Enzymatic Rebaudioside M Synthesis with DMSO Solubilizer
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Solution Overview
Problem
Current methods for preparing Rebaudioside M have low conversion rates and purity, making them unsuitable for industrial production due to high production costs and inefficient processes.
Innovation Solution
An enzyme method using Rebaudioside A or D as substrates, catalyzed by a mixture of UDP-glucosyl transferase and sucrose synthetase, in an aqueous-phase system with optimized conditions such as temperature, pH, and solubilizers, to produce Rebaudioside M with a purity greater than 98%, reducing production costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the prior art biological method is used to prepare Rebaudioside M, then the conversion rate reaches about 80% and purity reaches 95%, but the production cost is high and the process is not suitable for industrialized production
Solution Approach 1:
The patent optimizes multiple reaction parameters including substrate concentration (Rebaudioside A ≥10 g/L or Rebaudioside D ≥15 g/L), temperature (20-60°C), pH (5.0-9.0), and enzyme ratios to achieve both high conversion rate (>80%) and high purity (>98%) while reducing production cost. The optimized parameters enable industrialized production by balancing efficiency and cost-effectiveness
2Productivity
If the prior art biological method is used to prepare Rebaudioside M, then the conversion rate reaches about 80%, but the purity is only 95%
Solution Approach 1:
The patent employs a two-stage purification process: first using macroporous adsorbent resin to remove impurities and obtain crude Rebaudioside M, then using aqueous ethanol solution for recrystallization to achieve purity greater than 98%. This preliminary purification action ensures high purity product while maintaining high conversion rate
Solution Approach 2:
The patent optimizes crystallization parameters including ethanol concentration (30-70% v/v), temperature (0-25°C), and solvent ratios to maximize purity. By carefully controlling these parameters during the crystallization stage, the patent achieves purity greater than 98% while maintaining efficient production
3Productivity
If the substrate concentration is increased to improve productivity, then the conversion rate improves, but the solubility of the substrate decreases
Solution Approach 1:
The patent uses dimethyl sulfoxide (DMSO) as a solubilizing agent to enhance substrate solubility in the aqueous reaction medium. DMSO acts as an intermediary that allows high concentrations of Rebaudioside A (≥10 g/L) or Rebaudioside D (≥15 g/L) to remain dissolved, enabling high conversion rates without precipitation or solubility limitations
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly improves conversion rates and purity, making it suitable for industrialized production of Rebaudioside M with reduced costs.
Implementation Method 1
Rebaudioside M is generated by means of reaction of the substrate under the catalysis of a mixture of UDP-glucosyl transferase and sucrose synthetase (AtSUS1), or recombinant cells containing the UDP-glucosyl transferase and sucrose synthetase
Implementation Method 2
The reaction system further contains dimethyl sulfoxide at a concentration of 3% to 5% according to the ratio by volume for facilitating solubilization of the substrate
Implementation Method 3
the supernatant is taken, and separated with a macroporous adsorbent resin to obtain a solution of a crude product of Rebaudioside M
Implementation Method 4
which is crystallized in an aqueous ethanol solution, so as to obtain a Rebaudioside M product with a purity greater than 98%
Implementation Method 5
After the reaction, the reaction solution is centrifuged, then the supernatant is taken
Data Source
Figure 1
AI summary
Provided is a method for preparing Rebaudioside M by using an enzyme method. In the method, Rebaudioside A or Rebaudioside D is used as a substrate, and in the presence of sucrose and UDP, Rebaudioside M is generated by means of reaction of the substrate under the catalysis of a mixture of UDP-glucosyl transferase and sucrose synthetase, or recombinant cells containing the UDP-glucosyl transferase and sucrose synthetase. The reaction is carried out in an aqueous-phase system at a temperature of 20 to 60°C and pH 5.0 to 9.0. The reaction system further contains dimethyl sulfoxide at a concentration of 3% to 5% according to the ratio by volume for facilitating solubilization of the substrate.