Method for preparing rebaudioside M through enzyme method

A technology for enzymatic preparation and rebaudioside, which is applied in the field of biological preparation of rebaudioside M, can solve problems such as no commercial production of rebaudioside M, and achieves shortening production cycle, expanding application scope, and producing The effect of cycle shortening

Active Publication Date: 2013-11-20
PEPSICO INC
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Problems solved by technology

There is no commercial producti...
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Abstract

The invention relates to a method for preparing rebaudioside M through an enzyme method. According to the method, rebaudioside A or rebaudioside D is used as a substrate, and the substrate reacts to generate the rebaudioside M in the presence of a glucosyl donor under the catalytic action of UDP-glucosyltransferase and/or recombinant cells containing the UDP-glucosyltransferase. The method for preparing rebaudioside M through an enzyme method has important application value; and compared with the existing technology of extracting rebaudioside M from stevia rebaudian leaves, the method provided by the invention obviously shortens the production cycle, improves the productivity and lowers the cost, and can provide products having higher purity. Thus, the method can be used in the food and beverage industry in a more economical manner.

Application Domain

Microorganism based processesFermentation +2

Technology Topic

Enzyme methodUdp glucosyltransferase +5

Image

  • Method for preparing rebaudioside M through enzyme method
  • Method for preparing rebaudioside M through enzyme method
  • Method for preparing rebaudioside M through enzyme method

Examples

  • Experimental program(10)

Example Embodiment

[0045] Example 1: Preparation of recombinant E. coli cells containing UGT-A
[0046] According to sequence 1 and sequence 2, the UGT-A gene fragment was synthesized by gene, with NdeI and BamHI restriction sites added at both ends, and connected to the pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.). The UGT gene fragment was digested with restriction enzymes NdeI and BamHI, the purified fragment was recovered, and T4 ligase was added to connect the fragment to the corresponding restriction site of pET30a, and the BL21 (DE3) strain was transformed.
[0047] Inoculate UGT strains into 4ml liquid LB medium at a ratio of 1%, culture with shaking at 37°C (200rpm) overnight, transfer the overnight culture to 50ml liquid LB medium with 1% inoculation volume, and culture with shaking at 37°C (200rpm) To OD 600 When the value reaches 0.6-0.8, add final concentration of 0.4mM IPTG and culture overnight at 20°C with shaking. After the induction, 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-A for catalysis.

Example Embodiment

[0048] Example 2: Preparation of freeze-dried UGT-A powder
[0049] The UGT-A recombinant cells prepared in Example 1 were ultrasonically disrupted in an ice bath, the disrupted liquid was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a freeze-dried powder of UGT-A.

Example Embodiment

[0050] Example 3: Preparation of recombinant E. coli cells containing UGT-B
[0051] According to sequence 3 and sequence 4, the UGT-B gene fragment was synthesized by gene, with NdeI and BamHI restriction sites added at both ends, and connected to pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.). The UGT gene fragment was digested with restriction enzymes NdeI and BamHI, the purified fragment was recovered, and T4 ligase was added to connect the fragment to the corresponding restriction site of pET30a, and the BL21 (DE3) strain was transformed.
[0052] Inoculate UGT strains into 4ml liquid LB medium at a ratio of 1%, culture with shaking at 37°C (200rpm) overnight, transfer the overnight culture to 50ml liquid LB medium with 1% inoculation volume, and culture with shaking at 37°C (200rpm) To OD 600 When the value reaches 0.6-0.8, add final concentration of 0.4mM IPTG and culture overnight at 20°C with shaking. After induction, 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 UGT-B-containing recombinant cells for catalysis.
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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

  • Reduce manufacturing cost
  • Shorten the production cycle
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