Vytautas Reipa

Improving the Cytochrome P450 Enzyme System for Electrode-Driven Biocatalysis of Styrene Epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large‐scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min−1 by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, α‐hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The α‐hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Electrochemical Properties of Nanocrystalline Cadmium Stannate Films

Electrochemical properties of sol-gel nanocrystalline cadmium tin oxide electrodes (CTO) were investigated in 0.2 M potassium chloride buffered at pH 7.4 with phosphate. Films were found to he n-type degenerate semiconductors with charge carrier levels from 10 19 to 10 22 cm 3 depending on the thermal aftertreatment. X-ray diffraction analysis was used to reveal the appearance of the cubic cadmium stannate (Cd 2 SnO 4 ) phase at annealing temperatures above 600°C, and to indicate the extent of this dominant phase above 750°C. The flatband potential (E FB . ) of the film electrodes, as determined from capacitance measurements, was found to be around +0.25 V at pH 7.4. Electrochemical activity toward ten redox processes in the range -0.45 V < E < 0.45 V was investigated, and standard electron transfer rate constants were estimated from ac impedance measurements. The dominant factor in the charge-transfer rate on CTO electrodes is the bulk film charge carrier concentration. It was found that the charge-transfer rates were dependent on the separation of the redox carrier formal potential (E of ) from the CTO flatband potential. The slowest rates (10 5 cm s -1 were found for redox couples where E of E FB . For charge transfer from redox couples where E of is away from E FB , the rates can be several orders of magnitude greater and it is thought that the density of states in the conduction hand is rate limiting.

A non-perturbative relation between the mutual diffusion coefficient, suspension viscosity, and osmotic compressibility : application to concentrated protein solutions

Abstract We test a proposed relation between mutual diffusion coefficient and viscosity in suspensions which is essentially identical to a dynamic mode-coupling theory of diffusion in binary fluid mixtures near a critical mixing point. In contrast to conventional treatments of suspension hydrodynamics, this relation is non-perturbative in the sense that it is not necessary to assume that the volume fraction of suspended particles is a small parameter. In order to apply this theory to protein solutions, we measured the mutual diffusion coefficient of several globular proteins in aqueous solutions containing 2% ampholyte and protein up to 40% by weight. The viscosity was also measured in aqueous solutions of up to 30% protein by weight. The mutual diffusion coefficient was then related to the viscosity and osmotic compressibility of the concentrated suspension relative to the infinite dilution limit. We conclude that this non-perturbative relation provides a convenient estimate of protein diffusion coefficients as a function of protein concentration.

CONFORMATIONAL ALTERATIONS OF BOVINE INSULIN ADSORBED ON A SILVER ELECTRODE

Abstract Surface enhanced Raman spectra of bovine insulin, adsorbed on the silver electrode from aqueous solutions of micromolar concentrations, are presented for the potential range −0.2 to −1.2 V/AgCl. The data suggest that insulin is bound to silver through ionized tyrosine residues nd carboxy terminal groupings. Disulfide linkages are reduced sequentially upon increasing negative potential: A7–B7 at −0.3 to −0.5 V, and A20–B19 at −0.5 to −0.6 V. Rupture of disulfide bonds increases the portion of β/disordered conformation at the expense of the α helix.

Differential Surface Stress of a Tin Oxide Electrode

Differential surface stress measurements performed using the estance technique and simultaneous measurements of the differential capacitance provide experimental evidence for deviation of n-tin oxide electrodes properties from the electrocapillary relationship that has the form of the classical Lippmann equation. The differential stress curves consist of two distinct regions. At positive potentials, where the electrical properties of n-tin oxide electrode are consistent with the Mott-Schottky model, the differential stress does not depend upon polarization and is insensitive to solution pH. In this region, the main contribution to the surface stress originates from the ordered dipole layer, which could include water molecules. In the negative potential range, where deviations from the Mott-Schottky model occur, the differential stress curves exhibit nonmonotonic behavior and become pH sensitive. The similar behavior of differential surface stress and semiconductor electrode charge was observed in this potential range.

Covalent attachment of photoluminescent silicon nanoparticles to streptavidin

We have covalently attached multiple fluorescent silicon nanocrystals (SNs) to streptavidin molecule. Selective conjugation of SNs to a target protein is accomplished using sequential silicon surface termination chemistry. In the first step, freshly prepared hydrogen terminated surfaces of SNs are substituted with alkane monolayer that serves as a platform for chemical linkage to hetero bifunctional crosslinker (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester), and provides optical and chemical stabilities against oxidation and aggregation of nanoparticles. Next, an open end of bifunctional cross linker - diazirine succinimidyl ester forms an amide bond with a carboxyl of target protein. Gel electrophoresis of SNs labeled streptavidin clearly show separate elution of conjugation product and neat protein. Conjugate functionality was verified by allowing it to interact with biotinylated micro beads. A bright fluorescence, characteristic to SNs was observed from vigorously washed micro beads showing selective attachment of nanoparticle bearing streptavidin to biotinylated micro beads. High quantum yield of streptavidin-SN conjugate in combination with the biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescent tagging in diverse bioassays.

Observation of photon correlations in scattering from a silver electrode

Abstract Dynamic light scattering experiments were carried out on silver electrodes in aqueous solution. Two distinct time-scales were observed in the photon autocorrelation function: 1 ms and 100 ms. The 1 ms decay in the correlation was predominant during voltage cycling, while the slow decay is present at all times in varying strength. A possible source of the fast correlation is the generation of dynamic polarizability inhomogeneities on the silver surface while the slow correlation is associated with microscopic surface height modulation during electrocrystallization.