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Thermostable Biocatalytic Films of Enzymes and Polylysine on Electrodes and Nanoparticles in Microemulsions Peterson M. Guto,† Challa V. Kumar,† and James F. Rusling*,†,‡,§ Department of Chemistry, UniVersity of Connecticut, Department of Cell Biology, UniVersity of Connecticut Health Center, and Institute of Material Science, UniVersity of Connecticut, Storrs, Connecticut 06269 ReceiVed May 28, 2008. ReVised Manuscript ReceiVed July 10, 2008 Microemulsions of oil, water and surfactant were evaluated as media for biocatalysis at high temperatures employing films of polylysine (PLL) and the enzymes horseradish peroxidase (HRP), soybean peroxidase (SBP) and the protein myoglobin (Mb). PLL was covalently linked to oxidized pyrolytic graphite electrodes or carboxylated 500 nm diameter silica nanoparticles, then cross-linked by amidization to HRP, SBP and Mb. The resulting film systems were stable at 90 °C for >12 h in microemulsions. Characterization of the microemulsions by conductivity, viscosity and probe diffusion coefficients suggested that these media have bicontinuous microstructures from 25 to 90 °C. UV circular dichroism and visible spectroscopy confirmed that the enzymes retained near-native conformation in the films at temperatures as high as 90 °C. Oxidation of o-methoxyphenol to 3,3′-dimethoxy-4,4′-biphenoquinone by enzyme-PLL films on silica nanoparticles gave yields 3-5-fold larger in microemulsions at 90 °C compared to the same reaction at 25 °C. The best yields were in CTAB microemulsions and were 3-fold larger than in buffers at 90 °C.
Introduction Biocatalysis can provide inherent regio- and stereoselectivity, and is predicted to be a major factor in future industrial syntheses.1-3 There has been extensive research employing directed evolution, chemical modification, thermophyllic bacteria, and attachment on specialized supports to yield enzymes operational at high temperatures to improve catalytic efficiencies.4-14 While many of these approaches are effective, they rarely yield biocatalysts that can be used at temperatures above 70 °C, and often have much lower operating temperatures. * To whom correspondence should be addressed. E-mail: james.rusling@ uconn.edu. † Department of Chemistry, University of Connecticut. ‡ University of Connecticut Health Center. § Institute of Material Science, University of Connecticut. (1) Taylor, I. N.; Brown, R. C.; Bycroft, M.; King, G.; Littlechild, J. A.; Praquin, C.; Toogood, H. S.; Taylor, S. J. C Biochem. Soc. Trans. 2004, 32(2), 290–292. (2) Rozzell, J. D. Bioorg. Med. Chem. 1999, 7, 2253–2261. (3) Koeller, K. M.; Wong, C-H. Nature 2001, 409, 232–240. (4) (a) Sterner, R.; Liebl, W. Crit. ReV. Biochem. Mol. Biol. 2001, 36, 39–106. (b) Niehaus, F.; Bertoldo, C.; Ka¨hler, M.; Antranikian, G. Appl. Microbiol. Biotechnol. 1999, 51, 711–729. (c) Blair, E.; Greaves, J.; Farmer, P. J. J. Am. Chem. Soc. 2004, 126, 8632–8633. (d) Fitter, J.; Haber-Pohlmeier, S. Biochemistry 2004, 43, 9589–9599. (5) (a) Vieille, C.; Zeikus, G. J. Microbiol. Mol. Biol. ReV. 2001, 65, 1–43. (b) Joe, K.; Borgford, T. J.; Bennet, A. J. Biochemistry 2004, 43, 7672–7677. (6) Kocabiyik, K.; Erdem, B. Bioresour. Technol. 2002, 84, 29–33. (7) Saeki, K.; Hitomi, J.; Okuda, M.; Hatada, Y.; kayeyami, Y.; Takaiwa, M.; Kubota, M.; Hagihara, H.; Kobayashi, T.; Kawai, S.; Ito, S. Extremophiles 2002, 6, 65–75. (8) Small, A. O.; Leiros, H.