Spatially Partitioned Engineering of an Alginate Lyase Overcomes the Stability–Activity Trade-Off via Distal Cap Loop Rigidification and Network Remodeling

The stability–activity trade-off limits engineering of alginate lyases for alginate oligosaccharide production. Here, a spatially partitioned semirational strategy coupled with the greedy accumulated strategy for protein engineering (GRAPE) was applied to an alginate lyase from Vibrio sp. The optimized variant M13 achieved a 4.6-fold increase in catalytic efficiency (kcat/Km) and an 11.4-fold extension of half-life at 50 °C. Molecular dynamics simulations indicated that distal Cap Loop rigidification suppresses thermal unfolding while Gate Loop flexibility is retained for catalysis. A67K reversion increased Cap Loop flexibility and shifted the optimal temperature from 40 to 30 °C, supporting its thermostability contribution. Residue interaction network analysis identified a Val272–Tyr260–Arg73 communication pathway associated with active site preorganization into a near-attack conformation. V272A reversion reduced kcat/Km by 45.7%, supporting its catalytic contribution. These findings show that spatial partitioning can concurrently improve thermostability and catalytic efficiency, providing a design principle for polysaccharide lyase engineering.