Phytase Variants Enhancing Thermostability and Activity
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Solution Overview
Problem
Current phytases derived from Citrobacter braakii ATCC 51113 lack improved properties such as thermostability, pH profile, specific activity, performance in animal feed, protease-sensibility, and glycosylation pattern, which are essential for enhanced functionality in animal feed applications.
Innovation Solution
Development of phytase variants with at least 74% identity to the Citrobacter braakii ATCC 51113 phytase, incorporating specific alterations at defined positions to enhance thermostability, pH profile, specific activity, and reduced protease-sensibility, while modifying glycosylation patterns for improved stability when expressed in fungi.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wildtype Citrobacter braakii ATCC 51113 phytase is used, then the enzyme maintains natural sequence integrity, but it lacks improved thermostability, pH profile, specific activity, and protease-sensibility
Solution Approach 1:
The patent applies parameter changes by systematically modifying specific amino acid positions (1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411) in the phytase sequence to improve thermostability, pH profile, specific activity, and reduce protease-sensibility while maintaining at least 74% identity to the wildtype sequence
Solution Approach 2:
The patent applies local quality by making targeted local modifications at specific amino acid positions rather than globally altering the entire sequence. This allows improvement of specific properties (thermostability, pH profile, specific activity, protease-sensibility) through localized changes while preserving the overall structure and function of the enzyme
2Productivity
If phytase variants with multiple alterations are created to improve functionality, then thermostability and specific activity are enhanced, but the risk of reducing enzyme stability through unintended alterations increases
Solution Approach 1:
The patent applies preliminary action by conducting in silico modeling and simulation studies before actual variant creation and testing. This allows prediction and evaluation of potential stability impacts of multiple alterations, enabling selection of variants that improve productivity while maintaining or enhancing stability
Solution Approach 2:
The patent applies feedback through systematic evaluation of variant performance in terms of thermostability, pH profile, specific activity, and protease-sensibility. This feedback loop allows iterative optimization where results from one round of variant testing inform subsequent rounds, ensuring that improvements in specific activity do not compromise overall enzyme stability
3Reliability
If phytase is expressed in fungi to improve glycosylation pattern, then stability and performance in animal feed are enhanced, but the complexity of production systems increases
Solution Approach 1:
The patent applies intermediary by using fungi as an intermediate expression system to produce the phytase variant. The fungal expression system serves as a mediator that provides eukaryotic post-translational modifications (glycosylation) to improve enzyme stability and performance in animal feed, while the patent simplifies the overall production approach by selecting fungal hosts that are well-established and易于操作
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 phytase variants exhibit improved thermostability, enhanced performance in animal feed, and reduced protease-sensibility, leading to increased efficiency and stability in animal feed applications.
Implementation Method 1
The phytase variants exhibit improved thermostability, enhanced performance in animal feed, and reduced protease-sensibility
Data Source
AI summary
The present invention relates to a phytase which has at least 74% identity to a phytase derived from Citrobacter braakii and comprises at least one alteration as compared to this phytase. These phytase variants have amended, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensibility, and/or an amended glycosylation pattern. The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g., in animal feed and animal feed additives.