-K. S.; Os, V.; Willassen, N. P. Biotechnol. Annu. ReV. 2000, 6, 1–57. (9) Wanatabe, S.; Yamaoka, N.; Fukumaga, N. Extremophiles 2002, 6, 397– 405. (10) Van den Burg, B.; Eijsink, V. G. H. Curr. Opin. Biotechnol. 2002, 13, 333–337. (11) Gonzalez-Blasco, G.; Sanz-Apricio, J.; Gonzalez, B.; Hermoso, J. A.; Polaina, J. J. Biol. Chem. 2000, 275, 13708–13712. (12) Reading, N. S.; Aust, S. D. Biotechnol. Prog. 2000, 20, 326–333. (13) Bokhari, S. A.; Afzal, A. J.; Rashid, H. H.; Rojaka, M. I.; Siddiqui, K. S. Biotechnol. Prog. 2002, 18, 276–281. (14) (a) Kumar, C. V.; Chaudhari, A. Chem. Commun. 2002, 2382–2383. (b) Pessela, C. C.; Mateo, C.; Fuentes, M.; Vain, A.; Garcia, J. L.; Carrascosa, A. V.; Guisan, J. M.; Fernandez-Lafluente, R. Biotechnol. Prog. 2004, 20, 388–392. (c) Jagannadham, V.; Bhambhani, A.; Kumar, C. V. Microporous Mesoporous Mater. 2006, 88, 275–282.
Our goals in this area include combining microemulsions with stable biocatalytic films of common enzymes operable at temperatures of 90 °Candabove.Mostreactionsareacceleratedwithincreasedtemperature andanapproximateestimateisthatratesdoubleforevery10°Cincrease intemperature.However,amajorityofenzymesdenatureattemperatures wellbelow90°C,exceptionsbeingenzymesisolatedfromthermophilic bacteria or specially engineered mutants. Therefore, it is a major challenge to stabilize ordinary enzymes to achieve accelerated biocatalysis at high temperatures. Microemulsions are thermodynamically stable, clear dispersions of water, oil, surfactant and cosurfactant. They are less expensive, less toxic alternatives to organic solvents that solubilize both polar and nonpolar substrates and are capable of providing unique synthetic pathway control.15,16 Employing enzyme films in microemulsions facilitates dissolution of water insoluble organic compounds for biocatalytic reactions while providing a water-rich biocatalyst microenvironment within the PLL films.17,18 Herein we report high temperature stability, electrochemical reactivity, and activity of ordinary enzymes cross-linked within polylysine (PLL) films in contact with microemulsions made with cationic and anionic surfactants. We developed cross-linked enzyme-polylysine (PLL) films attached to carboxylated surfaces that provide excellent stability and biocatalytic activity.17,19 We recently demonstrated remarkable thermal stability at 90 °C for PLL films of peroxidases attached to pyrolytic graphite electrodes or silica nanoparticles in buffer solutions.20 Improved biocatalysis and increased electron transfer rates were obtained at 90 °C. Herein we extend these studies to microemulsions to evaluate their potential for biocatalysis at high temperature in these media. (15) Rusling, J. F. in Texter, Ed., Reactions and Synthesis in Surfactant Systems, Marcel Dekker: New York, 2001, pp. 323-335. (16) Rusling, J. F. In Encyclopedia of Electrochemisry; Vol. 2, Interfacial Kinetics and Mass Transport; Calvo, E., Ed.; Marcel Dekker: New York, 2003; pp 418-439. (17) Vaze, A.; Parizo, M.; Rusling, J. F. Langmuir 2004, 20, 10943–10948. (18) Vaze, A.; Rusling, J. F. Faraday Discuss. 2005, 129, 265–274. (19) Guto, P. M.; Rusling, J. F. J. Phys. Chem. B 2005, 109, 24457–24464. (20) Guto, P. M.; Kumar, C. V.; Rusling, J. F. J. Phys. Chem. B 2007, 111, 9125–9131.
