[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-194] Specific-activity–based directed evolution of laccase using a hydrogel bead–based cell-free screening platform

○Kensei Orita¹, Tomoyuki Ito², Mitsuo Umetsu², Noriho Kamiya^1,3 (1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University (Japan), 2. Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University (Japan), 3. Division of Biotechnology, Center for Future Chemistry, Kyushu University (Japan))

Keywords:

Directed evolution,Hydrogel,Cell free protein synthesis,Laccase

Laccases are a class of multicopper oxidases that catalyze substrate oxidation coupled with the four-electron reduction of molecular oxygen to water. These properties make laccases promising for applications in biomass processing, environmental remediation, and bioelectrocatalysis. However, engineering laccases remains challenging due to difficulties in achieving efficient functional expression and proper cofactor incorporation, particularly in high-throughput screening workflows.Directed evolution provides a general framework for enzyme optimization through iterative cycles of mutagenesis, genotype–phenotype linkage, and screening. In order to establish effective genotype–phenotype linkage, various compartmentalized screening platforms have been developed, including water-in-oil emulsions and hydrogel-based systems. In particular, the combination of compartmentalization with cell-free protein synthesis (CFPS) facilitates expeditious and cell-independent enzyme screening. Previously, we developed a high-throughput screening system that integrates hydrogel beads (HBs) with CFPS. However, conventional screening strategies frequently depend exclusively on activity-based readouts, which can lead to a selection bias toward variants with higher expression levels rather than enhanced intrinsic catalytic efficiency.To address this limitation, a screening platform was developed that enables selection based on enzyme-specific activity by integrating HBs with CFPS. In this system, laccase variants fused with a C-terminal SpyTag (ST) are expressed in emulsion droplets, followed by gelation of the aqueous phase to form HBs that encapsulate and immobilize the enzymes. Subsequent to the expression process, copper ions are introduced to activate laccase. The quantification of enzyme expression levels is achieved through the utilization of the SpyTag/SpyCatcher (SC) system, which employs covalent labeling with muGFP-fused SC. Concurrently, the detection of enzymatic activity is facilitated by employing Cy5-tyramide as a fluorescent substrate. This dual-fluorescence approach facilitates a two-parameter screening strategy, predicated on expression level (muGFP signal) and catalytic activity (Cy5 signal), thereby enabling direct evaluation of specific activity within individual compartments.The implementation of this system in laccase directed evolution led to the successful generation of variants that exhibited enhanced specific activity in comparison to the wild type. These results demonstrate that the proposed method provides an effective and versatile strategy for high-precision screening based on specific activity and has broad potential for applications in enzyme engineering.