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Method for preparing rebaudioside-M by enzymic method

A technology of enzymatic preparation and crude enzyme solution, which is applied in the direction of introducing foreign genetic material using carriers, recombinant DNA technology, fermentation, etc., can solve problems such as poor economy, cumbersome process, recrystallization, etc., and achieve reduced production costs and high reaction conversion rates Higher and shorter process steps

Active Publication Date: 2015-06-24
XINGHUA GL STEVIA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of Reb M by extraction requires a large amount of stevia raw materials. In addition, the process of enriching Reb M is cumbersome. After extraction, it needs to pass through the column for many times, desalting, decolorization, and recrystallization, and a large amount of waste water is generated in the production process. The production cost High, not suitable for industrialized mass production
In addition, the current bio-enzymatic method for synthesizing Reb M requires the addition of expensive UDP-glucose as a substrate. Through the action of UDP-glucosyltransferase, Reb A is catalyzed to generate Reb M, so that the economic efficiency of enzyme-catalyzed Reb A to synthesize Reb M is relatively low. Poor, lack of market competitiveness

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the construction of the escherichia coli engineering bacterium expressing double enzyme gene

[0023] Add NdeI and XhoI restriction sites to both ends of the UDP-glycosyltransferase gene (sequence 1), insert the gene fragment into the corresponding restriction sites of the expression vector pETDuet-1 through double restriction digestion and ligation, and place in Under the control of the T7 promoter, a recombinant plasmid pEUGT expressing UGT76C4 was constructed.

[0024] Using the CloneEZ? kit from Nanjing GenScript Biotechnology Co., Ltd., the sucrose synthase gene (sequence 3) sequence was cloned into pEUGT at the Xho I restriction site to construct the expression plasmid pEUGT-SUS. Transform pEUGT-SUS into E. coli In Rosetta (DE3), the engineered bacteria co-expressing the dual enzymes can be obtained E. coli Rosseta (pEUGT-SUS).

Embodiment 2

[0025] Embodiment 2: Escherichia coli engineering bacteria ferment and produce enzyme

[0026] Pick engineering bacteria E. coli Add Rosseta (pEUGT-SUS) to LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and culture overnight at 37°C with shaking. Then inoculate 2% inoculum into fresh induction medium (15 g / L peptone, 25 g / L yeast extract, 10 g / L NaCl, 2 g / L glucose and 3 g / L lactose) containing corresponding antibiotics, After induction of expression for 12 h, the bacterial solution was centrifuged at 8000 rpm, 4°C for 10 min, the supernatant was discarded, and the pellet was set aside.

Embodiment 3

[0027] Embodiment 3: the preparation of the Escherichia coli crude enzyme liquid expressing double enzyme

[0028] Take the cell pellet in Example 2, wash twice with potassium phosphate buffer (100 mmol / L, pH 7.2), suspend the washed precipitate in potassium phosphate buffer, and sonicate the cells (power 300W, ultrasonic 5 s , with an interval of 5 s, a total of 5 min), the crushed liquid was centrifuged at 8000 rpm, 4°C for 15 min, and the supernatant was taken as the crude enzyme solution.

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PUM

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Abstract

The invention discloses a method for preparing rebaudioside-M by an enzymic method. The method comprises the steps of utilizing tomato UDP-glycosyltransferase and potato sucrose synthetase, and using rebaudioside-A and sucrose as raw materials for producing rebaudioside-M. According to the method disclosed by the invention, the high-yield recombinant strain of UDP-glycosyltransferase and sucrose synthase is obtained by virtue of a genetic engineering technology at first, then a bio-enzyme crude product is collected, a catalytic reaction is directly carried out, UDP or UDP-glucose is not added in the reaction system, the rebaudioside-A and the sucrose are used as the raw materials, and the rebaudioside-A is catalyzed to generate the rebaudioside-M through the efficient circulation of catalyzing the UDP-glucose by the sucrose synthase, and through the action of the tomato UDP-glycosyltransferase. Compared with the existing preparation method for rebaudioside-M, the process is short in steps, low in cost, important in application value, and good in economical efficiency.

Description

technical field [0001] The invention utilizes tomato UDP-glycosyltransferase and potato sucrose synthase to efficiently catalyze rebaudioside A to prepare rebaudioside M, and belongs to the technical field of biocatalytic conversion. Background technique [0002] Stevia (Steviol glycosides) is derived from the Compositae herb stevia ( Stevia rebaudiana ) natural sweetener extracted from leaves. It is a mixture of more than 30 kinds of glycosides, and different glycosides have great differences in taste and quality. The content of the three main glycoside components in stevia leaves is usually: stevioside (Stevioside) accounts for 9.1% of the dry weight of the leaf, rebaudioside A (Reb A) accounts for 3.8%, rebaudioside C (Rebaudioside C, Reb C) accounted for 0.6%. Stevioside has high sweetness, low calorie, high stability, and potential curative effects such as anti-hyperglycemia, anti-hypertension, anti-inflammation, anti-tumor, anti-diarrhea, and diuretic, immune regu...

Claims

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Application Information

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IPC IPC(8): C12P19/56C12N15/70C12N15/81C12N15/74
Inventor 李艳严明陈量量
Owner XINGHUA GL STEVIA CO LTD
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