Thermostable TiT4E Epimerase for Stable Multi-Enzyme Tagatose Production
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Solution Overview
Problem
Current tagatose 6-phosphate 4-epimerases exhibit low thermal stability, making them unsuitable for large-scale production of tagatose, a rare sugar, as they are not stable enough for efficient use in multi-enzyme systems.
Innovation Solution
A thermostable tagatose 6-phosphate 4-epimerase (TiT4E) derived from Thermoanaerobacter indiensis, with specific amino acid sequences or their derivatives, is developed to catalyze the conversion between fructose 6-phosphate and tagatose 6-phosphate, integrated into a vector and expressed in host cells for enhanced stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If fructose 1,6-phosphate aldolase from E. coli is used as tagatose 6-phosphate 4-epimerase, then the enzyme can catalyze the conversion of tagatose 6-phosphate to fructose 6-phosphate, but the thermal stability is low making it unsuitable for large-scale production
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's thermal stability properties through directed evolution and rational design. Specifically, the enzyme's structural parameters were optimized to increase thermal stability while maintaining catalytic activity. This involved changing amino acid sequences and protein folding characteristics to enable the enzyme to function reliably at elevated temperatures suitable for large-scale industrial production.
Solution Approach 2:
The patent uses copying by creating engineered versions of the original enzyme through molecular cloning and protein expression systems. The wild-type fructose 1,6-phosphate aldolase was cloned into expression vectors and produced in host organisms, allowing for systematic modification and optimization of thermal stability properties while preserving the essential catalytic function for tagatose production.
2Adaptability or versatility
If the enzyme is used in multi-enzyme systems for tagatose production, then the conversion process can be integrated, but the low thermal stability limits its effectiveness
Solution Approach 1:
The patent modifies the enzyme's operational parameters by optimizing its thermal stability to match the temperature conditions required for efficient multi-enzyme system operation. This parameter optimization ensures that the epimerase remains stable and active when integrated with other enzymes in the tagatose production pathway, enabling reliable large-scale production.
Solution Approach 2:
The engineered enzyme is designed to serve multiple functions within the metabolic pathway: it maintains catalytic activity for tagatose 6-phosphate to fructose 6-phosphate conversion while simultaneously achieving thermal stability suitable for industrial multi-enzyme systems. This multi-functionality enables its effective integration into comprehensive tagatose production platforms.
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 thermostable TiT4E enzyme maintains high activity at elevated temperatures, enabling efficient large-scale production of tagatose by forming a stable multi-enzyme system with other enzymes like α-glucan phosphorylase, phosphoglucomutase, and tagatose 6-phosphate phosphatase, achieving a 55% conversion rate from starch to tagatose.
Implementation Method 1
The 4-position epimerization of 6-carbon sugar plays an important role in the production of rare sugars and their derivatives
Implementation Method 2
fructose 6-phosphate is subjected to epimerization at 4 position to be converted into tagatose 6-phosphate
Implementation Method 3
one set of catalytic amino acids catalyzes the condensation of fructose 1,6-diphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate
Data Source
AI summary
Disclosed is a new tagatose 6-phosphate 4-epimerase, which is capable of converting fructose 6-phosphate into tagatose 6-phosphate and vice versa. Also disclosed is an application of the enzyme in tagatose production.