Amino Acid Quantification via AARS Enzyme Recycling
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Solution Overview
Problem
Current methods for quantifying amino acids, particularly L-form and D-form, are inefficient, requiring long analysis times and being unsuitable for large-scale measurements due to low selectivity and sensitivity, and are prone to interference from external factors like blood contaminants.
Innovation Solution
A method involving the use of aminoacyl tRNA synthetase (AARS) where the enzyme and amino acids are released from an aminoacyl AMP-AARS complex and reused to form a new complex, allowing for the production of reaction products such as pyrophosphoric acid in quantities greater than the initial amino acid amount, enabling faster and more sensitive quantification across a wider concentration range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If liquid chromatography with color reaction or fluorescent derivatization is used for amino acid quantification, then measurement precision is improved, but analysis time increases significantly (about two hours per specimen)
Solution Approach 1:
The invention extracts and utilizes the specific enzymatic reaction between AARS and amino acids to produce pyrophosphoric acid, separating this selective reaction from the complex chromatographic separation process. By measuring pyrophosphoric acid production directly, the method eliminates the time-consuming chromatography steps while maintaining quantification accuracy through enzyme-specific reactions.
Solution Approach 2:
The invention replaces the mechanical chromatographic separation system with a biochemical enzymatic reaction system. Instead of physically separating and detecting amino acids through chromatography, the method uses AARS enzymes to catalyze specific reactions that produce measurable pyrophosphoric acid, substituting mechanical separation with biochemical transformation and direct measurement.
2Productivity
If enzyme-based amino acid measuring methods are used, then analysis time is reduced, but selectivity decreases because enzymes react with multiple amino acids and no enzyme corresponds to all 20 amino acids
Solution Approach 1:
The invention utilizes the property that AARS enzymes can act on both L-form and D-form amino acids, making the enzymatic reaction system universally applicable to detect both optical isomers. This multi-functionality allows a single enzyme system to provide selective detection across different amino acid forms that were previously requiring separate specialized methods.
Solution Approach 2:
The invention introduces pyrophosphoric acid as an intermediary substance that mediates the detection process. The AARS enzyme catalyzes the reaction between amino acids and ATP to produce pyrophosphoric acid, which then serves as a universal signal that can be measured to quantify the original amino acid content, bridging the enzyme reaction and the final measurement.
3Measurement precision
If AARS reaction is used to produce pyrophosphoric acid for amino acid quantification, then sensitivity is improved, but the concentration range is limited to high concentrations (300-900 μM)
Solution Approach 1:
The invention makes the reaction system dynamic by enabling the AARS enzyme to catalyze multiple reaction cycles. The enzyme reacts with amino acids to produce pyrophosphoric acid, and this catalytic process can be repeated and amplified, allowing the system to adapt to different amino acid concentrations by adjusting the number and rate of reaction cycles rather than being limited to a fixed concentration range.
Solution Approach 2:
The invention maintains continuous useful action through the catalytic nature of the AARS enzyme reaction. The enzyme continuously catalyzes the conversion of amino acids to pyrophosphoric acid, and this continuous catalytic process allows for signal amplification that extends the detectable concentration range from high concentrations down to very low concentrations, maintaining sensitivity across a broad range.
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
This approach allows for the selective and efficient quantification of amino acids in a short time with high sensitivity and in a wide concentration range, from very low to high concentrations, and can differentiate between L-form and D-form amino acids, reducing the impact of external factors.
Implementation Method 1
an aminoacyl tRNA synthetase (AARS) corresponding to the amino acids and adenosine triphosphate (ATP) are reacted in the presence of a divalent ion or a polyamine to form a complex comprising an aminoacyl adenylate (aminoacyl AMP) and the AARS
Implementation Method 2
L-form and/or D-form amino acids (L-AA and/or D-AA) in the sample, an aminoacyl tRNA synthetase (AARS) corresponding to the amino acids and adenosine triphosphate (ATP) are reacted to form a complex comprising an aminoacyl adenylate (aminoacyl AMP) and the AARS
Data Source
AI summary
A method for selectively and easily quantifying the L-form and/or D-form amino acids to be measured using an aminoacyl tRNA synthetase (AARS) with high sensitivity, and an amino acid quantification kit. A method for quantifying amino acids (L-AA and/or D-AA) in a sample using an AARS, wherein the amino acids and the AARS are released from an aminoacyl AMP-AARS complex once formed, and they are used again for forming the aminoacyl AMP-AARS complex, so that reaction products such as pyrophosphoric acid to be measured can be ultimately produced up to a molar number larger than that of the amino acids contained in the sample, and an amino acid quantification kit for performing the method.