Fructose-6-phosphate 3-epimerase for allulose production
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Solution Overview
Problem
Existing methods for producing allulose from fructose suffer from low conversion rates and require additional separation steps due to reaction equilibrium, and existing enzymes lack the thermal stability and conditions necessary for industrial production.
Innovation Solution
A fructose-6-phosphate 3-epimerase enzyme derived from Clostridium lundense, with specific amino acid and nucleotide sequences, exhibits high sequence homology and is optimized for a temperature range of 40 to 70°C and pH 6 to 8, achieving a maximum allulose production of 32% with enhanced activity from metal ions like Mn and Co.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If fructose is converted to allulose using fructose-6-phosphate 3-epimerase, then allulose production is achieved, but the conversion rate is limited to 20-35% due to reaction equilibrium
Solution Approach 1:
The patent extracts and removes the product allulose-6-phosphate from the reaction system using ion-exchange resin, shifting the reaction equilibrium toward complete conversion of fructose to allulose, thereby overcoming the 20-35% conversion limit imposed by equilibrium constraints
Solution Approach 2:
The patent changes the reaction conditions by controlling pH within 6.0-8.0 and temperature within 40-70°C to optimize enzyme activity and prevent side reactions, while using metal ions (Mn2+, Co2+, Ni2+) as activators to enhance the epimerase's catalytic efficiency and achieve near-complete conversion
2Productivity
If existing enzymes are used for industrial allulose production, then production can proceed, but thermal stability and suitability for industrial conditions are insufficient
Solution Approach 1:
The patent optimizes reaction parameters including temperature (40-70°C), pH (6.0-8.0), and metal ion concentration to match industrial production requirements, ensuring the enzyme maintains high activity and stability under scalable conditions
Solution Approach 2:
The enzyme system is designed to be self-sufficient by incorporating all necessary cofactors (metal ions) and operating under conditions where the enzyme naturally maintains its structure and activity without requiring complex additional systems
3Productivity
If saccharides are used as substrate under alkaline conditions to improve conversion, then reaction efficiency increases, but browning of saccharides occurs
Solution Approach 1:
The patent carefully controls pH within the range of 6.0-8.0 and temperature within 40-70°C to optimize enzymatic activity while avoiding conditions that would cause Maillard reaction or caramelization of fructose, thus preventing browning and maintaining product quality
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 enzyme achieves a high conversion rate of fructose-6-phosphate to allulose-6-phosphate, reducing production costs and enabling industrial-scale production with improved thermal stability and reaction conditions.
Implementation Method 1
Fructose-6-phosphate epimerases include 3-epimerase and 4-epimerase. Specifically, D-allulose-3-epimerase (EC 5.1.3.30) produces allulose-6-phosphate through 3-epimerization (epimerization at the C-3 position ) of fructose (D-fructose)
Implementation Method 2
ketohexose-6-phosphate epimerase can epimerize C3 or C4. The ketohexose may be one or more ketohexoses selected from the group consisting of fructose, allulose, sorbose, and tagatose
Data Source
Figure 1
Figure 2
Figure 3~4
AI summary
The present disclosure relates to an epimerase protein of fructose-6-phosphate, nucleic acid molecule encoding the epimerase protein, a recombinant vector and a transgenic microorganism which comprise the nucleic acid molecule, and a composition for producing allulose by using them.