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

Abstract

Living cells are highly organized with many functional units or organelles separated by membranes. The membrane is comprised of specific proteins and lipid components that enable it to perform its unique roles for that cell or organelle. At cellular membranes, lipid bilayers are stabilized laterally with the help of integral proteins. This stability is provided through a clustering of the hydrophobic core of both the lipid bilayer and the integral protein. Surface and interfacial phenomenon involving the activities of enzymes are wide spread in cellular systems and occur within the interfacial constraints of substrate accessibility, distribution and partitioning. Similar mechanisms can be used to enhance productivity of industrial biotransformations at oilwater interface. Detailed study and manipulation of interfacial enzyme catalysis is of great interest for biotechnology, chemical technology, biology, and offers new opportunities in protein and polymer chemistry, separation science, bio-renewable products, environmental science and waste minimization. Herein, novel self-assembling enzyme systems were developed by manipulation of microenvironment of the enzymes for interfacial biotransformations at oil-water interface. The enzyme molecules were modified to self-assemble at oil-water interface by conjugation with hydrophobic moieties like polymers. 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 iii 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. In a previous study, it was demonstrated that interface-assembled enzymes improved the reaction rate by two orders of magnitude. As a part of the present work, the important role of mobility and the assembly morphology of the interface-assembled enzyme on regulating the enzymatic liquid membrane fluidity at the interface were investigated. To characterize the surface assembly morphology of the interfaceassembled enzyme by surface pressure analysis, Langmuir film balance was used. The resulting surface pressure isotherm exhibited monolayer assembly, with intramolecular rearrangement of the interface-assembled enzymes. The mobility of the novel interfaceassembled enzymes was evaluated by using fluorescence recovery after photobleaching technique that gave the diffusion coefficient of 6.7×10-10 cm2/sec, three orders of magnitude less than that of native enzymes in aqueous solution, due to localization of the modified enzyme at the interface. Though modification of enzymes with polymer for interfacial assembly reduced its mobility, the conjugation of polymer to enzyme stabilized the enzymes against interfacial denaturation. The polymer stabilizes the three dimensional structure of enzymes and prevent it from unfolding at hydrophobic interfaces. Apart from the iv interfacial stabilization of interface-assembled enzyme by polymer, the localization of enzymes at the interface offered a unique opportunity to enhance the stability of the enzymes against the deactivation effect of compounds in bulk phase. 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 (H2O2) in the bulk phase, still limited the overall productivity of the enzyme. Two approaches to further improve the stability of interfaceassembled 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. On the other hand, glucose enhanced the operational stability by 2 folds, but exhibited no significant effect on storage stability. 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. The Damkohler number that gives the ratio between reaction rate and mass transfer rate was obtained to be 0.12 with interface assembled cofactor, compared to 87.5 with native enzymes and free cofactor, indicating mass transfer limitations with interface assembled cofactor. The kinetic analysis of interface-assembled cofactor gave the binding resistance of enzyme to cofactor at the interface, Kc, as 0.18 mM compared to 0.03 mM of native enzyme and free cofactor, which indicated that limited interfacial interaction between molecules and two-dimensional mobility of the enzymes contributed significant resistance towards interfacial reaction. A novel mechanism of nanostirring was developed to improve the twodimensional mobility of interface-assembled enzymes. Iron oxide (Fe3O4) 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 interfaceassembled 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-1 mgenzyme- 1 (or 0.39 A M-1 cm-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. The confinement of the enzyme within the body of nanofibers also stabilized the enzyme, such that the biosensor retained 80% of its sensitivity after 70 days. The interface-assembled enzymes with their improved interfacial stability can substitute soluble enzymes that are presently used for many industrial applications with biphasic systems. Also, Interface-assembled enzymes offer simultaneous access to reactants in both the bulk phases across the interface and thus improve the overall efficiency for the biotransformations between immiscible chemicals. The novel polymerenzyme conjugates and functional materials that were developed through this research with their unique structural, magnetic and mechanical properties can be used in broad range of applications like sensors, membrane technology, generate alternative strategies for encapsulation and delivery of therapeutic agents, and will enable minimum downstream processing for specialty chemical synthesis. The present work is of great interest in the search for production of different important industrial chemicals including bio-renewable products and for sustainable environmental quality.