Phytase Mutant Thermostability Amino Acid Substitution
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Solution Overview
Problem
Current phytase enzymes, particularly bacterial phytase APPA, have low thermostability, limiting their application in feed processing due to heat stability issues during pelleting, and existing methods for enzyme application are costly and lack uniformity.
Innovation Solution
Development of phytase mutants with improved thermostability through specific amino acid substitutions, such as W46E, Q62W, G70E, and others, encoded by DNA sequences like SEQ ID NO: 4, expressed in host cells like Pichia pastoris, enhancing heat resistance without compromising enzymatic properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If bacterial phytase APPA is used for feed processing, then high specific activity and good gastrointestinal stability are achieved, but heat stability is low with retention rate less than 30% after heating at 70°C for 5 minutes
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (46, 62, 70, 73, 75, 80, 114, 137, 142, 146, 159, 161, 176, 187, 255, 380) in the phytase APPA sequence to improve thermostability. Multiple mutant variants were created with different amino acid substitutions to optimize heat resistance while maintaining enzymatic activity.
2Ease of operation
If liquid spraying technology using phytase is applied, then phytase can be added to feed, but equipment cost is high and stability and uniformity of enzymes in feed are poor
Solution Approach 1:
The patent improves enzyme stability and uniformity in feed by creating thermostable phytase mutants that can withstand feed processing temperatures. This eliminates the need for expensive liquid spraying equipment and allows for more simple and cost-effective application methods while ensuring uniform distribution and stability of the enzyme in the feed.
3Reliability
If inorganic phosphorus is added to feed to ensure animals' phosphorus requirement, then phosphorus availability is improved, but feed costs increase
Solution Approach 1:
The patent enables feed to become self-sufficient in phosphorus utilization by adding phytase that breaks down phytate phosphorus, a naturally occurring compound in plant feeds. This allows animals to access the phosphorus already present in the feed without requiring additional inorganic phosphorus supplements, thereby reducing feed costs while maintaining phosphorus availability.
4Object-generated harmful factors
If phytase is added to improve phosphorus availability, then phosphorus excretion is decreased, but the enzyme must withstand high temperature during feed pelleting
Solution Approach 1:
The patent resolves this contradiction by creating phytase mutants with enhanced thermostability through amino acid substitutions at specific positions. These mutants can withstand the high temperatures of feed pelleting (80-90°C) while maintaining enzymatic activity, thereby reducing phosphorus excretion without being deactivated by heat during processing.
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 phytase mutants exhibit significantly improved heat resistance, maintaining high activity and stability at elevated temperatures, suitable for broader application in animal feed processing, reducing environmental pollution and feed costs.
Implementation Method 1
Phytase is a type of phosphatase enzyme and can hydrolyze phytate phosphorus (myo-inositol hexakisphosphate) into myo-inositol and inorganic phosphate
Data Source
Figure 1
AI summary
Provided are a phytase mutant, a preparation method therefor and a use thereof, a DNA molecule encoding the phytase mutant, a vector comprising the DNA molecule, and a host cell comprising the vector.