Enzyme stabilization in magnetically-separable mesoporous silica via ship-in-a-bottle approach for uses in nonaqueous solvent

Magnetically-separable mesocellular silica (Mag-MSU-F), with larger mesocellular pores of 38 nm connected by smaller window mesopores of 18 nm and deposited by magnetic nanoparticles, were used for the immobilization and stabilization of subtilisin Carlsberg (SC) from Bacillus licheniformis in the form of nanoscale enzyme reactors (NERs). NERs of SC (NER-SC) were prepared via a simple two-step process: enzyme adsorption and crosslinking. The crosslinked enzymes in large mesocellular pores cannot leach out through smaller window pores, creating a ship-in-a-bottle approach as one of the stabilization mechanisms. The other stabilization mechanism would be the multi-point covalent linkages that can effectively prevent the denaturation of enzymes. As a result, NER-SC maintained 66% of initial activity even after incubation under shaking (200 rpm) for 15 days while free and adsorbed SC showed less than 1% and 2.8% of initial activities, respectively. Stable NER-SC was successfully used for the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with n-propanol in isooctane. Magnetic separation of Mag-MSU-F facilitated the repeated uses of stable NER-SC. This is the first demonstration for the uses of stable and magnetically-separable NERs in organic solvents, which will create a great potential for various synthetic reactions including the biodiesel production.