Dual enzyme self-assembly cluster for high efficient asymmetric reduction of ethyl 6-oxo-8-chlorooctanoate

Abstract

(R)-ECHO is synthesized through the asymmetric reduction of ethyl 6-oxo-8-chlorooctanoate (ECOO) using carbonyl reductase(CR2) as a catalyst and co-enzyme NADPH as a hydrogen donor with addition of glucose dehydrogenase (GDH) for NADPH regeneration, thereby enhancing biotransformation efficiency. To improve the reaction efficiency further, a dual enzyme self-assembly cluster EutM-SC-ST-CR2(GDH) was developed specifically for this purpose. This co-immobilised system demonstrated a 50% increase in the conversion of ECOO compared to when SpyTag-CR2(ST-CR2) and SpyTag-GDH(ST-GDH) were co-catalysts. The optimal conditions for this increase were observed to be at a pH of 8.0, 30°C, when concentrations of 250 mM ECOO, 270 mM glucose and 0.1 mM NADPH, and a molar ratio of ST-CR2 to ST-GDH to EutM-SpyCatcher(EutM-SC) of 1:1:2 (molecular concentrations of ST-CR2, ST-GDH and EutM-SC were 3.5 mM, 3.5 mM and 7 mM). Under these conditions, the conversion of ECOO reached up to 95% and the enantiomeric excess (e.e.) of (R)-ECHO achieved 99.9%. Moreover, the EutM-SC-ST-CR2(GDH) construct displayed enhanced enzyme activity, stability, and kinetics, as well as improvements in electrostatic and ionic strengths. In conclusion, this study presents the development of a novel self-assembled protein scaffold, EutM-SC, for the co-immobilisation of ST-CR2 and ST-GDH, thus optimizing the biological reaction conditions and offering potential for the industrial production of (R)-α-lipoic acid.