세포 투과성 물질의 개발과 재조합 리파제의고정화에 관한 연구

Abstract

This thesis deals with preparation of cellular delivery system for protein, and biofuctionalization of nanoparticles and metal-organic frameworks for immobilization of recombinant lipase. In protein delivery, use of therapeutic proteins has been limited by their inability by pass the plasma membrane. To protein delivery into the cell, the carriers are needed. Liposome, cell permeable peptides, and polymer have been used. Recently, delivery carriers synthesis and applications have gained more attention due to increase of the demand for systems of drug delivery or stabilizer. Enzymes are very useful biocatalyst, and they can catalyze various and have highly enantio- and regioselectivitys and accept a wide range of substrate. However, enzymes are often easily inactivated. There are several stabilization strategies to improve catalytic stability of enzymes, for example, enzyme modification, genetic modification, and enzyme immobilization strategies. Among these methods, immobilization strategy using solid supports has been most extensively researched due to the specific advantages such as reduce costs by enabling the efficient separation and reuse.

First, the protein delivery system studies were investigated based on the delivery carriers such as nanoparticles and several known cell permeable peptides (CPPs). I prepared glucose-coated polymeric nanobeads by dispersion polymerization. The EGFP (enhanced green fluorescent protein) as a model protein for protein delivery was attached to the beads, and translocated into mouse embryonic stem cells and HeLa cells. The glucose-coated beads delivered the EGFP inside the cells whereas non-glucose-coated beads or the EGFP alone did not. In addition, I examined the transfection efficiency of several known CPPs into mouse embryonic stem cells under various conditions. The transfection of CPPs was independent on its concentration.

Second, immobilization of recombinant lipase were investigated. I achieved increase of the expression yield of CAL-B (lipase B from Candida antarctica) by using a codon-optimized synthetic gene and mutagenesis to introduce five aspartates on the surface of CAL-B. The 5D-CAL-B mutant showed three-fold higher expression yield (3.3 mg/L of culture) compared to the wild type. And I employed magnetic beads in refolding B. cepacia lipase. The chaperone-conjugated magnetic polystyrene beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after ten cycles of refolding B. cepacia lipase. In addition, biofuntionalization of nanoparticles and metal-organic frameworks was studied. The eight commercial enzymes were immobilized to the magnetic polymethylmethacrylate (PMMA) beads by carbodiimide activation. The lipase-conjugated magnetic beads were showed similar hydrolytic activity to the free enzyme. The recycling experiment showed that the lipase-conjugated magnetic beads can be used several times with maintaining the same activity. In addition, the Enhance green fluorescent protein (EGFP) and Candida antarctica lipase B (CAL-B) were covalently bound to the metal-organic frameworks (MOFs). The immobilized enzyme on MOFs has the higher specific activity compared to the free enzyme in organic solvent. It can be concluded that the functional proteins can be decorated on MOFs without losing their functions and they are recyclable without significant loss of the activity.