Proteins immobilized monoliths

Monolithic silica columns with various surface derivatization, including ion exchange (Xie et al., 2005), and chiral functionalities (Chen et al., 2002 Lubda et al., 2003 Chankvetadze et al., 2004), as well as protein-immobilized monoliths (Kato et al., 2005) have been reported. The latter will be an important part of an integrated multidimensional separation/identification system. 

Pan Z, Zou HF, Mo W, Huang X, Wu R. Protein A immobilized monolithic capillary column for affinity chromatography. Anal. Chim. Acta 2002 466 141-150. 

The positive effect of convection of the substrate solution on mass transfer can be observed even better with macromolecular substrates that undergo processes such as protein digestion. For example, Fig. 9 compares reversed-phase chromatograms of cytochrome c digests obtained by cleavage with trypsin immobilized in both packed and molded column reactors, and clearly demonstrates the much higher activity of the monolithic device under otherwise similar circumstances [90]. 

Bedair, M., and El Rassl, Z. (2004). Affinity chromatography with monolithic capillary columns I. Polymethacrylate monoliths with Immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography. /. Chromatogr. A 1044, 177-186. 

On-line hydrolysis of proteins catalyzed by trypsin or pepsin immobilized on monolithic silica beds was described by Kato et al. [86,195], whereas pepsin was encapsnlated into the silica-gel matrix (75 pm capillary column), without loss in enzymatic activity [195]. 

Svec, F. (2006) Less common applications of monoliths I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports. Electrophoresis, 27, 947. 

Peterson, D.S., Rohr, T., Svec, F., Frechet, J. M.J., Enzymatic microreactor-on-a-chip Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal. Chem. 2002, 74(16), 4081M088. 

E. Calleri, C. Temporini, E. Perani, C. Stella, S. Rudaz, D. Lubda, G. MeUerio, J. -L. Veuthey, G. Caccialanza, G. Massolini, Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins, J. Chromatogr. A, 1045 (2004) 99. 

Different high molecular mass affinity ligands were successfully immobilized to monolithic disks.Monolithic disks with immobilized heparin and collagen were used for the purification of membrane proteins and annexins. The heparin unit was also used in the quality control of the preparation of the plasma proteins Antithrombin 111 and clotting Factor IX. Purification of IgG was successfully performed by immobilizing Protein... 

The first report, in 1991, describes the immobilization of carbonic anhydrase. Interestingly, an increase in enzyme activity with the increase of flow rate through the bioreactor was observed. Recently, the immobilization of trypsin was reported. Contrary to the previous work, increased flow rate diminished the extent of protein degradation. In contrast to the previously mentioned experiments, where the immobilized enzyme was used for substrate degradation, the synthesis of polyriboadenylate from ADP was studied by polynucleotide phosphorylase immobilized on a monolithic disk. 

Comparison with Free Lipase and Novozyme To evaluate the catalyst performance, free lipase and a commercial immobilized lipase (Novozyme) were also tested. The results of the activity per gram of protein are plotted in Figure 11.10. The specific activity of immobilized lipase is lower than that of the free enzyme. It is known that the residual activity of an enzyme usually decreases significantly. A decrease in the rate observed can usually be ascribed to conformational changes, steric effects, or denaturation. For the monolithic biocatalysts, the immobilized activity was found to be 30 to 35%, and for Novozyme around 80% in the first run. Deactivation of the commercial sample in consecutive runs is probably due to the instability observed for the support matrix under reaction conditions... 

In addition to serving as the stationary phases, monolithic materials are also finding numerous other applications in the microfluidic world. Their use in on-chip solid-phase extraction and preconcentration,as supports for immobilization of enzymes to form enzymatic microreactors for protein mapping,static mixers,and valves represent just a few examples of modules in the microfluidic toolbox and further growth is inevitable. 

One solution to the challenges of mixing proteins in microfluidic systems is to immobilize proteins in microreactors [5]. These systems typically consist of chambers of enzymes immobilized on beads, micropillars [6], or porous polymer monoliths [7] (Fig. 2a and b). Such systems have large surface area-to-volume ratios, which minimize diffusion time for reactions with solution-phase reagents. Microreactors can be used either for the conversion of an analyte to another form that is more easily detected or for direct studies of the properties of enzymes and substrates. One of the most common uses is for the digestion of proteins for proteome profiling, but such systems can also be used for the removal of amino acid residues from peptides or proteins or for enzyme kinetic studies. 

Literature on enzyme microreactors for chemical synthesis is scarce. There is abundant literature, however, on the use of enzymes in microsystems for purposes of DNA analysis, e.g. using the polymerase chain reaction discussed before or DNA restriction fragment analysis [81], for proteomics, such as tryptic digestion of proteins with the enzyme free in solution [82], with trypsin-coated beads trapped in a microreactor [83] or trypsin immobilized on a porous polymer monolith (in a fused-silica capillary) [84]. Also, enzyme microreaction devices have been used extensively for medical diagnostics or immunoassays, e.g. using porous silicon as a support for immobilized enzymes [85]. 

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