Self-assembling multienzyme systems at oil-water interface for biphasic biotransformations.

摘要

The present work focused on (i) characterization of enzyme assembly morphology, (ii) stabilizing the enzymes at the interface, (iii) broadening the scope of interfacial biocatalysis with multienzyme-cofactor system and developing method to assemble cofactors at the interface, (iv) investigating the kinetic parameters of the interfacial reaction, (v) improving the activity of interfacial enzyme by interfacial mobility enhancement, and (vi) extending the hydrophobic manipulation of enzyme's microenvironment for development of biosensors based on nanofibers containing organic soluble enzymes. Four sets of model reactions system, a single enzyme system and three multiple enzyme system was employed for interfacial biocatalysis study and oxidation of glucose by glucose oxidase was chosen as a model system for biosensor development.;Chloroperoxidase (CPO) was chosen as a model enzyme to explore the factors that determine the stability of interface-assembled enzymes. Although the interface-assembled CPO showed improved stability as compared to native CPO, enzyme deactivation by peroxide reactants like hydrogen peroxide (H2 O2) in the bulk phase, still limited the overall productivity of the enzyme. Two approaches to further improve the stability of interface-assembled CPO were examined in this work. In one approach, several chemical stabilizers were used to prevent highly reactive intermediates from oxidizing the porphyrin ring active site of CPO; polyethylene glycol (PEG) was found exceptional in that it increased both the operational and storage stability of CPO with a productivity increase of 57%, an operational stability improvement by almost 2 folds and a storage stability of 60% activity retention after 24 hours incubation in 1 mM H2O2. While in a second approach, in situ generation of hydrogen peroxide (H2O2) by using glucose oxidase (GOx) to keep H2O2 concentration low was applied. It was found that the combined effect of presence of glucose and lowered concentration of H2O2, extended operational lifetime to 60 minutes for CPO with in situ generation of H2O2 by GOx.;To expand the scope of interfacial enzyme catalysis, multienzyme oxidoreductases-cofactor systems were employed. The structure of cofactors involves unique combination of functional groups that are required by oxidoreductases enzymes to carry out biotransformations and any modification to cofactor for interfacial assembly should not affect the enzyme-cofactor interaction. The challenge of modifying cofactors to assemble at the interface was overcome by structural manipulation of the adenine group of nicotinamide cofactor. The synthesis of interface-assembled cofactor gave a process yield of 67%, the modified cofactor was highly stable with a continuous operation of 2150 hours and turnover number of 2617 for a biphasic reaction involving reduction of acetophenone in organic phase and oxidation of glucose in aqueous phase.;A novel mechanism of nanostirring was developed to improve the two-dimensional mobility of interface-assembled enzymes. Iron oxide (Fe3O 4) superparamagnetic nanoparticles were coupled with polymer conjugated enzymes for interfacial assembling and applied to improve the mobility of the interface-assembled enzyme under external electromagnetic field. The enhanced mobility of the interface-assembled enzymes was quantified through fluorescent microscopic visualization, and enabled over 600% of improvement in the observed reaction rate for both single enzyme and multienzyme systems as compared to reactions in the absence of the magnetic field.;The combination of slow reactions and denaturation of dehydrogenase enzymes due to stirring posed a major constraint for realizing reactions with configuration of both cofactor and enzymes assembled at the interface. This limitation was overcome by development of a unique interfacial biotransformation with interface assembled cofactor and interface-assembled multiple enzymes was realized by employing relatively shear resistant dehydrogenase, ADH RS1, coupled with GluDH for faster NADH turnover. A maximum NADH turnover of 13 was achieved by optimizing the reaction conditions enzyme ratio, organic phase and aqueous phase substrates concentrations, and polymer modifier concentration added during modification of enzymes.;In another effort, the manipulation of microenvironment of enzymes for enhanced hydrophobicity was extended to develop completely organic-soluble enzymes. The organic soluble enzymes were utilized in the development of polymer-enzyme composite nanofibers for biosensing applications. Polyurethane nanofibers of diameters of 100-140 nm containing up to 20% (w/w) protein were prepared via electrospinning. The enzyme, glucose oxidase (GOx), was complexed with an ionic surfactant and was thus transformed into organic soluble prior to electrospinning. When examined for biosensor applications, such prepared nanofibers showed a sensitivity of up to 66 A M-1mg-enzyme -1 (or 0.39 A M-1cm-2), 100 times improvement from previous studies. The high enzyme loading coupled with the high specific surface area of the nanofibers enhanced the reaction kinetics and thus enabled strong responses for small changes in glucose concentration. (Abstract shortened by UMI.)