Thermostable Phytase Variants via Disulfide Bridges
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Solution Overview
Problem
Current phytases derived from Citrobacter braakii ATCC 51113 lack improved stability and specificity, particularly in thermal and pH conditions, limiting their effectiveness in animal feed applications.
Innovation Solution
Development of phytase variants with additional disulfide bridges, such as those formed between positions 52/99 and 31/177, enhancing thermostability and stability across various conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If additional disulfide bridges are introduced to enhance thermostability, then thermal stability improves, but protein structure complexity increases
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence to introduce additional disulfide bridges at specific positions (52/99 and 31/177). This changes the structural parameters of the protein, creating new covalent bonds that enhance thermostability without requiring a complete redesign of the protein architecture.
Solution Approach 2:
The patent creates a composite protein structure by combining the native disulfide bridge framework with additional engineered disulfide bridges. This results in a hybrid structure that integrates the original protein fold with supplementary stabilizing elements, achieving enhanced thermostability while maintaining functional integrity.
2Stability of the object's composition
If the amino acid sequence is modified to create additional disulfide bridges, then thermostability improves, but manufacturing complexity increases
Solution Approach 1:
The patent modifies manufacturing parameters by introducing specific amino acid substitutions that create additional disulfide bridges. These controlled changes to the amino acid sequence allow for systematic production of stabilized variants through standard protein engineering workflows, balancing improved heat-stability with manageable manufacturing complexity.
3Stability of the object's composition
If disulfide bridges are added to improve pelleting stability, then enzyme stability during processing improves, but protein structure complexity increases
Solution Approach 1:
The patent applies parameter changes by introducing additional disulfide bridges that specifically enhance pelleting stability. The engineered cysteine residues at positions 52, 99, 31, and 177 form new covalent connections that reinforce the protein structure during the mechanical stress of pelleting, achieving improved stability with controlled structural modifications.
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 variants exhibit improved thermal properties, including thermostability, heat-stability, and pelleting stability, leading to enhanced performance in animal feed by effectively degrading phytate.
Implementation Method 1
comprising the establishment of at least two disulfide bridges which are not among the four naturally occurring disulfide bridges as compared to this and closely related phytases
Data Source
Figure 1
AI summary
The present invention relates to a method for producing phytase variants which has at least 74% identity to a phytase derived from Citrobacter braakii and comprises at least two additional disulfide bonds as compared to this phytase. These phytase variants have modified, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensitiliby, and/or an modified glycosylation pattern. The invention also relates to the variants produced, DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.