10.1021/la801644e CCC: $40.75 2008 American Chemical Society Published on Web 08/09/2008
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This paper combines microemulsions with enzyme-PLL films using horseradish peroxidase (HRP), soybean peroxidase (SBP) and myoglobin (Mb). Cross-linked films of these biocatalysts were stable in SDS and CTAB microemulsions at 90 °C for over 12 h, and enzymes retained near native secondary structures in the films. Higher product yields for the oxidation of omethoxyphenol were realized at 90 °C compared to 25 °C in both microemulsions.
Experimental Section ChemicalsandSolutions.Poly-L-lysine(PLL,MW150 000-300 000), horseradish peroxidase (HRP), soybean peroxidase (SBP), hemin, horse heart myoglobin (Mb), o-methoxyphenol, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) were from Aldrich-Sigma. Mb was dissolved in acetate buffer, pH 5.5, filtered through YM30 filter (Amicon, 30 000 MW cutoff) to give 3.0 mg mL-1 Mb. HRP, SBP and hemin were dissolved in the pH 5.5 buffer to 3.0 mg mL-1. Bicontinuous SDS and CTAB microemulsions were prepared by mixing in appropriate ratios as described previously.19 Water was purified with a Hydro Nanopure system to specific resistance >15 MΩ-cm. All other chemicals were reagent grade. Film Preparation. Ordinary basal plane pyrolytic carbon (PG) disk electrodes (A ) 0.16 cm2) and fused silica slides were prepared as described previously.20 Electrode surface area determined by cyclic voltammetry (CV) of ferrocyanide oxidation was 0.21 cm2. Covalently linked enzyme-PLL films were made as described previously.19,20 For spectroscopy, films were made on poly(acrylic acid) (PAA) covalently attached with EDC to aminoalkylsilated fused silica slides. Slides were rinsed with water, then 10 µL 24 mM EDC and 10 µL 3 mg mL-1 of the desired enzyme in water was deposited on the PAA surface and reacted 12 h, followed by rinsing with water. A second enzyme/PLL bilayer was formed by repeating the above steps. Two-bilayer enzyme-PLL films on 500 nm diameter silica nanoparticles were prepared as described previously.20 Oxidation of o-Methoxyphenol. Biocatalyst nanoparticles were suspended in 1.0 mL of microemulsion at approximately 2.5 mg mL-1 of the silica nanoparticles based on initial concentrations. Total concentration of enzyme was ∼8 µM. To these dispersions, 5 mM tert-butylhydroperoxide (tBuOOH) and 10 mM o-methoxyphenol (o-MP) were added and the oxidation product (3,3′-dimethoxy4,4′-biphenoquinone) was monitored by visible spectroscopy at 480 nm with time at 25, 60 and 90 °C. Product concentrations were determined from Beer’s law, using extinction coefficient (ε) 26 000 M-1cm-1 at 480 nm.21 Instrumental Methods. Methods for voltammetry and spectroscopy were described previously.20 Bulk viscosities of SDS and CTAB bicontinuous microemulsion were measured with a kinematic viscometer calibrated with pure water at 25 °C. Electrical conductivities were measured with a YSI Model 35 conductance meter and YSI 3403 conductance probe calibrated with known solutions of sodium chloride.
Results Microemulsion Conductivity and Viscosity. The bicontinuous SDS and CTAB microemulsions used have been characterized previously at room temperature,22,23 but not at higher temperatures. Table 1 shows bulk viscosities (η) and conductivities (K) of the clear microemulsions at 25 and 90 °C. These properties are related to microemulsion nanostructure.24-26 Conductivities increased ∼36-fold for SDS, ∼58-fold for CTAB microemulsion, (21) Ozaki, S.-I.; Ishikawa, Y. React. Kinet. Catal. Lett. 2006, 89(10), 21–28. (22) Mackay, R. A.; Myers, S. A.; Bodalbhai, L.; Brajter-Toth, A. Anal. Chem. 1990, 62, 1084–1090. (23) Myers, S. A.; Mackay, R. A.; Brajter-Toth, A. Anal. Chem. 1993, 65, 3447–3453. (24) Ajith, C. J.; Animesh, K. R. J. Colloid Interface Sci. 1993, 156, 202–206. (25) Zhang, S.; Rusling, J. F. J. Colloid Interface Sci. 1996, 182, 558–563. (26) Digout, L.; Bren, K.; Palepu, R.; Moulik, S. P. Colloid Polym. Sci. 2001, 279, 655–663.
