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Monolithic Bioreactors for Macromolecules 259
2. CIM Convective Interaction Media ® imidazole carbamate-activated groups
containing poly (glycidyl methacrylate-co-ethylene dimethacrylate) monolithic
columns (BIA Separations) with a diameter of 12 mm and thickness of 3 mm
(monolith volume 0.34 ml) having median pore size of approximately 1.5 or
6 μm (Carbonyl diimidazole (CDI) CIM disk).
3. Trypsin from bovine pancreas, type XI, lyophilized powder, ≥6000, Nbenzoyl-L-arginine
ethyl ester hydrochloride (BAEE) units/mg protein (Sigma,
Taufkirchen, Germany).
4. Deoxyribonuclease I (DNase I) from bovine pancreas (Sigma).
5. Highly polymerized calf thymus DNA (0.006–0.08 g/l) (Sigma).
6. Ribonuclease A (RNase A) (Sigma).
7. BAEE, 3×10 −4 M (Sigma).
8. Cytidin-2,3-cyclic monophosphate (Sigma).
9. BCA protein assay (Sigma).
10. Benzamidine hydrochloride, 50 mM.
11. Tris–HCl buffer, 50 mM, pH 9.
12. Borate buffer, 0.1 M, pH 8.
13. Tris–HCl buffer, 20 mM, pH 8.
14. Acetate buffer, 50 mM, pH 5, containing 1 mM CaCl 2 .
15. Tris–HCl buffer, 50 mM, pH 7 and 9, containing 1 mM CaCl 2 .
16. Tris–HCl buffer, 10 mM, pH 7.5, 2 mM EDTA, 0.1 M NaCl.
17. Tris–HCl buffer, 40 mM, pH 8, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.1 M NaCl.
18. Tris–HCl buffer, 40 mM, pH 8, 1 mM MgCl 2 , 1 mM CaCl 2 .
2.1. Equipment
1. HPLC system KNAUER (Berlin, Germany).
3. Methods
Methods described below outline (i) static and dynamic immobilization
method on CDI and epoxy-activated monolith (see Subheading 3.1.); (ii) determining
biological activity of immobilized enzymes (see Subheading 3.2.); (iii)
immobilization of DNase (see Subheading 3.3.), RNase (see Subheading 3.4.)
and trypsin (see Subheading 3.5.); and (iv) application of DNase or RNase
reactor for removal of DNA or RNA from sample (see Subheading 3.6.).
3.1. Immobilization Methods
Two methods, static (see Subheading 3.1.1.) and dynamic (see Subheading
3.1.2.), could be used for enzyme immobilization (see Note 1). The monolith
has a disk shape (CIM disk) and can be immobilized as such or when placed
in a CIM housing forming CIM disk monolithic column (6,7).