Immobilization of growing Sphingomonas sp. HXN-200 to gelatin microspheres: Efficient biotransformation of N-Cbz-pyrrolidine and N-Boc-pyrrolidine into hydroxypyrrolidine derivatives

Sphingomonas sp. HXN-200 bacteria were immobilized onto gelatin and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres in an aqueous system for industrial scale biohydroxylation. Scanning electron microscopy and confocal laser scanning microscopy were used to confirm the bacterial immobilization. The gelatin microsphere-immobilized cells successfully transformed N-benzyloxycarbonyl-pyrrolidine and N-tert-butoxycarbonyl-pyrrolidine into R-N-benzyloxycarbonyl-3-hydroxypyrrolidine and R-N-tert-butoxycarbonyl-3-pyrrolidinol at various substrate concentrations showing an improved conversion yield and product efficiency with respect to the freely suspended cells system at each reaction interval time. Additionally, in the immobilized cells system, it showed a faster reaction rate as compared to the freely suspended cells system for reaching the same product concentration. The inhibition effect due to substrate and product concentrations was significantly lower after immobilizing the bacteria onto the microspheres, which is beneficial for continuous reaction. The efficient whole-cell biocatalytic process was feasibly conducted in a 500 mL preparative scale bioreactor and the conversion yield of N-benzyloxycarbonyl-pyrrolidine did not decrease after consecutive repeated use, suggesting that the microsphere-cells immobilization system was stable and reusable.