Method for preparing Rebaudioside E through enzyme method

A technology of enzymatic preparation and glycosyltransferase, which is applied in the field of enzymatic preparation of rebaudioside E, can solve the problems of low economic efficiency of rebaudioside E, simplify material preparation steps, improve preparation efficiency, and reduce work volume effect

Active Publication Date: 2019-05-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The purpose of the present invention is to provide an enzymatic method to prepare rebaudioside E for the problem that it is difficult to obtain and prepare rebaudioside E in large quantities directly from the dry leaves of Stevia rebaudiana only by means of leaf crushing, physical adsorption, purification and the like. The method of baudioside E uses the glycosylation of glycosyltransferase UGTSL2 to catalyze the synthesis of rebaudioside E from stevioside in one step, which provides a strong basis for the preparation and purification of rebaudioside E

Method used

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  • Method for preparing Rebaudioside E through enzyme method
  • Method for preparing Rebaudioside E through enzyme method
  • Method for preparing Rebaudioside E through enzyme method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the construction of recombinant escherichia coli genetically engineered bacteria

[0031] 1), acquisition of recombinant plasmids co-expressing UGTSL2 and StSUS1

[0032] The full sequence of UGTSL2 and StSUS1 genes was synthesized by Nanjing GenScript Company and constructed on the plasmid pRSFDuet-1 (Novagen Company), wherein the UGTSL2 gene fragment was inserted between the Nde I and Xho I restriction sites of the plasmid vector, and StSUS1 The gene fragment was inserted between the Nco I and EcoR I restriction sites of the plasmid vector, and finally a recombinant plasmid, pRSFDuet-UGTSL2-StSUS1, which can co-express glycosyltransferase UGTSL2 and sucrose synthase StSUS1 was provided.

[0033] 2), the acquisition of recombinant Escherichia coli genetically engineered bacteria

[0034] Add 40uL ddH 2 O into the recombinant plasmid pRSFDuet-UGTSL2-StSUS1 dry powder, fully dissolved, take 5ul of heat shock and transform into Escherichia coli BL21 (DE3) ...

Embodiment 2

[0035] Embodiment 2: the co-expression of genetically engineered bacteria double enzyme

[0036] Inoculate the recombinant genetically engineered bacteria containing plasmid pRSFDuet-UGTSL2-StSUS1 into LB medium containing 50mg / L kanamycin resistance (0.5g / L yeast powder, 1g / L sodium chloride, 1g / L tryptone) , shake overnight at 37°C and 200rpm, and then insert the culture bacteria into 100mL TB medium (2.5g / L yeast powder, 1g / L sodium chloride, 1.5g / L tryptone, 0.2g / L glucose, 0.05g / L lactose) in a 500mL shake flask, cultured at 200rpm at 37°C for 2h, then transferred to 25°C for 24h, and centrifuged to collect the bacteria. The cells were disrupted by ultrasonication, and the supernatant obtained by centrifugation was the crude cell extract, which was stored at 4°C until use.

Embodiment 3

[0037] The method of embodiment 3 enzymatic preparation rebaudioside E

[0038] 1), catalytic reaction system

[0039] Add 20g / L stevioside, 60g / L sucrose, 3mM Mg 2+ And an appropriate amount of crude cell extract (~6 mg / mL total protein) co-expressing UGTSL2 and StSUS1 in 50 mM potassium phosphate buffer (pH 7.2), and the total volume was quantified to 20 mL. React at 30°C and 200rpm for 24h, sample 500uL at regular intervals, heat in a water bath at 95°C for 15min, centrifuge at room temperature at 12000rpm for 1min, separate the supernatant into a new 1.5mLEP tube, store at 4°C, and wait for HPCL detection. From the results (Table 1), it is feasible to catalyze the synthesis of rebaudioside E from stevioside by the coupling of glycosyltransferase UGTSL2 to sucrose synthase, and at the time of reaction for 24 hours, the conversion rate of stevioside has reached 94.12%. The yield of diglycoside E was 66.32%.

[0040] 2), PHLC detection method

[0041] Chromatographic colu...

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Abstract

The invention discloses a method for preparing Rebaudioside E through an enzyme method. By means of the method, through tomato source UDP-glycosyl transferase and potato source sucrose synthetase, with stevioside as the raw material, the Rebaudioside E is produced through one-step glycosylation; meanwhile, by means of the fixed-point mutation technology, the UDP-glycosyl transferase is transformed, and the yield of the Rebaudioside E is further improved. By means of the method, no UDP-glucose or UDP needs to be added, the UDP-glucose is obtained as the raw material of glycosylation under the sucrose synthetase decomposition effect of the UDP in a crude extraction solution and the added sucrose, a double-enzyme circulating reaction system is established, and the stevioside is effectively catalyzed to generate the Rebaudioside E. The yield of the Rebaudioside E can reach 65% or above; through the fixed-point mutation technology in the later period, N358F beneficial mutants are obtained through screening, and the yield of the Rebaudioside E is improved through the catalysis and can reach 85% or above at most. Compared with an existing Rebaudioside E preparing method, the method is simple in processing step, low in cost and high in yield and has important application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing rebaudioside E by an enzymatic method. Background technique [0002] As the sweetest sweetener known so far, stevioside has a sweetness 250-450 times that of sucrose, and its calories are only 1 / 300 of sucrose, with a slight astringent taste. In addition, stevioside also has auxiliary pharmacological effects on hypertension, obesity, diabetes, etc. [1,2] , therefore, more and more people's attention. Stevioside is the general term for steviol glycosides, and more than 35 steviol glycosides have been isolated and identified from Stevia rebaudiana [3] , including stevioside (Stevioside) and rebaudioside A, B, C, D, E, M (Rebsudioside A / B / C / D / E / M), etc. The steviol glycoside structure revolves around a diterpene steviol as the backbone, in C 13 hydroxyl and C 19 The carboxyl groups are connected to different glucose groups, among them, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/56C12N9/10C12N15/54C12R1/19
Inventor 李艳陈量量贾红华潘华祎欧阳平凯
Owner NANJING UNIV OF TECH
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