Fig. 5. Lipase-catalyzed kinetic resolution of rac-2-pentanol.

3. To follow the reaction, 20 ||L aliquots should be taken from the reaction mixture at regular appropriate times and suspended in 1 mL of hexane (see Note 5). The resulting biphasic mixture is strongly shaken for 3 min to extract all substrates and products into the hexane phase. Finally, mix 200 |L of hexane extract with 100 |L of 300 mM ethyl propionate (internal standard) solution in hexane, and chromatographically analyze 1 |L of the resulting solution. Recover all the IL aliquots into a test tube for cleaning and reuse (see Subheading 3.6.).

4. Analysis of samples can be carried out by a GC P-DEX-120 capillary column (30 m x 0.25 mm x 0.25 |im; Supelco) and a flame ionization detector. Chromatographic conditions are as follows: carrier gas (He) at 1 MPa (205 mL/min total flow); temperature program: 60°C, 16 min, 15°C/min, 90°C; split ratio, 100:1; detector, 230°C. Retention times of the peaks are as follows: vinyl propionate, 6.3 min; ethyl propionate, 8.3 min; R-2-pentanol, 15.3 min; S-2-pentanol, 15.8 min; R-2-pentyl propionate, 22.2 min; S-2-pentyl propionate, 23.2; propionic acid, 25.1.

5. To extract all the product and remaining substrates, the reaction mixture should be washed with hexane. For this, add a 30-fold excess volume of hexane to the reaction mixture and shake vigorously for 15 min to give liquid-liquid extraction.

6. The resulting CALB-bmim+ NTF2- system can be reused in several reaction cycles by adding fresh substrates, remaining practically constant the enzyme activity.

3.4. P-Galactosidase-Catalyzed N-Acetyllactosamine Synthesis in mmim+ MeSO4- (22)

1. Start the reaction by adding 1 mL of 1 % (w/v) P-galactosidase solution in 0.1 M potassium phosphate buffer, pH 7.3 (see Fig. 6).

2. Mix mechanically the reaction mixture (e.g., by a rotary disk shaker) at 25°C for up to 2 h to reach as least a half product yield (see Note 6).

3. To follow the enzyme reaction, take 100-|L aliquots from the reaction mixture at appropriate intervals of time, and heat to 100°C for 10 min to stop the reaction by thermal deactivation (see Note 7). Filter samples to remove proteins with a Minisart RC 4 system (Sartorius). The resulting clear solution is analyzed chro-matographically.



Fig. 6. P-Galactosidase-catalyzed #-acetyllactosamme synthesis by transglycosilation.


Fig. 6. P-Galactosidase-catalyzed #-acetyllactosamme synthesis by transglycosilation.

4. Analyze samples by HPLC using a Aminex HPX-87H column (25-cm length and 3.9-mm internal diameter, 5-|im particle size, and 10-nm pore size, BioRad) in isocratic conditions at 0.8 mL/min flow rate and 60°C. The mobile phase will consist of 6 mM sulfuric acid solution in water. Elution profiles can easily be monitored by using RI or UV (208 nm) detectors.

3.5. Immobilized CALB-Catalyzed Polyester Synthesis in bmim+ PF6- (23)

1. Mix both momomers solutions and start the reaction by adding Novozym 435 (see Fig. 7 and Note 8).

2. Mix the reaction mixture mechanically (e.g., by a rotary disk shaker) at 50°C for less then 24 h to observe the precipitation of polymeric product.

3. Recover the polyester product from the IL reaction medium, and re-dissolve it with tetrahydrofuran (see Note 9). The molecular weight of re-dissolved polymeric products can be analyzed by gel permeation chromatography.

4. Analysis of samples can be carried out by a HPLC PL-gel MIXED E column (250-mm length and 4.6-mm internal diameter, 3-|im particle size; Polymer Laboratories) in isocratic conditions at 1 mL/min flow rate and 35°C, using tetrahy-drofuran as mobile phase. Elution profiles can easily be monitored by RI detector (see Note 10).

3.6. Recovery and Cleaning of Ionic Liquids

1. For each of the proposed reactions, all the respective IL fractions, containing IL, enzymes, traces of reagents, and so forth, should be collected into a specifically labeled vessel.

2. For ILs immiscible with water, dissolve the IL fraction with a volume of acetoni-trile (dilution 1:1, to reduce viscosity), and then filter the resulting solution through an ultrafiltration cell (Amicon, Millipore) equipped with a YM 3 membrane (cut-off 3000 Da.) at 6 bar (pressurized with N2) and room temperature to separate the enzyme molecules (see Fig. 2).

3. Remove solvents by vacuum evaporation to concentrate the IL.

4. Add water in a 10-fold excess. Shake strongly the IL-water biphasic mixture for 3 min and decant, to extract any water-soluble compounds. Repeat this washing step twice.


O Divinyl Adipate +

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