Guto et al. Table 1. Conductivity (K) and Viscosity (η) of SDS and CTAB Bicontinuous Microemulsions medium 13.3% SDS 13.3% SDS 17.5% CTAB 17.5% CTAB 0.1 M NaCl 0.1 M NaCl 0.1 M NaCl
temp, K, mΩ-1 °C cm-1 90 °C/25 °C 25 90 25 90 25 90 25
8.2 297 1.9 110 115 280 107 (27)
36.2 58.0 2.4
η (cP) 8.2 1.7 11.0 1.8 1.00 0.37
90 °C/25 °C 0.21 0.16 0.37
Table 2. Apparent Diffusion Coefficients (D′o) and ηD/T in the SDS and CTAB Microemulsionsa medium 13.3%SDS 13.3%SDS 17.5%CTAB 17.5%CTAB 0.1 M NaCl 0.1 M NaCl
temp 106 D′o (A) 103ηD/T 106 D′o (B) 103ηD/T cm2 s-1 (La K-1) cm2s-1 (La K-1) (°C) 25 90 25 90 25 90
0.23 0.40 0.50 2.90 2.14 8.54
6.4 2.0 19 14 7.0 8.5
1.5 4.9 6.1 7.1 -
40 23 57 36 -
a L represents the units Cp cm2 s-1; A is 1.0 mM Potassium ferricyanide, B is 1.0 mM Ferrocene.
and ∼2 fold for 0.1 M NaCl when temperature increased from 25 to 90 °C. The microemulsion K’s are consistent with increased mobility of the surfactant ions at the higher temperature, suggesting the possibility of smaller or more dynamic aggregate units. The decrease in viscosity for this increase in temperature was similar for all fluids. Low temperature K and η values were reproduced when media at 90 °C were cooled to 25 °C. Diffusion Coefficients. Diffusion coefficients of probes were used to help characterize microemulsion structure.16 Potassium ferricyanide dissolves in the water phase, and ferrocene dissolves in the oil phase. These electroactive species were used to probe diffusion coefficients in their respective nanophases. Cyclic voltammograms of these probes up to 1 V/s had shapes of a reversible one electron redox reaction with reduction peak currents (ip) linear when plotted versus square root of scan rate (ν), consistent with the Randles-Sevcik equation.28 Slopes of ip vs ν1/2 were used with this equation to estimate apparent diffusion coefficients of the probes at 25 and 90 °C.16 Table 2 shows that diffusion coefficients of all the probes were larger at 90 °C compared to 25 °C in all fluids. The Stokes-Einstein relation (eq 1)28 indicates that (Dη/T) ) k/6rπ should remain constant for the same fluid.
D)
kT 6πrη
(1)
where k is Boltzmann’s constant, T is the temperature in K, r is the hydrodynamic radius of the probe and η is the bulk viscosity. Table 2 shows diffusion coefficients and Dη/T increased slightly in 0.1 M NaCl, but decreased somewhat in the microemulsions. However, if the increase in temperature had caused a change to a fully homogeneous fluid, an expected large increase in D for the probes would increase Dη/T. The fact that this is not observed suggests that both detergent-based fluids remain microemulsions at 90 °C. Spectroscopy. The effect of temperature on the electronic environment around the heme prosthetic group of SBP, HRP and Mb in films were examined by visible spectroscopy (Figure 1 (27) Lide, R. D. (Ed), Handbook of Chemistry and Physics; 82nd ed.; CRC Press: New York, 2001-2002. (28) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. 2nd ed., John Wiley and Sons, Inc.: New York, 2001.
