Modified Phytase Enzyme Thermostability Mutations
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Solution Overview
Problem
Current microbial phytases used in food and animal feed industries face challenges such as high production costs, poor stability at elevated temperatures, and insufficient activity in acidic environments, limiting their industrial application and bioavailability of phosphate in animal nutrition.
Innovation Solution
A recombinant DNA molecule encoding a polypeptide with enhanced thermostability and proteolytic stability is developed by modifying the E. coli phytase sequence with specific mutations and N- or C-terminal extensions from Aspergillus niger acidic phosphatase, maintaining the enzyme's essential characteristics and improving its performance at higher temperatures and in acidic conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current microbial phytases are used in animal feed, then phosphate can be released from phytic acid, but the enzymes show poor stability at elevated temperatures during pelleting processes
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid positions in the phytase protein sequence. Mutations at positions 74, 139, 142, 145, 198, and 200 were introduced to alter the enzyme's thermal stability parameters while maintaining its catalytic function. This allows the enzyme to withstand elevated temperatures during pelleting processes.
Solution Approach 2:
The patent creates a composite enzyme structure by combining the E. coli phytase backbone with stabilizing elements from Aspergillus niger acidic phosphatase. This composite approach integrates functional domains from different sources to achieve both catalytic activity and enhanced thermostability.
2Reliability
If exogenous phytases are added to animal feed to improve phosphate bioavailability, then phosphate release from phytate is enhanced, but production costs increase
Solution Approach 1:
The patent modifies the enzyme's stability parameters through site-directed mutagenesis, creating variants with improved durability. This reduces the amount of enzyme needed per unit of feed, thereby lowering overall production costs while maintaining effective phosphate release.
Solution Approach 2:
The patent introduces multiple amino acid mutations (positions 74, 139, 142, 145, 198, 200) that collectively provide sufficient stability enhancement. This partial modification approach achieves the desired performance improvement without requiring complete redesign of the enzyme, reducing development and production costs.
3Quantity of substance
If phytic acid is present in animal feed, then phosphate storage in plant seeds is maintained, but phosphate and metal ions become unavailable for absorption
Solution Approach 1:
The modified phytase enzyme acts as an intermediary that bridges the contradiction between phosphate storage and availability. It selectively hydrolyzes phytate to release phosphate and metal ions in the animal's gastrointestinal tract, making them bioavailable without compromising the phosphate content in the feed原料.
Solution Approach 2:
The enzyme extracts phosphate and metal ions from the phytate complex through hydrolysis. This extraction process separates the bound nutrients from the phytic acid structure, making them available for absorption while leaving the carbon backbone of inositol for further metabolism.
4Object-generated harmful factors
If phytic acid is not metabolized in the gastrointestinal tract, then phosphate pollution of the environment is reduced, but nutritional value of the diet is reduced
Solution Approach 1:
The phytase enzyme serves as a mediator that enables controlled metabolism of phytic acid in the animal's digestive system. This localized hydrolysis releases phosphate for nutritional use while preventing its excretion, thus maintaining nutritional value without increasing environmental pollution.
Solution Approach 2:
The modified phytase enables the animal's own digestive system to process phytic acid, making the phosphate and metal ions bioavailable. This self-service approach allows the animal to utilize the phosphate stored in plant seeds, improving nutritional value without requiring external phosphate supplementation that would lead to pollution.
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 exhibits increased residual activity at high temperatures and improved stability against proteolytic decomposition, making it suitable for industrial applications, including pelleting processes and animal feed, where it enhances phosphate bioavailability and reduces environmental phosphate pollution.
Implementation Method 1
Phytases catalyse the hydrolysis of phytate to myoinositol and/or mono-, di-, tri-, tetra- and/or pentaphosphate as well as inorganic phosphate
Implementation Method 2
the DNA sequence has been obtained by variation of the mature wild-type E. coli phytase sequence with defined amino acid positions being modified in comparison to the wild-type sequence... increased temperature stability
Implementation Method 3
increased proteolytic stability of the enzyme activity... improved stability against proteolytic decomposition
Data Source
AI summary
The invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity. The DNA sequence has been obtained by variation of the mature wild-type E. coli phytase sequence with defined amino acid positions being modified in comparison to the wild-type sequence or with the sequences having N- and/or C-terminal extensions, respectively. The invention further relates to a method for expressing the recombinant phytase as well as its use in the food and animal feed technologies.