Phytase Thermostability via Disulfide Bond Mutations
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Solution Overview
Problem
Current phytases, such as Escherichia coli phytase (EcAppA), lack thermostability, which limits their industrial application in animal feeds, leading to reduced phosphorus availability and increased environmental phosphorus pollution.
Innovation Solution
Modification of the phytase by introducing disulfide bonds through site-directed mutagenesis at specific positions, such as amino acids 143 and 262, 259 and 312, 205 and 257, and 264 and 309, to enhance protein stability and thermostability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If phytase is used to hydrolyze phytate and release inorganic phosphate, then phosphorus availability is improved and phosphorus pollution is reduced, but the enzyme lacks thermostability which limits its industrial application
Solution Approach 1:
The patent introduces disulfide bonds at specific positions (143-262, 259-312, 205-257, or 264-309) in the phytase protein sequence to change the structural parameters of the enzyme. This modification enhances the protein's thermostability by forming cross-linked structures that resist thermal denaturation, allowing the enzyme to maintain its catalytic activity at higher temperatures during industrial feed processing.
2Reliability
If directed evolution with random mutagenesis is used to improve enzyme performance, then useful mutants can be obtained, but the process is laborious and has low efficiency
Solution Approach 1:
Instead of random mutagenesis affecting the entire protein, the patent applies local quality modification by introducing disulfide bonds at specific predetermined positions (143-262, 259-312, 205-257, or 264-309) in the protein sequence. This targeted approach focuses the modification on specific structural regions known to enhance stability, significantly reducing the time and effort required compared to comprehensive random mutagenesis and screening.
3Loss of time
If rational design is used to modify enzyme performance, then modification efficiency is improved, but it requires extensive protein structure information and bioinformatics tools which may be limiting
Solution Approach 1:
The patent applies preliminary action by pre-identifying and pre-selecting the optimal positions for disulfide bond introduction (143-262, 259-312, 205-257, or 264-309) based on established protein structure principles and thermal stability characteristics. This preliminary determination of modification sites eliminates the need for extensive real-time bioinformatics analysis during the modification process, making the rational design approach more accessible and less resource-intensive.
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 modified phytases exhibit improved thermostability, increasing their residual activity after heat treatment and reducing production costs, thereby enhancing their industrial value and phosphorus availability in animal feeds.
Implementation Method 1
the modification is one of mutations A to D. The mutation A is to substitute amino acids at positions 143 and 262 with cysteine, the mutation B is to substitute amino acids at positions 259 and 312 with cysteine, the mutation C is to substitute amino acids at positions 205 and 257 with cysteine, and the mutation D is to substitute amino acids at positions 264 and 309 with cysteine
Data Source
AI summary
A phytase having improved thermostability is disclosed. The phytase has a modified amino acid sequence of SEQ ID NO: 2, wherein the modification is one of mutations A to D. The mutation A is to substitute amino acids at positions 143 and 262 with cysteine, the mutation B is to substitute amino acids at positions 259 and 312 with cysteine, the mutation C is to substitute amino acids at positions 205 and 257 with cysteine, and the mutation D is to substitute amino acids at positions 264 and 309 with cysteine.