A biosensor for all D-amino acids using evolved D-amino acid oxidase

Area of Science:

• Biotechnology
• Analytical Chemistry
• Enzyme Engineering

Background:

• Accurate determination of D-amino acids is crucial in food and biological analyses.
• Existing methods may lack specificity or efficiency for complex mixtures.

Purpose of the Study:

• To develop a robust biosensor for quantifying total D-amino acid content.
• To overcome challenges posed by varying D-amino acid compositions in samples.

Main Methods:

• Utilized flavoenzyme D-amino acid oxidase (DAAO) from Rhodotorula gracilis.
• Employed rational design and directed evolution to engineer DAAO mutants.
• Developed a screen-printed electrode amperometric biosensor.

Main Results:

• Identified specific DAAO mutants (T60A/Q144R/K152E and M213G) with limited response dependence on substrate composition.
• Achieved accurate total D-amino acid determination when D-alanine comprised at least 20% of the mixture (SE 5-9%).
• Established a detection limit of 0.25 mM and a response time of 10-15 minutes.

Conclusions:

• The developed DAAO biosensor is the first to enable determination of entire D-amino acid content using an amperometric approach.
• This biosensor provides an inexpensive, simple, and rapid assay for D-amino acid analysis in diverse biological samples.