D-psicose 3-epimerase Mutant Thermostability Amino Acid Substitution
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Solution Overview
Problem
D-psicose 3-epimerase derived from Agrobacterium tumefaciens has poor thermostability, limiting its utility for large-scale industrial production of D-psicose, a rare sugar with potential health benefits.
Innovation Solution
A D-psicose 3-epimerase variant with improved thermostability is developed by substituting specific amino acids in the enzyme's sequence, along with a recombinant expression vector and immobilized reactor system for continuous production of D-psicose.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Power
If D-psicose 3-epimerase from Agrobacterium tumefaciens is used for industrial production, then high enzymatic activity is achieved, but poor thermostability limits continuous operation and large-scale production
Solution Approach 1:
The patent applies parameter changes by substituting specific amino acid residues (e.g., Serine at position 213 with Cysteine, or Isoleucine at position 33 with Leucine/Valine/Cysteine) in the D-psicose 3-epimerase sequence. These amino acid substitutions modify the enzyme's structural parameters to enhance thermostability while preserving catalytic activity, enabling continuous operation at industrial temperatures.
Solution Approach 2:
The invention applies local quality by making targeted amino acid substitutions at specific positions (residues 33 and 213) within the enzyme sequence. Rather than modifying the entire enzyme, localized changes at these critical positions improve thermostability while maintaining the overall enzymatic function and activity.
2Device complexity
If wild type D-psicose 3-epimerase is used, then simple enzyme structure is maintained, but short half-life at reaction temperature prevents continuous production
Solution Approach 1:
The patent modifies the enzyme's amino acid sequence parameters by substituting residues at positions 33 and/or 213. These parameter changes extend the enzyme's half-life at reaction temperatures from less than 1 hour (wild type) to over 100 hours (mutant), enabling continuous production operations while maintaining relatively simple enzyme structure.
3Reliability
If amino acid substitutions are made to improve thermostability, then extended half-life and improved thermostability are achieved, but enzymatic activity may be affected
Solution Approach 1:
The invention carefully selects specific local positions (residues 33 and 213) for amino acid substitution that do not interfere with the catalytic active site. The substitutions at these distant positions improve thermostability through structural stabilization without affecting the local catalytic function, thus maintaining high enzymatic activity.
Solution Approach 2:
The patent employs specific amino acid substitutions (Serine-213 to Cysteine, or Isoleucine-33 to Leucine/Valine/Cysteine) that change the chemical parameters at these positions to enhance thermal stability. The selected substitutions are specifically chosen to improve thermostability while preserving the enzyme's catalytic parameters and activity.
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 variant exhibits significantly extended half-life at reaction temperatures, enabling efficient and cost-effective large-scale production of D-psicose with improved thermostability and maintaining enzymatic activity over time.
Implementation Method 1
D-psicose 3-epimerase variant with improved thermostability... converting fructose into D-psicose
Data Source
Figure 1~2
Figure 3(a)~3(f)
Figure 4
AI summary
The present invention relates to a D-psicose 3-epimerase mutant of which the thermal stability is improved by substituting an amino acid of a specific sequence number. In addition, the present invention relates to a recombinant vector comprising the gene of the D-psicose 3-epimerase mutant, and a recombinant strain transformed with the recombinant vector. Further, the present invention relates to an immobilized reactor prepared using the enzyme mutant or the recombinant vector, and a method for producing D-psicose using the immobilized reactor.