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Biocatalytic method for synthesis of rebaudioside M

A technology of biocatalysis and catalytic reaction, applied in the field of bioengineering, can solve the problems of low economic efficiency of rebaudioside M

Pending 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

[0014] The purpose of the present invention is to address the problem of low economic efficiency in the preparation of rebaudioside M at present, and to provide a new route for the biocatalytic synthesis of rebaudioside M. First, the glycosylation of glycosyltransferase UGTSL2 is used to catalyze the synthesis of stevioside Rebaudioside E, and then use glycosyltransferase UGT76G1 to catalyze the synthesis of rebaudioside M from rebaudioside E, providing a strong basis for the preparation and purification of rebaudioside M

Method used

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  • Biocatalytic method for synthesis of rebaudioside M

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the construction of recombinant escherichia coli genetically engineered bacteria

[0033] 1) Obtaining recombinant Escherichia coli genetically engineered bacteria co-expressing UGTSL2 and StSUS1

[0034] The full sequence of UGTSL2 and StSUS1 genes was synthesized by Nanjing GenScript Company and constructed on the plasmid pRSFDuet-1, wherein the UGTSL2 gene fragment was inserted into the plasmid vector RSFDuet-1 (Novagen Company) Nde I and xho Between the I restriction sites, the StSUS1 gene fragment was inserted into the plasmid vector pRSFDuet-1 (Novagen) Nco I and Eco Between the R I restriction sites, a recombinant plasmid, pRSFDuet-UGTSL2-StSUS1, which can exogenously co-express glycosyltransferase UGTSL2 and sucrose synthase StSUS1 was provided.

[0035] Add 40 uL ddH 2 O into the recombinant plasmid pRSFDuet-UGTSL2-StSUS1 dry powder, fully dissolved, take 5 ul of heat shock and transform into E. coli BL21 (DE3) competent cells, and the ...

Embodiment 2

[0039] Embodiment 2: the co-expression of genetically engineered bacteria double enzyme

[0040] Inoculate recombinant genetically engineered bacteria containing plasmid pRSFDuet-UGTSL2-StSUS1 (or pRSFDuet-UGT76G1-StSUS1) into LB medium containing 50 mg / L kanamycin resistance (0.5 g / L yeast powder, 1 g / L Sodium chloride, 1 g / L tryptone), shake overnight at 37°C and 200 rpm, then transfer the cultured bacteria into 100 mL of TB medium (2.5 g / L yeast powder, 1 g / L sodium chloride, 1.5 g / L tryptone, 0.2 g / L glucose, 0.05 g / L lactose) in a 500 mL shake flask, at 200 rpm, 37°C for 2 h, then transferred to 25°C for 24 h, Bacteria were collected by centrifugation. 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

[0041] Example 3: Synthesis of rebaudioside E from stevioside by enzymatic method

[0042] 1) Catalytic reaction system

[0043] Add 20 g / L stevioside, 60 g / L sucrose, 3 mM 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), the total volume was quantified to 20 mL. React at 30°C and 200 rpm for 24 h, sample 500 uL at regular intervals, heat in a water bath at 95°C for 15 min, centrifuge at room temperature at 12,000 rpm for 1 min, separate the supernatant into a new 1.5 mL EP tube, store at 4°C, and wait for HPCL detection. According to the results (Table 1), it is feasible to catalyze the synthesis of rebaudioside E from stevioside by glycosyltransferase UGTSL2 coupled with sucrose synthase StSUS1, and the conversion rate of stevioside reached 94.12% after 24 hours of reaction. The yield of rebaudioside E was 66.32%.

[0044] 2), PHLC detection method

[0045] Chromat...

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Abstract

The invention discloses a biocatalytic method for synthesis of rebaudioside M. Firstly, rebaudioside E is synthesized by a glycosylation reaction of stevioside with UDP-glycosyltransferase from tomatos and sucrose synthetase from potatoes; then, rebaudioside M is synthesized by a glycosylation reaction of rebaudioside E with UDP-glycosyltransferase from stevia rebaudiana and sucrose synthetase from potatoes. The method uses a molecular cloning technique, escherichia coli genetically engineered bacteria for heterologous expression of UDP-glycosyltransferase and sucrose synthetase are obtained,after fermentation and enzyme production, crude extract of cells is directly used for a catalytic reaction, addditionally added saccharose is decomposed with sucrose synthetase to obtain UDP-glucose,UDP in the crude extract and the UDP-glucose serve as raw materials for the glycosylation reaction, a double-enzyme cycle reaction system is established, and the rebaudioside M is produced by effectively catalyzing stevioside. The cost of raw materials is lower, the processing steps are simple, and the method has an important application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for biocatalytically synthesizing rebaudioside M. Background technique [0002] Stevia, as a perennial herbaceous plant native to South America, can extract rich steviol glycosides from its stems and leaves, and has been used as a natural sweetener by South American people for centuries [1] . After the successful planting of stevia introduced from Japan in 1976, my country began to promote planting to the whole country after the 1980s. At present, Fujian, Anwei, Jiangsu and other places have been planted in large areas, with a total area of ​​1 million mu, and has become the world's largest producer and exporter of stevia sugar [2, 3] . 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. Its solubility is not low, and it can exi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/56C12N15/70C12N9/10
Inventor 贾红华李艳陈量量潘华祎欧阳平凯
Owner NANJING UNIV OF TECH
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