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Preparation method of rebaudioside A, enzyme for preparing rebaudioside A, and use thereof

A technology of rebaudioside and transferase, applied in the field of biomedicine, can solve the problems of complicated process steps, low purity and yield, and high energy consumption, and achieve the effects of simple process, few purification steps and high yield

Pending Publication Date: 2019-01-11
BONTAC BIO ENG SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above process steps are cumbersome, high energy consumption, large pollution, low purity and yield

Method used

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  • Preparation method of rebaudioside A, enzyme for preparing rebaudioside A, and use thereof
  • Preparation method of rebaudioside A, enzyme for preparing rebaudioside A, and use thereof
  • Preparation method of rebaudioside A, enzyme for preparing rebaudioside A, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A preparation method of rebaudioside A, comprising:

[0058]In sodium phosphate buffer, add stevioside 100g, sucrose 80g and UDP 1g respectively, stir until completely dissolved; continue to add 50g of glucosyltransferase RA07 enzyme powder and 50g of sucrose synthase (derived from Arabidopsis thaliana, NP_197583), react The total system volume is 1L, 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 200rpm for 16h, and the conversion rate is measured to be 99%. The protein was denatured and filtered to remove the protein, and the filtrate was collected and spray-dried to obtain a crude product of rebaudioside A. After the crude product of rebaudioside A was purified by silica gel resin separation, crystallization and other post-treatments, 96.22 g of rebaudioside A was obtained. Purity >95%.

Embodiment 2

[0060] A preparation method of rebaudioside A, comprising:

[0061] In the sodium phosphate buffer, add stevioside 100g and UDPG 1.5g respectively, stir until completely dissolved; continue to add 50g of glucosyltransferase RA07 enzyme powder, the total volume of the reaction system is 1L, adjust the pH of the system to 7.4; , 250rpm stirring rate under the reaction 16h, the conversion rate is 99% measured by experiments, after the reaction is completed, the reaction solution is heated to 100 ℃ heat treatment for 0.5h, the RA07 protein denatured and filtered to remove the protein, the filtrate is collected and then spray-dried to obtain Laibao Rebaudioside A crude product, the rebaudioside A crude product was purified by silica gel resin separation, crystallization and other post-treatments to obtain 95.82 g of rebaudioside A, with a purity of >95%.

Embodiment 3

[0063] A preparation method of rebaudioside A, comprising:

[0064] In the sodium phosphate buffer, add stevioside 100g and UDPG 1.5g respectively, stir until completely dissolved; continue to add 50g of glucosyltransferase RA07 enzyme powder, the total volume of the reaction system is 1L, adjust the pH of the system to 6.0; , 200rpm stirring rate under the reaction 16h, the conversion rate is 97% measured by experiments, after the reaction is completed, the reaction solution is heated to 100 ℃ heat treatment for 0.5h, the RA07 protein is denatured and filtered to remove the protein, after the filtrate is collected, it is spray-dried to obtain Laibao Rebaudioside A crude product, the rebaudioside A crude product was purified by silica gel resin separation, crystallization and other post-treatments to obtain 93.17 g of rebaudioside A, with a purity of >95%.

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Abstract

The invention provides a preparation method of rebaudioside A, which comprises: preparing a reaction liquid by using a stevioside raw material as a substrate in the presence of uridine diphosphate glucose and glucosyltransferase, and adjusting the pH of the reaction solution to 6.0-8.0, after stirring reaction at a constant temperature of 20-45 DEG C, collecting rebaudioside A; the stevioside rawmaterial comprises stevioside, and the amino acid sequence of the glucosyltransferase comprises SEQ ID NO: 1 The amino acid sequence shown. The preparation method is a high-efficiency, simple, low-cost, high conversion rate, green and environmentally friendly biological enzymatic method, and can be widely applied to industrial scale production. The invention also provides an enzyme and applicationfor the preparation of rebaudioside A.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method of rebaudioside A, an enzyme for preparing rebaudioside A and its application. Background technique [0002] Steviol glycoside (Steviol glycoside), also known as steviol glycoside, is a low-calorie high-intensity sweetener, its sweetness is 150-300 times that of sucrose, and its calories are about 1 / 300 of sucrose. Studies have shown that it has no toxic side effects and is safe to eat. Studies have shown that stevioside can be used to prevent hypertension, diabetes, obesity, heart disease and dental caries. It is an ideal sweetener that can replace sucrose. However, the sweetness multiples and mouthfeel of each component of stevioside are uneven; for example, stevioside (Stevioside, St), one of the main components, has a bitter taste and aftertaste, which greatly limits its application; and rebaudioside A (Rebaudioside A, RA) is superior to stevioside i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/56C12N15/54C12N9/10
CPCC12N9/1051C12P19/56C12P19/18
Inventor 傅荣昭李振伟刘文山刘玉凤
Owner BONTAC BIO ENG SHENZHEN
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