Design, Fabrication And Application Of Lipase Nanogel For Chemical Synthesis In Non-aqueous Media

The research into enzyme modification and stabilization is of fundamental importance to expend the application spectrum of enzymatic synthesis, originally accomplished in aqueous phase, to non-aqueous phase where conventional organic synthesis is performed and yields the majority of chemicals today. This dissertation started with an overview of the recent advances in enzymatic catalysis in non-aqueous phase, followed by the design and synthesis of lipase nanogel by aqueous in situ polymerization, and the demonstrative application of lipase nanogel in the synthesis of dextran-based surfactant in anhydrous organic solvent, the non-solvent synthesis of polyester, and the synthesis of biodiesel in oil-water biphasic media. A molecular insight into the fabrication and the application of enzyme nanogel was pursued via a complementary input of structure characterization, molecular simulation, and enzymatic reaction. With these the mechanism underlying the strengthened stability and enhanced catalytic performance at high temperature and in the presence of various organic solvents was elucidated, which serves as molecular fundamental for the design, fabrication and application of lipase nanogel for chemical synthesis in non-aqueous phase.Encapsulation of Candida rugosa lipase into nanogel with average diameter of 25 nm was accomplished via the aqueous two-step in situ polymerization, in which lipase nanogel retained 85% of the hydrolytic activity of its native counterpart. The assembly of monomer around enzyme as the essential step in the fabrication of enzyme nanogel was illustrated by molecular dynamic simulation and evidenced by dynamic light scattering and fluorescence resonance energy transfer spectrum. The multipoint linkage with the hydrophilic polymer network enhanced not only the stability of enzyme at high temperature but also the tolerance of enzyme to those hydrophilic solvents such as dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), methanol, and ethanol.Application of Candida rugosa lipase nanogel in anhydrous DMSO, a universal solvent for organic synthesis, was exemplified by catalyzing the transesterification between dextran and vinyl decanoate, yielding dextran-VD as a biodegradable surfactant. The lipase nanogel behaved as a stable catalyst in anhydrous DMSO at 60°C for 10 day and showed an overwhelming regioselectivity towards the C-2 hydroxyl group in the glucopyranosyl unit of dextran. The dextran-VD micelle effectively accommodated the hydrophobic substance including both small molecules and protein. The delivery of the encapsulated substances was triggered once lipase was applied to hydrolyze ester bonds of dextran-VD micelle. The controlled assembly and dissociation behavior indicated potential application of dextran-VD micelle as a novel drug delivery system.The polycondensation of succinic acid and 1,4-butanediol catalyzed by lipase nanogel at 95 oC under high vaccum (<10 mmHg) yielded biodegradable poly(butylene succinate) with Mn of 1820 g/mol and Mw of 1900 g/mol and melting point of 107.2 oC. The effects of monomer and the molar ratio of reactants, and solvents were examined, respectively. The demonstrative synthesis indicated the potential of lipase nanogel for non-solvent synthesis of polyesters.The lipase (NS81006) nanogel was synthesized and tested for the production of biodiesel by methanolysis of soybean in oil-water biphasic media. Compared to its native counterpart, free lipase NS81006, the lipase nanogel showed an extension of operation time in this process by 2.5-fold at 55 oC and a significantly enhanced tolerance to methanol.