Supporting Information

Transcription

1 Supporting Information Precipitation-Based Nanoscale Enzyme Reactor with Improved Loading, Stability and Mass Transfer for Enzymatic CO 2 Conversion and Utilization Han Sol Kim, Sung-Gil Hong, Kie Moon Woo, Vanesa Teijeiro Seijas, Seongbeen Kim, Jinwoo Lee, and Jungbae Kim*, Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea Corresponding Author * jbkim3@korea.ac.kr 1

2 Theoretical Maximum Loading of Carbonic Anhydrase in Mag-S-MCF Theoretical maximum loading of carbonic anhydrase (CA) in Mag-S-MCF was calculated by following the method of Hwang et al. 1 In details, the pore volume of Mag-S- MCF was estimated as 1.23 x nm 3 g -1. The volume of CA was calculated from the unit cell parameters of 5.06 x 4.47 x 5.69 nm 3. 2 The perfect packing model was assumed with no void space between enzyme molecules. The number of CA molecules that can be loaded in the pore of Mag-S-MCF was calculated by dividing the pore volume with the unit volume of a CA molecule. The number of CA was converted into the weight of CA by using Avogadro s number (6.02 x mol -1 ) and molecular weight of CA (30,000 Da). 3 The weight percent of CA loading was calculated by following equation. Weigth percent of CA loading in Mag S MCF = Weight of loaded CA Weight of Mag S MCF + Weight of loaded CA 100 The theoretical maximum loading of CA in Mag-S-MCF was calculated as 32.24% (w/w). 2

3 Bovine Serum Albumin Pre-Loading in Mag-S-MCF The theoretical maximum weight of bovine serum albumin (BSA) that can fill the pores of Mag-S-MCF was calculated by the aforementioned method with modified parameters. For example, 14.0 x 4.0 x 4.0 nm 3 and 66,000 Da were used for the unit volume and molecular weight of BSA, respectively. 4 The maximal BSA loading in Mag-S-MCF was calculated to be 0.6 mg of BSA in 1.0 mg of Mag-S-MCF, which can be represented by 37.6% (w/w). 3

4 Table S1. Enzyme kinetics of the immobilized α-chymotrypsin (CT) in Mag-S-MCF. Sample KM (mm) kcat* (sec -1 ) kcat/km** (mm -1 sec -1 ) Free CT 0.11 ± ± ± ADS 0.33 ± ± ± 0.05 NER 0.48 ± ± ± 0.18 p-ner 0.27 ± ± ± % (v/v) BSA pre-loaded p-ner 0.23 ± 0.00 n.d.*** n.d.*** * The kcat values of free CT, ADS, NER and p-ner were 18.61, 2.66, 0.90 and 1.19 sec -1, respectively. Immobilized CT resulted in decreased kcat values when compared to free CT due to the reduced enzyme flexibility upon the immobilization. Smaller kcat value of NER than ADS can be explained by the enzyme denaturation during the crosslinking. Marginally increased kcat value of p-ner than NER can be explained by the prevention of enzyme denaturation during the immobilization process by forming closely-packed CLEAs. ** The overall catalytic efficiencies, kcat/km, of free CT, ADS, NER and p-ner were , 8.12, 1.90 and 4.35 mm -1 sec -1, respectively. All the immobilized CT samples exhibited lowered catalytic efficiencies than free enzyme due to the decreased mass transfer and enzyme flexibility. *** kcat and kcat/km of p-ner with 100% (v/v) BSA pre-loading could not be determined because CT was mixed with BSA, and the elemental analysis cannot give information of each protein amount. 4

5 Figure S1. N2 adsorption-desorption isotherm (a), Barrett-Joyner-Halenda (BJH) adsorption profile (b) and BJH desorption profile (c) of Mag-S-MCF for the determination of pore shape, pore diameter and window diameter, respectively. (d) Pictures showing the magnetic capture of Mag-S-MCF. The N2 adsorption-desorption isotherms followed the configuration of type IV, which represents the formation of mesopores. The shape of hysteresis implies that the pore was formed in the bottleneck-like shape (Classification of International Union of Pure and Applied Chemistry). The BJH adsorption and desorption profiles reached their highest volume around the diameters of 40 nm and 12 nm, respectively, which correspond to pore and window diameters, respectively. 5

6 Figure S2. FE-SEM images of Mag-S-MCF (a), ADS (b), NER (c) and p-ner (d). 6

7 Figure S3. Enzyme adsorption (a) and desorption (b) during the preparation of ADS sample. 7

8 Figure S4. FE-SEM images of p-ner (a), p-ner-r1 (b) and p-ner-r2 (c) with two different magnifications of 3,000 (upper) and 10,000 (bottom). 8

9 Figure S5. Stability of free carbonic anhydrase (x), ADS ( ), NER (blue ) and p-ner (red ) at room temperature under shaking (200 rpm) up to 25 days. Both NER and p-ner showed the biphasic inactivation kinetics while free CA and ADS showed monotonous inactivation of enzyme activity. 9

10 Figure S6. Schematic illustration for the preparation of p-ner and p -NER. p-ner was prepared by simultaneously adding ammonium sulfate and glutaraldehyde, while p -NER was prepared with the sequential addition of ammonium sulfate and glutaraldehyde. 10

11 Figure S7. FE-SEM images of p -NER with three different magnifications of 1,000 (a), 10,000 (b) and 30,000 (c). p -NER showed less population of external CLEAs on Mag-S- MCF compared to p-ner. 11

12 Figure S8. Hydratase activities of NER (blue ) and p-ner (red ) with the BSA preloading. In the first reactor, NER and p-ner produced the bicarbonate solution by their hydratase activities hydrating the bubbling CO2 into the bicarbonate for 1 min. In the second reactor, the calcium carbonate was formed from the reaction of calcium ions with the bicarbonate solution for 90 min. The dry weight of calcium carbonate was used to represent the hydratase activity of NER and p-ner. The results of hydratase activities were more fluctuating than those of esterase activities in Figure 4b due to more complicated activity measurements. 12

13 Figure S9. Two reactor system for enzymatic CO2 conversion and utilization. The first CO2 bubbling reactor was equipped with a ph titrator to maintain ph at 7.6, and immobilized carbonic anhydrase was captured using a magnet after the reaction. The bicarbonate solution was transferred to the second reactor with calcium ions for the formation of calcium carbonate. 13

14 References (1) Hwang, E. T.; Lee, B.; Zhang, M.; Jun, S. H.; Shim, J.; Lee, J.; Kim, J.; Gu, M. B. Immobilization and Stabilization of Subtilisin Carlsberg in Magnetically-Separable Mesoporous Silica for Transesterification in an Organic Solvent. Green Chem 2012, 14, (2) Eriksson, E. A.; Liljas, A. Crystallization of and Preliminary X-ray Data for Bovine Carbonic Anhydrase III. J Biol Chem 1986, 261, (3) Nyman, P. O.; Lindskog, S. Amino Acid Composition of Various Forms of Bovine and Human Erythrocyte Carbonic Anhydrase. Biochim Biophys Acta 1964, 85, (4) Wright, A. K.; Thompson, M. R. Hydrodynamic Structure of Bovine Serum-Albumin Determined by Transient Electric Birefringence. Biophys J 1975, 15,