[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-015] SMART: An in vitro single-molecule display system for engineering D-amino acid oxidase

○Kalhari Munaweera¹, Hannah Halim¹, Maurizio Camagna¹, Hideo Nakano¹, Jasmina Damnjanovic¹ (1. Nagoya university (Japan))

Keywords:

SMART,in vitro enzyme engineering,D-amino acid oxidase,NGS,Bioinformatics

In enzyme engineering, traditional methods for selecting desired enzyme variants are often slow, laborious, or expensive. Aiming to improve the efficiency of the selection process, we developed the SMART system (Single Molecule Assay on Ribonucleic acid by Translated product), enabling fast, simple, and cost-effective cell-free selection of enzymes using a single-molecule display technique coupled with next-generation sequencing and bioinformatics.
Given the importance of D-amino acid oxidase (DAAO) as a biocatalyst for the measurement of D-amino acids, we focused on protein engineering of Schizosaccharomyces pombe DAAO for the specific detection of D-Alanine (D-Ala), a biomarker of diseases such as gastric cancer, and viral infections¹. We first established the SMART system for oxidase selection and validated its efficiency by selecting a small library made by site-saturation mutagenesis at the catalytic residue Y232. The results demonstrated effective enrichment of active enzyme variants². Subsequently, two combinatorial libraries targeting multiple positions of the catalytic pocket and the substrate access tunnel were constructed and subjected to multiple rounds of SMART selection to yield DAAO variants with improved activity and specificity towards D-Ala.