Proteolytic enzyme reactors

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

Macromolecular substrates such as proteins offer unique opportunities in processing modes with enzymes. Ultrafiltration membrane reactors (10) can be used to retain the protein substrate and the proteolytic enzyme in the reactor, while the hydrolytic products escape through the membrane to be collected. Using an ultrafiltration reactor, Cheftel (11) was able to solubilize 95% of FPC in 24 hr using pronase digestion. 

Immobilized enzymes and whole cells have found well-documented applications in industry, medicine, and analytical chemistry. Theoretically, it should be possible to carry out any enzymatic reaction with the help of the respective immobilized enzyme or whole cell containing the enzyme. The technique of using an immobilized enzyme for a chemical transformation is not basically different from using the soluble enzymes. In commercial applications, the immobilized enzymes can be used in a continuous-flow reactor. However, the optimum conditions for a specific reaction will have to be redetermined before maximum turn-over can be achieved. Thus, proteolytic enzymes such as trypsin, when immobilized on an anionic matrix such as cofpolyethylene-maleic anhydride), require a much lower pH for reaction than in solution. Some typical applications of immobilized enzymes that are currently being made, or are in the process of development, are mentioned in Table 15-1. 

To fulfill such requirements, attempts have been made in the past decade by researchers working on peptide mapping and proteomics through development of immobilized microfluidic enzymatic reactors. Microfluidic enzymatic microreactors are an alternative to in-solution method employing immobilization of proteases on microchaimels of chip-based reactors or surfaces of capillaries. The microreactors that enable proteolytic digestion by enzymes immobilized on solid supports are also referred to as immobilized enzyme reactors, IMERs. The great potential of IMERS for proteomic applications comprise rapid and enhance... 

Figure 10.18 Graphical representation of an experiment on the development of an enzyme reactor using electric field-mediated orientation and immobilization of proteolytic enzymes (t7psin/chymot7psin) on PVDF membranes in...

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