Inmovilización de la carbonil reductasa en microesferas de polihidroxialcanoato de ingeniería para la síntesis...

An in vivo self-assembly strategy was developed to overcome the limitation of traditional enzyme immobilization methods. An engineered E. coli system was employed to immobilize carbonyl reductase (LcCR) onto intracellular polyhydroxyalkanoate (PHA) microspheres, which was realized by fusion expression of LcCR with covalent PhaC or non-covalent PhaP. The fusion of LcCR and PHA surface binding proteins (Phac, PhaP) was optimized by adjusting the fusion direction (C/N end) and replacing peptide linkers (GSA, (G4S)₃, (G4S)₄). The expression form for fusion of N-terminal of LcCR with three series connected PhaP, LcCR@PHA-3PhaP, showed the highest enzyme load and enzyme activity recovery rate of 36.8 % while maintaining the intact morphology of PHA particles. This immobilized enzyme was used for the catalytic reduction of the carbonyl compound 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO) to the corresponding chiral alcohol, (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), achieving both high enantioselectivity (98.5 % e.e.) and specific operational stability (75.0 % activity retention after eight reuse cycles). These findings establish fundamental design principles for engineered self-assembled biocatalysts while providing a robust and economic platform for industrial biotransformation.