Supporting Information

Transcription

1 Supporting Information Wiley-VCH Weinheim, Germany

2 Homogeneous Biocatalysis in both Fluorous Biphasic and Supercritical Carbon Dioxide Systems H. R. Hobbs, H. M. Kirke, M. Poliakoff, N.R. T ho mas * School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK Fax: (+) neil.thomas@nottingham.ac.uk Materials Cytochrome c (Cc) from bovine heart, α-chymotrypsin (CMT) from bovine pancreas (EC ; 6 U/mg) and N-acetyl-L-phenylalanine ethyl ester (APEE) were purchased from Sigma and used without further purification. Aerosol T (AT) was obtained from Fluka, KDP 466 and Krytox 17 FSL surfactants were kindly donated by DuPont Chemicals (Deepwater, NJ, USA). Perfluoromethylcyclohexane (PFMC) was purchased from Fluorochem. SCF grade carbon dioxide (99.99 % purity) was obtained from BC gases. All other chemicals were of reagent grade and obtained from Fisher Scientific. Protein Concentration Determination Many protein samples contain cryoprotectants, such as glycerols or sugars which can interfere with protein absorbance around nm, hence the bicinchoninic acid assay was the method of choice for protein concentration determination in aqueous solution. However, this method is inappropriate for nonaqueous protein solutions, therefore protein-surfactant complexes in perfluoromethylcyclohexane were measured by UV/Vis absorption at 28 nm. Interference by cryoprotectants was not an issue in perfluoromethylcyclohexane since they are not extracted by ion pairing into the fluorous solvent, hence this ion pairing mechanism could also be used as a purification method for some impure proteins.

3 Circular Dichroism (CD). Spectra were obtained on a Chirascan * spectropolarimeter using a bandwidth of 1 nm, a wavelength increment of. nm and a response time of 1. s. The optical path length of the quartz cell was 1 mm. The temperature was maintained at 2 C with a Peltier device. Three scans were averaged per spectrum. The resulting data were smoothed and baseline corrected by solvent subtraction Molar Ellipticity M -1 cm Cc-KDP (PFMC) Cc (aq) -12 Wavelength (nm) Supplementary figure 2. CD spectra for native Cc (aq) and Cc-KDP (perfluoromethylcyclohexane, PFMC). The CD spectrum for Cc-KDP in PFMC demonstrates that the Cc retains its α-helical secondary structure. Cc (aq) shows a larger band at 222 nm, indicative of an α-helical structure in an aqueous environment in the absence of surfactant, [26] whereas Cc-KDP in PFMC shows a larger band at 28 nm indicative of an α-helical protein in the presence of surfactant (more hydrophobic environment) as previously reported. [2]

4 Dynamic Light Scattering (DLS). DLS measurements were carried out on a Zetasizer Nano S at Malvern Instruments. The instrument contains a 4 mw He-Ne laser operating at a wavelength of 633 nm. Each sample was filtered through the smallest appropriate filter prior to measurements which were made at a detection angle of 173 (i.e. backscatter) and 2 C. The particle size was taken as a mean value of three measurements. Protein and protein surfactant concentrations for Cc and CMT were measured between.4.8 mm. The data was analysed using Auto:CNTIN. [3] 2 Volume (%) Cc 2.3 nm CcKDP 21.2 nm Size (d.nm) Supplementary Figure 3. Size distribution of Cc (aq) and CcKDP in perfluoromethylcyclohexane. Cc in aqueous buffer was investigated and its hydrodynamic diameter determined at around 2.3 nm with a narrow width of distribution. This suggests that Cc is a single molecular species in aqueous media and is in reasonably good agreement with that recently reported in the literature (~3 nm). [4] The slightly lower value obtained for this research may be due to the different aqueous buffers used (ph 3.8 sodium acetate buffer for this research versus ph 7.4 phosphate buffer). Cc-KDP has a diameter of 21.2 nm. This suggests that within Cc-KDP complexes there is more than one Cc molecule surrounded by surfactant, and aggregation of Cc occurs. The presence of a very small amount of higher diameter particles (ca 1 nm) is also noted and this could be due to even further aggregation of Cc within the KDP complex. ne reason for the aggregation of Cc within HIP complexes could be that Cc has a highly polar surface. Therefore when it is extracted into a hydrophobic fluorous solvent by a hydrophobic surfactant it is very likely that Cc will aggregate forming a multi-molecular species surrounded by surfactant.

