Mutated Sortase A Enzyme Enhancing Catalytic Activity
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Solution Overview
Problem
Current sortase enzymes, such as Staphylococcus aureus Sortase A, have limitations in enzymatic activity and substrate specificity, which hinder efficient protein conjugation and ligation reactions, particularly at low substrate concentrations and in preventing reverse reactions.
Innovation Solution
Introducing specific mutations, such as D160S and K196S, into the Staphylococcus aureus Sortase A amino acid sequence enhances enzymatic activity by increasing reaction kinetics and altering substrate preferences, resulting in a sortase with improved catalytic properties and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type Sortase A is used, then the enzyme maintains natural substrate recognition, but enzymatic activity is limited and reaction efficiency is low
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (D160S and K196S) in the Sortase A enzyme sequence. These parameter changes at the molecular level alter the enzyme's catalytic properties, increasing enzymatic activity while preserving the ability to recognize LPXTG substrates. The mutation transforms aspartate to serine at position 160 and lysine to serine at position 196, optimizing the enzyme's catalytic efficiency.
2Quantity of substance
If sortase is used at low substrate concentrations, then reaction cost is reduced, but reaction efficiency decreases
Solution Approach 1:
The mutated Sortase A enzyme with D160S and K196S substitutions exhibits enhanced catalytic efficiency that enables effective protein conjugation reactions even at low substrate concentrations. The parameter changes in the enzyme's amino acid sequence translate to improved kinetic parameters, allowing the enzyme to maintain high productivity under conditions of low substrate availability.
3Ease of manufacture
If sortase reaction is performed, then protein conjugation is achieved, but reverse reactions occur reducing stability
Solution Approach 1:
The patent utilizes parameter changes in the enzyme structure (amino acid mutations) to create a sortase variant that promotes irreversible or highly stable conjugation reactions. The D160S and K196S mutations alter the enzyme's catalytic mechanism or product release kinetics, favoring stable amide bond formation between the substrate and target protein, thereby minimizing reverse reactions and enhancing reaction stability.
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 mutated sortase exhibits a five-fold increase in enzymatic activity compared to wild-type Sortase A, with improved reaction efficiency at low substrate concentrations and enhanced stability, facilitating more effective protein conjugation and ligation reactions.
Implementation Method 1
Sortase A (SrtA) is a membrane bound enzyme which attaches proteins covalently to the bacterial cell wall. The reaction proceeds through a thioester acyl-enzyme intermediate, which is resolved by the attack of an amine nucleophile from the oligoglycine, covalently linking peptidoglycan to a protein substrate and regenerating SrtA.
Implementation Method 2
Herein is reported a sortase that has improved enzymatic activity and methods of using the same in transamidation reactions.
Data Source
AI summary
Reported herein is a sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 and that comprises the mutations D101S and K137S.