Modified E. coli Phytase for Enhanced Secretion
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Solution Overview
Problem
Current methods for producing phytase, an enzyme that breaks down phytic acid, are inefficient, leading to inadequate bioavailability of phosphate and essential ions in food and feed, resulting in nutritional deficiencies and environmental phosphate pollution.
Innovation Solution
A recombinant DNA molecule encoding a modified E. coli phytase with increased activity and secretion efficiency is expressed in host cells, specifically mutating amino acids in regions 189-211 and 137-152, enhancing enzyme activity in the culture supernatant and improving phosphate availability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type E. coli phytase is used, then the enzyme maintains essential properties for phosphate availability, but the activity in culture supernatant and secretion efficiency are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid positions (189-211 and 137-152) in the phytase protein sequence. These targeted substitutions alter the enzyme's properties to enhance secretion efficiency and activity in culture supernatant while preserving the essential catalytic function for phosphate release from phytate.
Solution Approach 2:
The invention applies local quality by making specific localized modifications to the phytase molecule at defined positions rather than global changes. The amino acid substitutions are confined to specific regions (positions 189-211 and 137-152), allowing the enzyme to maintain its overall structure and essential properties while improving local secretion characteristics.
2Quantity of substance
If phytic acid is present in food and feed, then phosphate storage is achieved, but phosphate and metal ion bioavailability are reduced
Solution Approach 1:
The modified phytase enzyme acts as an intermediary that bridges the contradiction between phosphate storage and bioavailability. By secreting higher levels of active phytase into the culture supernatant, the enzyme mediates the breakdown of phytate complexes, releasing bound phosphate and metal ions into bioavailable forms without compromising the storage function of phytic acid in the original seed material.
3Ease of operation
If exogenous phytase is added to food and feed, then phosphate bioavailability is improved, but production costs increase
Solution Approach 1:
The patent reduces production costs through parameter changes that enhance the enzyme's secretion efficiency. By modifying amino acid positions to improve secretion into the culture supernatant, the patent achieves higher yields of active phytase per unit of biomass, thereby reducing the overall cost of enzyme production and making exogenous phytase addition more economically viable.
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 achieves significantly higher activity and secretion efficiency compared to wild-type, effectively increasing phosphate bioavailability and reducing environmental phosphate pollution.
Implementation Method 1
Phytic acid or phytates can be degraded by phytases. Phytic acid containing plant seed contain endogenous phytase enzymes. Upon their intake the phytates in food and feed are theoretically hydrolysable by the endogenous plant phytases
Implementation Method 2
Apart from this phytic acid also binds proteins by electrostatic interaction to a certain extent
Data Source
AI summary
The present invention features a recombinant DNA molecule which, upon expression in a prokaryotic or eukaryotic host cell, encodes a polypeptide having phytase activity. In preferred embodiments, the recombinant DNA molecule comprises a DNA sequence selected from DNA sequences which have been obtained by variations of the mature wild-type E. coli phytase sequence, wherein at least one amino acid at position 200 or position 207 is mutated as compared to the wild-type sequence, where the recombinant DNA molecule is, upon expression in a suitable host cell, associated with an increased activity of the thus encoded protein in the culture supernatant.