Presentation Information
[P02-199]Development of a Hydrogel Bead-Based Screening System for Selecting Transglutaminase Mutants with Enhanced Specific Activity
○Taisei Koga1, Kensei Orita1, Noriho Kamiya1,2 (1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University (Japan), 2. Division of Biotechnology, Center for Future Chemistry, Kyushu University (Japan))
Keywords:
Microbial transglutaminase,Cell-free protein synthesis,High-throughput screening,Hydrogel beads
[Purpose]
Microbial transglutaminase (MTG) catalyzes the formation of isopeptide bonds through crosslinking between glutamine (Q) and lysine (K) residues, and is widely used in food processing and biomaterial applications. Improving its specific activity is important for enhancing reaction efficiency and reducing enzyme usage. However, conventional screening methods often fail to distinguish intrinsic enzymatic activity from differences in protein expression levels, making it difficult to accurately identify superior mutants 1. To address this limitation, we developed a novel screening system that enables selection of MTG mutants based on specific activity by simultaneously evaluating enzymatic activity and protein expression within individual compartments.
[Method]
MTG fused with either SpyTag (ST) or SpyCatcher (SC) at the C-terminus was encapsulated in water-in-oil (W/O) emulsions together with substrates and fluorescent probes. Hydrogel beads (HBs) were generated by gelation of the inner aqueous phase, allowing compartmentalization of individual enzyme variants. Fluorescently labeled ST or SC probes were used to detect enzyme presence via covalent ST–SC interaction, while separate fluorescent substrates reported MTG-catalyzed crosslinking activity. The resulting HBs were analyzed by fluorescence microscopy and sorted using an On-chip® sort, enabling high-throughput screening.
[Results and Discussion]
HB fluorescence correlated with MTG activity in both ST- and SC-fused constructs, demonstrating that enzymatic activity could be evaluated within individual beads. In addition, fluorescence derived from the ST–SC interaction enabled detection of enzyme presence, allowing discrimination between increased activity due to higher expression and intrinsically improved catalytic efficiency. However, in ST-fused MTG, lysine (K) residues within SpyTag were recognized as substrates by MTG, resulting in the interference of accurate activity measurement. In contrast, SC-fused MTG showed that this format provides a more reliable platform for evaluating specific activity.[Conclusion]We established a hydrogel bead-based screening system that enables simultaneous evaluation of enzymatic activity and expression, allowing selection of MTG mutants based on specific activity. SC-fused MTG avoids self-labeling artifacts and provides a robust and accurate strategy for efficient mutant screening.
[References]
T. Koga et al., ACS Synth. Biol., 14, 995-1001 (2025).
Microbial transglutaminase (MTG) catalyzes the formation of isopeptide bonds through crosslinking between glutamine (Q) and lysine (K) residues, and is widely used in food processing and biomaterial applications. Improving its specific activity is important for enhancing reaction efficiency and reducing enzyme usage. However, conventional screening methods often fail to distinguish intrinsic enzymatic activity from differences in protein expression levels, making it difficult to accurately identify superior mutants 1. To address this limitation, we developed a novel screening system that enables selection of MTG mutants based on specific activity by simultaneously evaluating enzymatic activity and protein expression within individual compartments.
[Method]
MTG fused with either SpyTag (ST) or SpyCatcher (SC) at the C-terminus was encapsulated in water-in-oil (W/O) emulsions together with substrates and fluorescent probes. Hydrogel beads (HBs) were generated by gelation of the inner aqueous phase, allowing compartmentalization of individual enzyme variants. Fluorescently labeled ST or SC probes were used to detect enzyme presence via covalent ST–SC interaction, while separate fluorescent substrates reported MTG-catalyzed crosslinking activity. The resulting HBs were analyzed by fluorescence microscopy and sorted using an On-chip® sort, enabling high-throughput screening.
[Results and Discussion]
HB fluorescence correlated with MTG activity in both ST- and SC-fused constructs, demonstrating that enzymatic activity could be evaluated within individual beads. In addition, fluorescence derived from the ST–SC interaction enabled detection of enzyme presence, allowing discrimination between increased activity due to higher expression and intrinsically improved catalytic efficiency. However, in ST-fused MTG, lysine (K) residues within SpyTag were recognized as substrates by MTG, resulting in the interference of accurate activity measurement. In contrast, SC-fused MTG showed that this format provides a more reliable platform for evaluating specific activity.[Conclusion]We established a hydrogel bead-based screening system that enables simultaneous evaluation of enzymatic activity and expression, allowing selection of MTG mutants based on specific activity. SC-fused MTG avoids self-labeling artifacts and provides a robust and accurate strategy for efficient mutant screening.
[References]
T. Koga et al., ACS Synth. Biol., 14, 995-1001 (2025).
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