Thermostable Biocatalytic Films of Enzymes and Polylysine
Figure 1. Absorption spectra of (a) SBP/PLL films in SDS microemulsion (b) Mb/PLL films in CTAB microemulsion (c) SBP/PLL films in buffers, pH 5.5 + 0.1 M NaCl and (d) Mb/PLL films in buffers, pH 5.5 + 0.1 M NaCl at 25 and 90 °C. (Spectra offset on y-axis for clarity). Films were attached to aminoalkylsilated fused silica slides and spectra taken after 30 min of incubation in the respective media. Table 3. Soret Absorption Maxima (nm) for SBP/PLL, HRP/ PLL and Mb/PLL Films in SDS and CTAB Microemulsions and pH 6.5 buffer at 25 and 90 °Ca SDS SDS CTAB CTAB buffer buffer in soln. enzyme 25 °C 90 °C 25 °C 90 °C 25 °C 90 °C 25 °C SBP HRP Mb
406 405 412
406 405 412
406 405 413
404 406 412
404 406 412
403 406 412
402 404 409
a Mb ) myoglobin, HRP ) horseradish peroxidase, SBP ) soybean peroxidase and PLL ) polylysine.
and Table 3). The Soret absorption maxima did not shift significantly when temperature was increased from 25 to 90 °C in microemulsions or buffer, and values were consistently a few nm to the red compared to the enzyme dissolved in buffer at 25 °C, as found in these films previously.20 Such small red shifts were found to be characteristic of heme proteins in films of several types, 29 and may be due to minor distortions of enzyme secondary structure in the heme vicinity. UV circular dichroism (CD) of enzyme-PLL films (Figure 2) provided information on protein secondary structure.30 The 210 and 222 nm minima and a maximum at 193 nm characteristic of the large fraction of R-helix secondary structure in these enzymes appear in all CD spectra. Except for changes in intensity related to poor control of film thickness, the CD spectra in buffers and microemulsions are similar to those for the enzymes dissolved in neutral buffer solutions.20 HRP films in CTAB microemulsion shows a change in ratio of 210/222 nm minima which may reflect small conformational differences. These data show the ensemble average of the conformational status of all the microenvironments of the enzymes. In general, however, the CD spectra are consistent with a significant retention of native enzyme conformation in the films at temperatures up to 90 °C in both microemulsions. Voltammetry. Square wave voltammetry (SWV) was used to monitor the stability of enzyme-PLL films on PG electrodes. Figure 3a shows representative square wave voltammograms (29) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363–369. (30) (a) Holzwarth, G.; Doty, P. J. Am. Chem. Soc. 1965, 87, 218–228. (b) Johnson, W. C. Annu. ReV. Phys. Chem. 1978, 29, 93–114.
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Figure 2. UV circular dichroism spectra of films on fused silica slides: (a) HRP/PLL films in SDS (b) Mb/PLL films in SDS (c) HRP/PLL films in CTAB (d) SBP/PLL films in CTAB microemulsions at 25 and 90 °C. Spectra taken in pH 5.5 buffers + 0.1 M NaCl at 90 °C are included for comparison.
