Immobilization and stabilization of subtilisin Carlsberg in magnetically-separable mesoporous silica for transesterification in an organic solvent

Abstract

Subtilisin Carlsberg (SC) was immobilized and stabilized on magnetically-separable mesoporous silica (Mag-MSU-F) in the form of nanoscale enzyme reactors (NERs) based on the ship-in-a-bottle mechanism. Stabilized NERs of SC (NER-SC) were freeze-dried and successfully used for the transesterification of N-acetyl-L-phenylalanine ethyl ester with n-propanol in isooctane. Magnetic separation of Mag-MSU-F facilitated the repeated usages of stable NER-SC. This is the first demonstration for the use of stable and magnetically-separable NERs in an organic solvent, which has the potential for environmentally-friendly synthesis using enzymes in organic solvents.Enzymes are environmentally-friendly biocatalysts that can be used for chemical transformation processes including the production of biochemicals, bioalcohols and biodiesels. [1][2][3][4][5] Enzymes offer chemo-, regio-and stereo-selectivities under mild reaction conditions, such as ambient temperature and pressure, and physiological pH. [6][7][8] In particular, the change of medium from water to organic solvents enables synthetic reactions such as esterification, transesterification, aminolysis and thiotransesterification that are suppressed in aqueous solution. 9 As a result, the organic solvent enzymology has provided numerous synthetic and processing advantages. 10 Despite these advantages, one of the potential problems is the structural denaturation of native enzymes in organic solvents, leading to the poor enzyme stability and lowered enzyme activity. 11 As a potential solution, the stabilization and immobilization of enzymes has been proposed for successful nonaqueous enzymology with enhanced activity by preserving its native conformation. 12,13 † Electronic supplementary information (ESI) available. See

Molecular mechanism of carbon nanotube to activate Subtilisin Carlsberg in polar and non-polar organic media

“…The catalytic triad residues exhibit much lower RMSD (vide Supplementary Table S1) and RMSF values (see Fig. 4) than most residues of the SC in all systems, indicating their relative rigidities to the solvent environments and the carrier materials, which should contribute to the fact that serine protease could maintain catalytic activity in organic solvents102122.…”

“…The first one is SC immobilized to one single walled carbon nanotube in water, labelled as CNT-wat. Similar to experimental operations91030, in which the enzymes were first immobilized on carbon nanotubes in aqueous solution and then transferred to the organic solvents, the initial conformations of SC adsorbed onto CNT in the organic solvents were extracted from the final snapshot of the first 100-ns MD trajectories of CNT-wat system. Then, we transformed its solvent environment into acetonitrile (labeled as CNT-acn) and heptane (labeled as CNT-hep) media.…”

“…Subtilisin Carlsberg and α-chymotrypsin adsorbed onto silica chromatography gel support gave 1000-fold greater catalytic activities in acetonitrile media than freeze-dried powders9. The immobilized subtilisin Carlsberg with magnetically-separable mesoporous silica was successfully recycled for iterative synthetic model reactions in isooctane10.…”

“…As a result, understanding of immobilization mechanism in the organic media has been lagged and is highly desired. Subtilisin Carlsberg is a member of serine protease family and converts a large number of substrates to perform hydrolytic and transesterification reactions1021. Also, it has been widely used for syntheses of peptides and amino acid esters in various organic solvents2122.…”

Stabilized glycerol dehydrogenase for the conversion of glycerol to dihydroxyacetone

“…In this process, chemical linkages formed upon enzyme crosslinking effectively inhibit enzyme denaturation, while enzyme leaching is prevented via ship-in-abottle mechanism where crosslinked enzymes in larger mesocellular pores cannot leach through smaller bottle-neck mesopores [11,12]. In addition, magnetically-separable mesoporous silica with a bottle-neck pore structure has exhibited potential for use in a stabilized enzyme system with the option of facile magnetic separation of immobilized enzymes via the NER approach [11,13].…”