Immobilization of tryptophan synthase on a magnetic bead-T4 capsid biocarrier for multi-round biocatalysis producing high-value L-tryptophan analogs.

Abstract

Enzyme immobilization is pivotal for industrial biocatalysis yet often suffers from activity loss, structural distortion, operational instability, and high production costs. This study presents a magnetic bead-T4 capsid biocarrier (MBTCB) platform for efficient immobilization of an engineered tryptophan synthase Pf0A9 to enable sustainable synthesis of high-value tryptophan analogs. Soc-fused Pf0A9 was immobilized onto T4 phage capsids via high-affinity Soc-capsid binding under mild physiological conditions, preserving native enzyme conformation, and combining with magnetic bead conjugation for rapid separation. The immobilized form of the enzyme, Pf0A9@MBTCB, retained near-native enzyme activity and efficiently synthesized diverse analogs. The system demonstrated exceptional stability with full activity retention over five consecutive catalytic cycles and 84.5% activity after 9-day storage at 4°C, attributed to the robust T4 capsid scaffold, spatially ordered enzyme array preventing aggregation, and ultra-stable Soc-capsid anchoring. Economically, the process utilized unpurified crude lysate, eliminated centrifugation/filtration steps via magnetic recovery, and enabled reuse of unbound enzymes and capsids. In scaled biocatalysis, Pf0A9@MBTCB produced 443mg of L-6-Cl-tryptophan over three cycles, which was a 2.75-fold yield enhancement versus single-use free enzyme. This platform merges high catalytic efficiency, operational robustness, and cost accessibility, offering a scalable solution for green manufacturing. The modular MBTCB strategy holds broad potential for multi-enzyme cascades and continuous-flow bioreactors, advancing sustainable biocatalysis for chiral chemical synthesis.