Enhancing Electrochemical Biosensor Selectivity with\nEngineered d‑Amino Acid Oxidase Enzymes for d‑Serine and d‑Alanine Quantification

Abstract

d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of\nselective DAAO-based enzymatic biosensors remains a challenge for\nreal-world biosensor application. Herein, a methodology for developing\nbiosensors with varying substrate selectivity is presented. First,\nwe address DAAO-based biosensor selectivity toward d-serine\nby introducing point mutations into DAAO using rational design. Next,\nthe wild-type yeast DAAO (<i>Rg</i>DAAO WT) and variants\nhuman DAAO W209R and yeast M213G are characterized for their selectivity\nand activity toward d-serine and d-alanine, the\npreferred DAAO substrates. The DAAO enzymes have been immobilized\nfor final biosensor design, where they demonstrate selectivity comparable\nto free DAAO. The cross-linking procedure impacts on DAAO structure\nand function and the use of a regeneration strategy allows the biosensor\nresponse to be improved.