphosphate glucose and form the uridine diphosphate glucose regeneration system;
  • the stevioside is prepared by the catalysis of the glucosyltransferase, and the glucosyltransferase is capable of reintroducing a glucosyl group on the sugar group of the carbon atom at the 13 position of the stevioside
  • the sucrose produces fructose catalyzed by the sucrose synthase.
  • the gene coding sequence of the glucosyltransferase comprises the nucleotide sequence set forth in SEQ ID NO: 2.
  • the gene coding sequence of the amino acid sequence of the glucosyltransferase should consider a degenerate base, ie, the coding gene of the amino acid sequence shown in SEQ ID NO: 1 includes the nucleus as shown in SEQ ID NO: The nucleotide sequence, the protective range should also protect the nucleotide sequence having the base degenerate property of SEQ ID NO: 2, and the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO: 1.
  • the mass ratio of the uridine diphosphate, the sucrose and the sucrose synthase is (0.0001-0.04): (0.1 -2): (0.1-1). Further, optionally, the mass ratio of the uridine diphosphate, the sucrose and the sucrose synthase is (0.005-0.01): 1: (0.2-1).
  • the mass ratio of the uridine diphosphate glucose to the stevioside is (0.001-1) :1. Further, optionally, the mass ratio of the uridine diphosphate glucose to the stevioside is (0.01-0.3):1.
  • the mixed raw material includes the uridine diphosphate glucose, sucrose, and sucrose synthase
  • the uridine diphosphate glucose is catalyzed by the glucosyltransferase to obtain urine in the reaction solution.
  • the glycoside diphosphate, the sucrose, the sucrose synthase, and the uridine diphosphate can be reacted to form uridine diphosphate glucose, and a cyclic regeneration system of the uridine diphosphate glucose is formed in the reaction solution.
  • the sucrose synthase is derived from one or more of Arabidopsis thaliana, soybeans, and potatoes. Further, optionally, the sucrose synthase is derived from Arabidopsis thaliana.
  • the stevioside raw material has a mass fraction of 10%-30% in the reaction solution. Further, optionally, the mass fraction of the stevioside raw material in the reaction liquid is 20% to 30%. For example, the stevioside raw material has a mass fraction of 10%, or 18%, or 25%, or 30% in the reaction liquid.
  • the mass ratio of the stevioside raw material to the glucosyltransferase is 1: (0.1-1);
  • the added form of the glucosyltransferase comprises a crude enzyme solution or an enzyme powder.
  • the crude enzyme solution of the glucosyltransferase refers to a microorganism disruption buffer containing a glucosyltransferase, and the microorganism can synthesize the glucosyltransferase.
  • the enzyme powder of the glucosyltransferase refers to a freeze-dried enzyme powder obtained by purifying the glucosyltransferase.
  • the reaction temperature of the reaction liquid is maintained at 20 to 45 °C. Further, optionally, the reaction temperature of the reaction liquid is maintained at 35 to 40 °C. For example, the reaction temperature of the reaction liquid is maintained at 20 ° C, or 30 ° C, or 35 ° C, or 38 ° C, or 40 ° C.
  • the glucosyltransferase has a high catalytic activity at the temperature range, and the conversion rate of the stevioside is high.
  • the pH of the reaction solution is adjusted to be 7.0 to 8.0.
  • the pH of the reaction solution is adjusted to 7.2, or 7.4, or 8.0.
  • the reaction time of the stirring reaction is 4-25 hours.
  • the stirring reaction is stirred at a rate of 200-300 rpm.
  • the reaction time of the stirring reaction is 10-20 hours.
  • the reaction time of the stirring reaction is 4 hours, or 10 hours, or 15 hours, or 20 hours.
  • the process of collecting the rebaudioside A comprises: heating the reaction solution to denature the glucosyltransferase, filtering and collecting the filtrate, and purifying the filtrate to obtain Lai Didi A, wherein the heating temperature is 85-100 ° C and the time is 0.3-1 hour.
  • the rebaudioside A may have partial loss during the purification treatment, and the sweetness, heat and physical and chemical properties of the rebaudioside A obtained after the purification treatment are in accordance with actual theoretical parameters.
  • the mass ratio of the stevioside to the sucrose is 1: (0.2-1).
  • the reaction solution further includes a buffer
  • the buffer includes one or more of a phosphate buffer solution, a borate buffer solution, and a Tris-HCl buffer solution.
  • the concentration of the buffer is 10-500 mmol/L.
  • the concentration of the buffer is 100-500 mmol/L.
  • the concentration of the buffer is 100 mmol/L, or 200 mmol/L, or 500 mmol/L.
  • the buffer also includes other types of buffers.
  • the preparation method of the rebaudioside A provided by the first aspect of the invention has the advantages of simple process, low cost and green environmental protection; and the rebaudioside A has an extremely high yield.
  • the conversion rate of the preparation method of the present invention is as high as 90% or even 99%.
  • the preparation method of Lai Di Di A can only use a substrate concentration of a few thousandths, and the conversion rate is low; even if the raw material is added by a method such as adding raw materials, the conversion rate is still limited, and therefore, the prior art as a whole
  • the high cost is not conducive to industrial production.
