[P007] Poster

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

[P04-503] Development of an Enzyme Activity Assessment System for Acquisition of Medium-Scale High-Precision Data

○Seiya Mori¹, Teppei Niide¹, Yoshihiro Toya¹, Shunsuke Kato³, Takashi Hayashi², Hiroshi Shimizu¹ (1. Grad. Sch. IST,UOsaka (Japan), 2. Grad. Sch. Eng.,UOsaka (Japan), 3. EGBRC,Kobe Univ. (Japan))

Keywords:

Substrate Specificity,Enzyme Engineering,Screening

Enzymes have substrate specificity and react only with specific substrates. Artificial engineering of accommodation with non-natural substrates would enable novel reactions to be introduced into microbial metabolic pathways. However, enzymes are composed of hundreds of sequences of twenty different types of amino acids. Identifying variants with the desired functions within this vast sequence space requires a time consuming and laborious work.To address this challenge, we have developed a method for identifying amino acid residues responsible for substrate specificity (S. Mori et al., Protein Sci. 2025). This approach compares conserved residues between two groups of enzymes that share homologous structures. By focusing on residues conserved within each group but differ between groups, the method identifies amino acid residues that are involved in substrate recognition but are not essential for structural stability. Introducing saturation mutagenesis at these key residues is expected to generate enzyme variants with altered substrate specificity toward novel substrates. However, such approach requires an efficient screening system to handle evaluation of large mutant libraries.In this study, we constructed an efficient enzyme activity evaluation system by integrating mutant library construction with the 96-well plate–based assay workflow. In this system, we balance efficiency and labor cost by utilizing a purification system employing Chitin and Strep-tag II (S. Kato et al., Angew. Chem., 2023), the BL21gold(DE3) strain (Agilent Technologies) that supports both expression and cloning, and the OT-2 automated liquid handler (Opentrons Labworks Inc.). This system can evaluate mutant libraries comprising several hundred variants, from library preparation to quantitative measurement of enzyme activity, within four days. Moreover, this system achieves high reproducibility, with a coefficient of variation less than 10%.We applied this system to evaluate a mutant library targeting predicted key residues in lactate dehydrogenase derived from Geobacillus stearothermophilus. As a result, we successfully identified variants exhibiting enhanced activity toward substrate analogs of the native substrate pyruvate, including 2-oxoglutarate and 4-methyl-2-oxovalerate. These findings demonstrate that combining rational identification of key residues and targeted saturation mutagenesis is an effective strategy for engineering enzyme substrate specificity.