Synthesis of chiral alcohol (S)-CHBE by co-immobilization of double enzymes based on organic-inorganic hybrid nanoflower.

The chiral alcohols (S)-4-chloro-3-hydroxy-butyric acid ethyl ester ((S)-CHBE) is a critical intermediate in the synthesis of various active pharmaceutical ingredients. This study presents the first investigation of the efficient production of (S)-CHBE using organic-inorganic hybrid nanoflowers (GDH-CR@HNFs) for the co-immobilization of glucose dehydrogenase (BsGDH) and carbonyl reductase (BsCR). By optimizing immobilization conditions, we significantly enhanced the catalytic activity and immobilization efficiency of the hybrid nanoflowers. The GDH-CR@HNFs exhibited superior catalytic performance compared to the free dual enzyme system, demonstrating a higher affinity for the substrate COBE (47-fold lower K value), increased maximum reaction rate (V), and improved catalytic efficiency (K/K). Additionally, the GDH-CR@HNFs displayed enhanced temperature adaptability, pH stability, and storage stability. The GDH-CR@HNFs retained over 60 % of their initial catalytic activity after 8 cycles of reuse. The hydrophobic nature of the substrate COBE can lead to substrate inhibition of the free enzyme. However, GDH-CR@HNFs exhibited excellent substrate tolerance, maintaining a high conversion rate (65 %) even at a substrate concentration of 200 mM, significantly outperforming the free enzyme system (13.8 % conversion rate). The hybrid nanoflower co-immobilization strategy offers a novel approach to addressing substrate and product inhibition issues in enzyme-catalyzed reactions, paving the way for the industrial production scale of (S)-CHBE.