D-amino acid oxidase mutant for L-phosphinothricin production
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Solution Overview
Problem
Existing D-amino acid oxidase (DAAO) enzymes exhibit low enzyme activity and poor thermal stability, limiting their effectiveness in the preparation of L-phosphinothricin through microbial catalysis.
Innovation Solution
A DAAO mutant is developed through single-point or multi-point mutations at specific positions in the amino acid sequence, enhancing catalytic performance and thermal stability. The mutant enzymes are then used in genetically engineered bacteria for the microbial production of L-phosphinothricin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type DAAO is used for catalysis, then the enzyme can convert D-PPT to PPO, but the enzyme activity is low and thermal stability is poor
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues at positions 54, 58, 213, 239, 73, 77, 79, 147, 185, 43, 45, 206, 207, 215, 122, 132, 195, and 234 of the DAAO protein sequence. These mutations alter the physical and chemical parameters of the enzyme structure, resulting in improved catalytic activity and thermal stability while maintaining the ability to convert D-PPT to PPO
Solution Approach 2:
The patent applies local quality by making targeted mutations at specific positions within the DAAO protein sequence rather than uniform modifications. Each mutation at positions such as 54, 58, 213, and 239 addresses local structural characteristics that influence overall enzyme performance, allowing optimization of both activity and stability without compromising the catalytic function
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 DAAO mutant enzymes demonstrate improved enzyme activity and thermal stability, facilitating more efficient production of L-phosphinothricin, which is crucial for reducing usage amounts and environmental impact.
Implementation Method 1
the D-PPT is catalyzed by DAAO to obtain an L-PPT precursor 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO)
Implementation Method 2
the D-PPT is catalyzed by DAAO to obtain an L-PPT precursor
Data Source
AI summary
A D-amino acid oxidase mutant with significantly improved catalytic performance, a gene encoding the gene, a vector containing the gene, a genetically engineered bacterium, and the application of the said mutant in the microbial catalytic preparation of L-ammonium glufosinate. The D-amino acid oxidase mutant was obtained from the amino acid shown in SEQ ID NO.1 by single-point mutation or multi-point combined mutation. The beneficial effects are mainly reflected in the following: the D-amino acid oxidase mutant with improved enzyme activity and thermal stability can be used in the microbial catalysed preparation of L-ammonium glufosinate, which is conducive to industrial production and has a better application prospect.