Interfacial Activation Of Lipase And Fabrication Of Lipase-immobilized Membrane Bioreactor

The technology of enzyme immobilization efficiently enables enzyme re-use as well as offers better catalytic stability. Lipases are a group of hydrolytic enzymes capable of catalyzing various reactions, therefore having a wide range of industrial applications including enzymatic membrane biorector. Up to now, increasing attempts aimed at improving the economics of lipase-based technology have been implemented through immobilization. As the properties of supports strongly influence the activities of immobilized enzymes, a theoretical understanding of how lipases interact with supports is a necessary prerequisite in order to obtain highly efficient immobilized lipases.In this thesis, we attempted to draw a comprehensive picture on lipase from Candida rugosa (CRL) interfacial activation behavior, and how surface wettability influences CRL immobilization state and activities was throughly researched in order to develop rational design strategies for a high performance CRL immobilization/ separation system. Our works are mainly concentrated on the following aspects:1. CRL was immobilized on self-assembled monolayers (SAMs) with wettabilities varied from high hydrophilicity to high hydrophobicity by adsorption in order to clearly elucidate the activation and deformation character of lipases. The SAMs were made of 11-hydroxyundecane-l-thiol and 1-dodecanethiol. The adsorption behavior was monitored in situ by quartz crystal microbalance with dissipation. Results showed that a hydrophobic support can activate the immobilized CRL and lay the protein evenly and regularly, while CRLs existed in aggregation and underwent deformation on a hydrophilic support, which was unfavorable for their activities.2. Poly(acrylonitrile-co-butyl acrylate), poly(acrylonitrile-co-lauryl acrylate) and poly(acrylonitrile-co-octadecyl acrylate) nanofiber membranes were prepared for CRL adsorption. The influences of hydrophobic chain length and hydrophobic component content on enzyme loading and activity were evaluated. For the same support, increasing the enzyme loading can result in a decrease in the activities of immobilized CRLs, but this decrease can be abated by increasing the hydrophobic component content. Moreover, free CRLs in solution were activated by surfactant Triton X-100 and undergone immobilization process. It was found that an improved surface hydrophobicity of the support can stabilize the activated conformation of CRLs.3. A CRL-immobilized membrane bioreactor was assembled with polysulfone hollow fiber membranes. In this bioreactor, CRLs were immobilized by filtration. The effects of pore size, mass transfer diffusion and operation variables (pressure, substrate concentration and temperature) on the performance of this bioreactor were investigated with the hydrolysis of triacetin. A bioreactor activity as high as 1.07×104 U/g was obtained under optimum conditions.