obtained for SBP/PLL in a bicontinuous microemulsions. All peaks were larger at 90 °C than at 25 °C, and peaks at the lower temperature were quite stable over 12 h. Figure 3b and c shows stability plots for HRP/PLL, SBP/PLL, Mb/PLL and a Hemin/ PLL control in SDS and CTAB microemulsions at 90 °C. HRP, SBP and Mb showed ∼10% decrease in peak current over 12 h and were reasonably stable in both microemulsions. SBP was the most stable followed by Mb then HRP while hemin lost 30% of its peak height over 12 h. Cyclic voltammetry (CV) of these films on PG electrodes at 25 and 90 °C in both SDS and CTAB bicontinuous microemulsions gave a single set of oxidation-reduction peaks representing the quasireversible reduction of the FeIII/FeII redox couple for Mb, HRP, SBP and Hemin. All the cyclic voltammograms were nearly symmetric in all the media and temperature investigated (Figure 4). Formal redox potentials (Eo′) (Table 4) depend on the enzyme, the microemulsion type, and temperature. At 90 °C, CTAB had the most positive formal redox potential (Eo′), while SDS had the most negative Eo′. In both media, peak current measured by square wave voltammetry increased linearly with temperature suggesting faster electron transfer28 at higher temperatures. Redox potentials did not shift very much from 25 to 90 °C in SDS microemulsion and buffer, but shifted significantly positive in the CTAB microemulsion. In general, redox potentials for hemin and the enzymes in PLL films differ significantly in the microemulsions in most cases. Following Hirst and Armstrong,31 we measured peak separations (∆Ep) by CV and subtracted the constant peak separation found at low scan rates to correct for nonkinetic influences. The corrected ∆Ep vs ν data were fit onto the Laviron model for nondiffusing redox sites on an electrode32 to obtain best values of surface electron transfer rate constant ks (Figure 4). The corrected ∆Ep values gave very good fits to theory, and yielded reproducible values ks (Table 4). Roughly a 2- to 5-fold increase in ks was found at 90 °C compared to 25 °C in all cases. Generally, ks values for the films were somewhat smaller when in contact with either microemulsion than when they were in contact with neutral buffer solutions.
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Figure 3. Square wave voltammetry data in anaerobic fluids for SBP/PLL, HRP/PLL, Mb/PLL and Hemin/PLL films covalently bound onto pyrolytic graphite electrodes (A) difference SWVs of SBP/PLL in SDS microemulsions; and influence of time at 90 °C on SWV peak current (µA) for immersion of films in (B) SDS and (C) CTAB microemulsions. (frequency 25 Hz, 25 mV pulse height).
Figure 4. CV data for enzyme-PLL films in microemulsions: (a and d) CVs at 0.5 V s-1. (b, c, e and f) corrected peak separation versus scan rate (ν) plots used to determine apparent electron transfer rate constants (ks). Points are experimental data; solid lines were calculated from the Laviron model for the best ks with R ) 0.5.
Biocatalytic Oxidation of o-Methoxyphenol. To assess synthetic potential at higher temperatures, enzyme-PLL films on 500 nm diameter silica nanoparticles was used to oxidize o-methoxyphenol to 3,3′-dimethoxy-4,4′-biphenoquinone.33,34 The oxidation product absorbs at 470-480 nm (Figure S1, Supporting Information). In this reaction, o-methoxyphenol is converted into a radical by the peroxide-activated enzymes (Scheme 1). The intermediate hydroquinone dimer 2 is then oxidized presumably by the activated peroxidase to the colored quinone 3.33 Our previous results in buffer20 suggested that the second oxidation of the multistep pathway (Scheme 1) was preferentially activated by thermal energy to provide larger yields at higher temperatures. To evaluate the unique features of this reaction in microemulsions that would be unobservable with the unstable peroxidases in solution at 90 °C, we ran the reaction with peroxide as the limiting reagent rather than using traditional (31) Hirst, J.; Armstrong, A. Anal. Chem. 1998, 70, 5062–5070. (32) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28. (33) Adam, W.; Lazarus, M.; Saha-Mo¨ller, C. R.; Weichold, O.; Hoch, U.; Ha¨ring, D.; Schreier, P. AdV. Biochem. Eng. Biotech. 1999, 63, 73–108. (34) Doerge, D. R.; Divi, R. L.; Churchwell, M. I. Anal. Biochem. 1997, 250, 11–17.