5 2 Volume (%) CMT 3.6 nm CMT KDP 11.7 nm CMT AT 9.2 nm Size (d.nm) Supplementary Figure 4. Size distribution of CMT (aq) and CMT-KDP in perfluoromethylcyclohexane. CMT in aqueous buffer was noted to have a diameter of 3.6 nm with a reasonably narrow distribution. CMT-AT was measured at 9.2 nm. This is slightly higher than the literature report of 6.8 nm [] and the increase could be due to a variation in buffer and ph used (sodium phosphate buffer, ph 6., c.f. Bis-Tris propane buffer, ph 7.8, in the literature [] ). For single molecules of CMT within a KDP complex a diameter of 8.2 nm would be expected. From Figure 4 it can be seen that a slightly higher diameter of 11.7 nm is obtained suggesting that a small amount of CMT aggregation is occurring within the complex. Transesterification of APEE by n-propanol Ph HN Ac CMT-AT (hexane) + nprh Ph + EtH or CMT-KDP (FBS) HN Ac Supplementary Figure. Conversion of N-acetyl-L-phenylalanine ethyl ester (APEE) into its propyl ester (APPE) by catalysed by CMT-AT in hexane or CMT-KDP in a fluorous biphasic system.

6 ptimum ph for complexation and enzyme activity Complexation efficiency % Yield % ph Complexation efficiency Yield Supplementary Figure 6. Complexation efficiency for CMT-KDP within the ph range Activity is also shown as yield for APPE. Complexation efficiency remains high between ph but CMT activity is highest when CMT is extracted from ph 7.2 buffer. It can be seen that ph > 7.2 will give good complexation efficiencies ( %). The activity of CMT-KDP was studied using the complexes extracted from ph Concentrations of CMT-KDP extracted from lower ph s were too low to give comparable activity data. From Figure 6 it can be seen that ph 7.2 is the best ph to extract CMT from to give the highest yield of APPE (16.8 %). A compromise between high complexation efficiency and good activity is required therefore ph 7.3 was chosen for CMT-KDP complexation and activity. Ability to recycle CMT-KDP for transesterification reaction Yield % CMT-KDP native CMT Cycle Supplementary Figure 7. Recycling of CMT-KDP and native CMT in a fluorous biphasic system. CMT-KDP has much better activity in comparison with native CMT under fluorous biphasic conditions. Good catalytic activity is retained over at least four cycles.

7 Solubility in scc 2. A rig was designed for on-line UV/Vis spectroscopy under supercritical conditions for the determination of protein concentration in C 2. A small volume high pressure IR cell has previously been described [6] which incorporates two small windows which allow an IR beam to pass through the cell. The cell was adapted for the purpose of high pressure UV/Vis spectroscopy. Cc-Krytox (4.2 mg) in PFMC (1 µl) was transferred to a fixed volume high pressure cell (. cm path length) with two calcium fluoride windows and containing a magnetic stirrer bar. The cell was sealed, heated to 4 C, pressurized with C 2 and stirred for 1 minutes before a UV/Vis measurement was taken. More C 2 was introduced into the cell to increase the pressure and the procedure repeated. 8 Concentration (mg/ml) Pressure (psi) Supplementary Figure 8. Solubility of Cc-Krytox in C 2. Results for two experiments are shown: 4.2 and 8.4 mg Cc- Krytox in perfluoromethylcyclohexane (1 µl). An increase in pressure and in mass of protein increases solubility in C 2. References 1. K. Chattopadhyay, and S. Mazumdar, Stabilization of partially folded states of cytochrome c in aqueous surfactant: Effects of ionic and hydrophobic interactions. Biochemistry 23, 42, N. Sanghera, N. and T.J.T. Pinheiro, Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles. Protein Sci. 2, 9, S.W. Provencher, Eigenfunction expansion method for analysis of exponential decay curves. J. Chem. Phys. 1976, 64, S. Roy, S. Singha, J. Bhattacharya, R. Ghosh-Moulick, A.K. Dasgupta A size dependent folding contour for cytochrome c. Biophys. Chem. 26, 119, V.M. Paradkar, and J.S. Dordick, Aqueous-like activity of alpha-chymotrypsin dissolved in nearly anhydrous organic-solvents. J. Am. Chem. Soc. 1994, 116, S.M. Howdle, Spectroscopy in liquefied and supercritical noble gases. Ph.D. Thesis University of Nottingham, UK 1989.