Mutant Protein Deamidase Stability via Amino Acid Substitution
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Solution Overview
Problem
Current protein deamidases require extensive labor to modify properties and have low stability to oxygen, leading to increased costs and inefficiencies in industrial applications.
Innovation Solution
A mutant enzyme with specific amino acid substitutions, such as Tyr82Ser or Val84Asp, is developed based on X-ray crystal structure analysis, improving substrate specificity and hydrogen peroxide stability, allowing for reduced enzyme usage and expanded applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If protein deamidase is used in industrial applications, then enzyme activity is achieved, but stability to oxygen is low requiring stabilizing agents
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at positions 82 and 84 in the protein deamidase sequence. Specifically, Tyr82 is changed to Ser or Thr, and Val84 is changed to Asp or Glu. These parameter changes at the molecular level directly improve the enzyme's stability to oxygen and hydrogen peroxide, eliminating the need for external stabilizing agents while maintaining enzyme activity.
2Adaptability or versatility
If mutant enzyme is prepared to modify properties, then substrate specificity is improved, but labor required is large
Solution Approach 1:
The patent employs preliminary action by using X-ray crystal structure analysis to identify and specify the exact amino acid positions (82 and 84) that control substrate specificity and stability before mutation. This preliminary structural characterization allows for rational design of mutants with desired properties, significantly reducing the time and labor required for screening and evaluation compared to random mutagenesis approaches.
3Productivity
If enzyme concentration is increased to improve yield, then productivity increases, but cost increases
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's catalytic efficiency through amino acid substitution at positions 82 and 84. These changes enhance the enzyme's substrate specificity and catalytic activity, allowing for reduced enzyme concentration to achieve the same productivity level, thereby lowering costs while maintaining or improving yield.
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 mutant enzyme enhances enzyme efficiency, reduces reaction time, and improves preservation stability, enabling novel applications and cost savings by minimizing the need for stabilizing agents.
Implementation Method 1
A protein deamidase is an enzyme that hydrolyzes amide groups of glutamine and asparagine in a protein to convert to glutamic acid and asparaginic acid
Implementation Method 2
by fully using a technique of an X-ray crystal structure analysis. That is, the inventors succeeded in crystallization of the mature form and the pro-enzyme for the protein-glutaminase and also obtaining their conformational information
Data Source
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AI summary
An object is to provide a novel method of improving an enzyme capable of deamidating a protein. A mutant enzyme is designed by the following steps: (1) specifying one or more amino acids selected from the following group, namely, consisting of an amino acid corresponding to the amino acid at position 35, an amino acid corresponding to the amino acid at position 38, an amino acid corresponding to the amino acid at position 39, an amino acid corresponding to the amino acid at position 40, an amino acid corresponding to the amino acid at position 41, an amino acid corresponding to the amino acid at position 42, an amino acid corresponding to the amino acid at position 43, an amino acid corresponding to the amino acid at position 45, an amino acid corresponding to the amino acid at position 46, an amino acid corresponding to the amino acid at position 49, an amino acid corresponding to the amino acid at position 79, an amino acid corresponding to the amino acid at position 80, an amino acid corresponding to the amino acid at position 81, an amino acid corresponding to the amino acid at position 82, an amino acid corresponding to the amino acid at position 83, an amino acid corresponding to the amino acid at position 84, an amino acid corresponding to the amino acid at position 103, an amino acid corresponding to the amino acid at position 104, an amino acid corresponding to the amino acid at position 105, an amino acid corresponding to the amino acid at position 106, an amino acid corresponding to the amino acid at position 117, an amino acid corresponding to the amino acid at position 142, an amino acid corresponding to the amino acid at position 143, an amino acid corresponding to the amino acid at position 146, an amino acid corresponding to the amino acid at position 166, and an amino acid corresponding to the amino acid at position 185 in an amino acid sequence set forth in SEQ ID NO: 2, in a protein deamidase (an enzyme to be mutated); and (2) constructing an amino acid sequence having substitution of the amino acid(s) specified in the step (1) by another amino acid(s) or having deletion of the amino acid(s) specified in the step (1) using the amino acid sequence for an enzyme to be mutated as a base sequence.