Carrier-free immobilized enzymatic reactor based on CipA-fused carbonyl reductase for efficient synthesis of chiral alcohol with cofactor self-sufficiency.
Int J Biol Macromol
; : 133873, 2024 Jul 14.
Article
em En
| MEDLINE
| ID: mdl-39013505
ABSTRACT
In this study, based on the self-assembly strategy, we fused CipA with carbonyl reductase LXCARS154Y derived from Leifsonia xyli by gene coding, and successfully performed the carrier-free immobilization of LXCARS154Y. The immobilized enzyme was then characterized using scanning electron microscope (SEM), dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). Compared with the free enzyme, the immobilized LXCARS154Y exhibited a 2.3-fold improvement in the catalytic efficiency kcat/km for the synthesis of a chiral pharmaceutical intermediate (R)-3,5-bis(trifluoromethyl)phenyl ethanol ((R)-BTPE) by reducing 3,5-bis(trifluoromethyl)acetophenone (BTAP). Moreover, the immobilized enzyme showed the enhanced stability while maintaining over 61â¯% relative activity after 18â¯cycles of batch reaction. Further, when CipA-fused carbonyl reductase was employed for (R)-BTPE production in a continuous flow reaction, almost complete yield (97.0â¯%) was achieved within 7â¯h at 2â¯M (512.3â¯g/L) of BTAP concentration, with a space-time yield of 1717.1â¯g·L-1·d-1. Notably, we observed the retention of cofactor NADH by CipA-based enzyme aggregates, resulting in a higher total turnover number (TTN) of 4815 to facilitate this bioreductive process. This research developed a concise strategy for efficient preparation of chiral intermediate with cofactor self-sufficiency via continuous flow biocatalysis, and the relevant mechanism was also explored.
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01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Int J Biol Macromol
Ano de publicação:
2024
Tipo de documento:
Article