  • the preparation method of the invention does not use a large amount of organic reagents, and the concentration of the substrate of the invention can be as high as 10-30%, and can be used to recycle the diphosphate urine with high raw material cost through the uridine diphosphate glucose regeneration system.
  • Glycoside Glucose (UDPG) Glycoside Glucose (UDPG); Therefore, the preparation method of the invention has low cost, is environmentally friendly, has few purification steps, and has great application prospects.
  • the present invention provides an enzyme for preparing rebaudioside A, wherein the enzyme for preparation is a glucosyltransferase, and the gene coding sequence of the glucosyltransferase comprises SEQ ID NO: 2 The nucleotide sequence shown.
  • the amino acid sequence of the glucosyltransferase comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • the glucosyltransferase is produced by microbial expression, including one or more of Escherichia coli, Pichia pastoris, and Bacillus subtilis. Further, optionally, the glucosyltransferase is produced by expression in E. coli. The glucosyltransferase is heterologously expressed in the system in which the E. coli expression is expressed.
  • the present invention preferably has an E. coli expression system which is simple and feasible, has a short culture period, low fermentation cost, and high enzyme yield.
  • the glucosyltransferase of the present invention may be added to the reaction solution in the form of an enzyme powder or a crude enzyme solution.
  • the glucosyltransferase of the present invention is derived from gardenia, and the gene coding sequence of the glucosyltransferase is screened by a large number of experiments and purified by optimizing the microbial expression system.
  • the enzymatic properties of glucosyltransferases from different species are different, including the specific activity of the enzyme, the substrate range of the enzyme, the optimum pH, the optimum temperature, the action time and the stability of the enzyme.
  • the present invention finds that another glucosyltransferase derived from gardenia does not have the catalytic conversion of stevioside to Laiwudi. ⁇ A's function.
  • the glucosyltransferase of the present invention has extremely strong specificity, and the rebaudioside A can be efficiently catalyzed by the stevioside.
  • the stevioside raw material may further be a mixture comprising stevioside as a main component, and the glucosyltransferase of the present invention can efficiently and specifically catalyze the conversion of the stevioside to rebaudioside A.
  • the glucosyltransferase is expressed in a microorganism by constructing a recombinant plasmid whose vector plasmid is a pET28a(+) vector plasmid. Inserting the gene coding sequence of the glucosyltransferase into the pET28a(+) vector plasmid to obtain a recombinant plasmid capable of efficiently and efficiently producing heterologous expression in a microbial cell to obtain the glucosyltransferase .
  • the nucleotide sequence of the His-tag (histidine tag) is added to the gene coding sequence of the glucosyltransferase, and the expressed protein is tagged with His tag, and the His tag is favorable for separation of the expressed protein. Purification, and analysis and tracing in experiments, such as for analysis in immunoblot experiments.
  • the gene coding sequence for the glucosyltransferase is inserted between the BamH I and Hind III restriction sites of the pET28a(+) vector plasmid.
  • the gene coding sequence of the glucosyltransferase is inserted into the pET28a(+) vector plasmid, the 5' end of the gene coding sequence of the glucosyltransferase may be added with a start codon (such as ATG) and a pET28a (+) vector.
  • the BamHI restriction site is ligated in the plasmid, and the stop codon (such as TAA) can be added to the 3' end to be ligated with the Hind III restriction site in the pET28a(+) vector plasmid.
  • the stop codon such as TAA
  • the present invention also provides a biocatalytic use of a glucosyltransferase and a microorganism strain comprising the gene of the glucosyltransferase, the amino acid sequence of the glucosyltransferase comprising SEQ ID NO: The amino acid sequence shown; the glucosyltransferase catalyzes the conversion of stevioside to rebaudioside A.
  • the glucosyltransferase is encoded by a glucosyltransferase gene derived from gardenia, and the gene coding sequence of the glucosyltransferase comprises the nucleotide sequence set forth in SEQ ID NO: 2.
  • the preparation method of the rebaudioside A according to the invention is a biological enzymatic method, the preparation method is simple and efficient, low in cost, high in conversion rate and green in safety, and can be widely applied to industrial scale production;
  • the final concentration of the substrate is far greater than the prior art, and the final concentration of the substrate can reach 10%-30%;
  • the Laiwudi A obtained by the preparation method of the invention has high purity and high yield and can be widely applied in the food industry and the pharmaceutical field;
  • the preparation method of the preparation method of the rebaudioside A of the present invention is characterized in that the enzyme-glucosyltransferase is highly specific and can efficiently convert the stevioside to the rebaudioside A.
  • FIG. 1 is a plasmid map of a recombinant plasmid pET28a-RA07 according to an embodiment of the present invention.
  • the gene coding sequence of the glucosyltransferase (RA07) comprises a nucleotide sequence as shown in SEQ ID NO: 2,
