L-aspartate Decarboxylase Mutants for Thermal Stability
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Solution Overview
Problem
Current L-aspartate α-decarboxylase enzymes derived from prokaryotes have low enzyme activity, while those from eukaryotes like Tribolium castaneum have high activity but poor stability, making them unsuitable for industrial β-alanine synthesis due to thermal instability.
Innovation Solution
Development of L-aspartate α-decarboxylase mutants K221R and G369A, where lysine at position 221 is mutated to arginine and glycine at position 369 is mutated to alanine, respectively, which are expressed in E. coli using a PET24a+ vector, enhancing thermal stability and enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Power
If L-aspartate α-decarboxylase is derived from eukaryotes (Tribolium castaneum) to achieve high enzyme activity, then enzyme activity is improved, but thermal stability deteriorates
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the enzyme sequence. Two specific mutations are introduced: K221R (lysine to arginine at position 221) and G369A (glycine to alanine at position 369). These parameter changes at the molecular level modify the enzyme's thermal stability while preserving its catalytic activity, resolving the contradiction between high activity and thermal stability.
2Ease of operation
If L-aspartate α-decarboxylase is derived from prokaryotes to achieve simplicity in operation, then ease of operation is improved, but enzyme activity deteriorates
Solution Approach 1:
The patent applies local quality by making specific localized changes to the enzyme structure rather than changing the entire enzyme source. By introducing point mutations at specific positions (K221R and G369A) in the prokaryotic enzyme sequence, the patent locally modifies the enzyme's properties to enhance activity while maintaining the simplicity of using prokaryotic expression systems.
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 mutants exhibit improved thermal stability with 44% and 40% residual enzyme activity after 50°C treatment, achieving high enzyme specific activity and efficient β-alanine production, with yields reaching 162.15 g/L through optimized fermentation and catalytic processes.
Implementation Method 1
Biosynthesis methods mainly use L-aspartate α-decarboxylase, and with L-aspartic acid as a substrate, an α-carboxyl group is removed to generate β-alanine
Implementation Method 2
The mutants exhibit improved thermal stability with 44% and 40% residual enzyme activity after 50°C treatment
Implementation Method 3
the L-aspartate α-decarboxylase mutant, where lysine at position 221 is mutated to arginine and glycine at position 369 is mutated to alanine, respectively, which are expressed in E. coli using a PET24a+ vector
Implementation Method 4
with sodium L-aspartate as a substrate, the cell is used for fermentation, and a bacteria solution after the fermentation broth is used for whole cell transformation to produce β-alanine
Data Source
AI summary
The disclosure discloses an L-aspartate α-decarboxylase mutant and application thereof, and belongs to the technical field of enzyme engineering. In the disclosure, lysine at position 221 of L-aspartate α-decarboxylase is mutated to arginine, glycine at position 369 is mutated to alanine, and the obtained new mutant enzymes have better temperature tolerance and are beneficial to industrial production. The K221R and G369A recombinant strains are subjected to high-density fermentation, and with sodium L-aspartate as a substrate, a whole cell catalytic reaction is carried out to prepare β-alanine. Compared with a chemical production method, the method has the advantages that the production process is safe and clean, and has no environmental pollution. Compared with a pure enzyme catalysis method, the method has the advantages that the operation is simple and convenient. The yield of the final product β-alanine reaches 91% and 90% respectively, and the concentration reaches 162.15 g/L and 160.42 g/L respectively.