initial rate studies. This approach also avoided using excess peroxide which might damage the enzymes at higher temperatures. Analysis of reaction media by colorimetric assay after reaching steady state showed that all the t-BuOOH was consumed.20 Using these conditions, we monitored the efficiency of peroxide use in the catalytic reaction in terms of the product yield. Figure 5 shows that increasing the temperature from 25 to 90 °C increased the yield of product 3 by ∼5 times for both HRP and SBP in CTAB bicontinuous microemulsions and ∼3 times in SDS bicontinuous microemulsions. Compared to buffer solutions, the CTAB microemulsion supported ∼3-fold higher product yields than in buffer at 90 °C, while the SDS microemulsion increased the yield by 10-20% when compared to the reaction when the film was in contact with the buffer at 90 °C. We also examined reusability of the enzyme biocatalyst nanoparticles after an initial reaction in microemulsions. For this purpose, enzyme beads that had been used to oxidize omethoxyphenol were collected by centrifugation, dried, weighed to assess losses, and used to oxidize o-methoxyphenol a second time. Results were expressed as absorbance normalized for bead
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Table 4. Apparent Surface Electron-Transfer Rate Constants (ks) and Formal Potentials (E°′) for Enzyme-PLL Films at 25 and 90 °C in CTAB and SDS Microemulsions and Buffer Solutions enzyme HRP/PLL HRP/PLL HRP/PLL HRP/PLL HRP/PLL HRP/PLL SBP/PLL SBP/PLL SBP/PLL SBP/PLL SBP/PLL SBP/PLL Mb/PLL Mb/PLL Mb/PLL Mb/PLL Mb/PLL Mb/PLL Hemin/PLL Hemin/PLL Hemin/PLL Hemin/PLL Hemin/PLL Hemin/PLL
medium SDS SDS CTAB CTAB buffer, buffer, SDS SDS CTAB CTAB buffer, buffer, SDS SDS CTAB CTAB buffer, buffer, SDS SDS CTAB CTAB buffer, buffer,
pH 6.5 pH 6.5
pH 6.5 pH 6.5
pH 6.5 pH 6.5
pH 6.5 pH 6.5
temp, °C
ks, s-1
-E°′, V vs SCE
25 90 25 90 25 90 25 90 25 90 25 90 25 90 25 90 25 90 25 90 25 90 25 90
9.7 ( 0.3 39 ( 2 5.0 ( 0.5 10.3 ( 0.2 22 ( 420 44 ( 420 7.3 ( 0.2 16 ( 1 5.3 ( 0.6 27 ( 1 31 ( 320 45 ( 620 6.3 ( 0.3 18 ( 3 3.3 ( 0.4 12.0 ( 0.5 19 ( 220 41 ( 720 5.8 ( 0.7 23 ( 4 1.7 ( 0.2 19 ( 4 11 ( 220 18 ( 220
0.460 ( 0.008 0.480 ( 0.007 0.415 ( 0.010 0.350 ( 0.008 0.315 ( 0.009 0.315 ( 0.002 0.520 ( 0.010 0.515 ( 0.010 0.402 ( 0.007 0.316 ( 0.005 0.310 ( 0.003 0.330 ( 0.003 0.400 ( 0.010 0.402 ( 0.008 0.432 ( 0.005 0.300 ( 0.010 0.306 ( 0.004 0.310 ( 0.001 0.470 ( 0.010 0.490 ( 0.008 0.412 ( 0.010 0.260 ( 0.005 0.304 ( 0.001 0.320 ( 0.010
Scheme 1. Simplified Pathway for Peroxidase Catalyzed Oxidation of o-Methoxylphenol34
mass to correct for losses in recovery. After first use, these biocolloids were separated from the mixture by centrifugation, washed, collected, and used to oxidize o-methoxyphenol a second time. Figure S2 (Supporting Information) shows negligible change in enzyme activity at 25 °C for both HRP- and SBP-PLL nanoparticles upon second use. At 90 °C, roughly 5% decrease in yield was found on the second use (Figure 6) in the microemulsions. These data indicate that the peroxidase-PLL films remain relatively stable under catalytic conditions. No significant differences were noticed between SDS and CTAB bicontinuous microemulsions in these reusability studies.
Discussion Figures 1, 2, and 3 demonstrate the remarkable thermal stability of the enzyme-PLL films in microemulsions by two types of spectroscopy and by voltammetry. Square wave voltammetry revealed ∼10% decrease in peak current over 12 h at 90 °C for HRP, SBP and Mb in the films (Figure 3). Extrapolation of these data suggest half-lives of ∼60 h for the enzyme-PLL films at 90 °C, compared to half-lives of