  • the amino acid sequence of the glucosyltransferase comprises the amino acid sequence set forth in SEQ ID NO: 1; the glucosyltransferase is derived from gardenia.
  • FIG. 1 is a representation of the recombinant plasmid pET28a-RA07.
  • the constructed recombinant plasmid pET28a-RA07 was transferred into E. coli BL21 (DE3), and inoculated into 4 mL of LB medium at a 1% inoculation amount, maintaining a constant 37 ° C, shaking rate of 200 rpm, after overnight culture. Transfer the bacterial solution to a 2L flask containing 1L of LB medium (50 ⁇ g/mL kanamycin) at 1% inoculation, and continue to incubate at 37 °C until the OD600 value in the medium reaches 0.6 or so.
  • the obtained crude enzyme solution containing RA07 was identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the molecular size of the RA07 is in agreement with a theoretically calculated value, and the theoretical molecular weight of the RA07 is 53 KD.
  • the collected enzyme solution is freeze-dried to further prepare an enzyme powder of RA07.
  • glucosyltransferases RA01-RA11 derived from different species obtained by experiments; wherein, the specific source of each of the glucosyltransferases is shown in the following table; wherein RA01 is derived from Stevia rebaudiana, RA02 From sunflower (Helianthus annuus), RA03 from rice (Oryza sativa), RA04 from Brachypodium distachyon, RA05 from Stevia rebaudiana, RA06 from Rauvolfia serpentina RA07 is derived from Gardenia jasminoides, RA08 is derived from Catharanthus roseus, RA09 is derived from Gardenia jasminoides, RA10 is derived from Picrorhiza kurrooa, and RA11 is derived from Lactobacillus. Reuteri 180), RA12 is derived from barley (Hordeum vulgare subsp. Vulgare);
  • RA02-RA06 and RA08-RA12 had no enzymatic activity and could not catalyze the conversion of stevioside to Laiwudi A, RA01.
  • the enzyme activity is low, the catalytic effect is poor, and RA07 has extremely high enzymatic activity, strong catalytic performance, and the conversion rate is as high as 99%, and the stevioside can be efficiently catalyzed to obtain the rebaudioside A;
  • the enzymatic properties of the enzymes of the same species are also greatly different.
  • both RA01 and RA05 are derived from stevia, and both RA07 and RA09 are derived from gardenia, but the catalytic properties of the two are greatly different.
  • the conversion rate is calculated by measuring the stevioside content in the reaction liquid by liquid chromatography after completion of the stirring reaction.
  • a method for preparing Lai Di Di A comprising:
  • the product was denatured and filtered to remove the protein, and the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A.
  • the crude product of Lycopidiside A was separated by silica gel resin, crystallized, etc., and purified by post-treatment to obtain a lyophilized A 96.22 g. >95%.
  • a method for preparing Lai Di Di A comprising:
  • a method for preparing Lai Di Di A comprising:
  • a method for preparing Lai Di Di A comprising:
  • the protein of RA07 was denatured and filtered to remove the protein.
  • the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A.
  • the crude product of rebaudioside A was separated by silica gel resin, crystallized, etc., and then purified to obtain a lyophilized A 94.37 g. , purity >95%.
  • a method for preparing Lai Di Di A comprising:
  • the protein of RA07 was denatured and filtered to remove the protein.
  • the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A.
  • the crude product of rebaudioside A was separated by silica gel resin, crystallized, etc., and purified by post-treatment to obtain Lai Di Di A 95.01 g. , purity >95%.
  • a method for preparing Lai Di Di A comprising:
  • boric acid buffer 200 g of stevioside, 120 g of sucrose and 1.5 g of UDP were added, respectively, and stirred until completely dissolved; glucosyltransferase RA07 enzyme powder 80 g and sucrose synthase (from Arabidopsis thaliana, NP_197583) 60 g were continuously added.
  • the total volume of the reaction system is 1 L, and the pH of the system is adjusted to 7.4; the reaction is carried out at a constant temperature of 37 ° C and a stirring rate of 250 rpm for 20 hours, and the conversion rate is 98% by the experiment. After the reaction is completed, the reaction liquid is heated to 100 ° C for 0.5 h.
  • the protein of RA07 was denatured and the protein was removed by filtration, and the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A.
  • the crude product of rebaudioside A was separated by silica gel resin, crystallized, etc., and then purified to obtain a lycopene A 190.02. g, purity > 95%.
  • a method for preparing Lai Di Di A comprising:
  • the boric acid buffer solution 300 g of stevioside, 150 g of sucrose and 2 g of UDP were added, respectively, and stirred until completely dissolved; 100 g of glucosyltransferase RA07 enzyme powder and 80 g of sucrose synthase (derived from Arabidopsis thaliana, NP_197583) were continuously added.
  • the total volume of the reaction system was 1 L, and the pH of the system was adjusted to 7.4.
  • the reaction was carried out for 20 h at a constant temperature of 37 ° C and a stirring rate of 300 rpm. The conversion was 96% by the experiment. After the reaction was completed, the reaction solution was heated to 100 ° C for 0.5 h.
  • the protein of RA07 was denatured and filtered to remove the protein.
  • the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A.
  • the crude product of rebaudioside A was separated by silica gel resin, crystallized, etc., and then purified to obtain a lycopene A 290.25g. , purity >95%.

