Proline-Modified Phytase Enhancing Thermal Stability
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Solution Overview
Problem
Conventional animal feed enzymes, such as phytase, lack thermostability and protease resistance, limiting their application in feed pelleting and starch liquefaction processes, where high temperatures and enzymatic stability are required, leading to inefficient phosphorus utilization and increased environmental phosphorus burden.
Innovation Solution
Introduction of six prolines at specific sites in the phytase enzyme sequence (S80, S151, T161, N176, S187, and A380) to enhance thermal stability, steam stability, and protease resistance, resulting in improved thermostability and specific activity, enabling effective phytate hydrolysis in animal feed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional phytase is used in animal feed, then phosphorus release from phytic acid is achieved, but the enzyme lacks thermostability and protease resistance required for feed pelleting and starch liquefaction processes
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (S80, S151, T161, N176, S187, A380) in the phytase protein sequence. These positional parameter changes were designed to enhance thermostability while maintaining enzymatic function, directly resolving the contradiction between stability and reliability under processing conditions.
Solution Approach 2:
The patent implements local quality by introducing proline substitutions at six specific local positions within the protein sequence rather than uniform modification throughout. This localized approach targeted regions critical for thermal stability while preserving overall enzyme structure and catalytic activity, enabling the enzyme to withstand feed pelleting temperatures.
2Productivity
If phytase is exposed to high temperature during feed pelleting (70-95°C) and starch liquefaction (75-120°C), then processing efficiency is improved, but the enzyme loses activity due to insufficient thermostability
Solution Approach 1:
The patent applies preliminary action by pre-modifying the phytase protein structure through site-directed mutagenesis before exposure to high-temperature processing conditions. The six proline substitutions were introduced in advance to create a thermally stable enzyme variant that could withstand subsequent feed pelleting and starch liquefaction processes without losing activity.
Solution Approach 2:
The patent implements beforehand cushioning by introducing proline residues at critical positions to create structural buffers that protect the enzyme from thermal denaturation. This preliminary structural reinforcement acts as a cushion against the harsh temperature conditions during processing, maintaining enzyme activity duration throughout the process.
3Stability of the object's composition
If six prolines are introduced at specific sites to enhance thermostability, then thermal stability is improved, but protein structure complexity increases
Solution Approach 1:
The patent resolves this contradiction by making precise parameter changes at six specific positions in the protein sequence, changing only the amino acid type (to proline) rather than adding complex structural elements. This minimal parameter change approach enhances thermal stability while maintaining relatively simple protein structure and facilitating straightforward production through recombinant expression.
Data Source
Figure 1~2
AI summary
The present invention relates to the field of genetic engineering, in particular, the present invention relates to a method for producing a phytase variant with an improved thermal stability, and a phytase variant and the use thereof. The phytase variant contains at least one proline modification, compared to the phytase from Escherichia coli and other mutants thereof. The phytase variants with the modification have preferably improved properties, such as the thermal stability, optimal reaction temperature, pH property, specific activity, protease resistance and performance in animal feeds.