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Abstract

L'invention concerne un procédé de préparation de rébaudioside A, ledit procédé consistant à : en présence d'uridine diphosphate glucose et d'une glycosyltransférase, utiliser une matière première de stéviol glycoside comme substrat pour préparer une solution réactionnelle, et à agiter et faire réagir celle-ci à une température constante de 20 à 45 °C pour obtenir du rébaudioside A. La matière première de stéviol glycoside comprend du stévioside, et la glycosyltransférase comprend la séquence d'acides aminés représentée dans SEQ ID NO 1. L'invention concerne également une enzyme pour la préparation de rébaudioside A, et une application.
PCT/CN2018/096211 2018-07-19 2018-07-19 Procédé de préparation de rébaudioside a, enzyme pour la préparation de rébaudioside a, et application Ceased WO2019161634A1 (fr)

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CN201880001964.4A CN109196110A (zh) 2018-07-19 2018-07-19 莱苞迪甙a的制备方法、莱苞迪甙a制备用酶及应用
PCT/CN2018/096211 WO2019161634A1 (fr) 2018-07-19 2018-07-19 Procédé de préparation de rébaudioside a, enzyme pour la préparation de rébaudioside a, et application

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WO2023030065A1 (fr) * 2021-08-30 2023-03-09 弈柯莱生物科技(上海)股份有限公司 Glycosyltransférase et son application

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CN113862319A (zh) * 2021-09-16 2021-12-31 华南理工大学 人参糖基转移酶在合成甜菊糖中的应用

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CN105164270A (zh) * 2013-02-28 2015-12-16 Cj第一制糖株式会社 由甜菊苷制备莱苞迪甙a的方法

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KR20150000258A (ko) * 2013-06-24 2015-01-02 한국생명공학연구원 효소전환법을 이용한 천연 고감미료의 제조방법

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DU TING ET AL.: "Synthesis, recombinant expression of arabidopsis sucrose synthase gene and its preliminary application in enzymatic synthesis of rebaudioside s", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, vol. 36, no. 23, 1 December 2015 (2015-12-01), pages 157 - 161 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023030065A1 (fr) * 2021-08-30 2023-03-09 弈柯莱生物科技(上海)股份有限公司 Glycosyltransférase et son application

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