Immobilization of Proteases

A protocol to improve pepsin immobilization was proposed by Busby et al. [17], Rather than directly using pepsin, they immobilized pepsinogen to POROS AL-20 support under optimal coupling conditions of pH 6.7 where pepsinogen is stable (in contrast with pepsin). Subsequently, they converted the coupled pepsinogen into active pepsin. Protein assays demonstrated that more enzyme was bound to the POROS AL-20 resin coupled with pepsinogen at pH 6.7 than pepsin at pH 5. 

Compared to X-ray crystallography or NMR spectroscopy, the spatial resolution obtained with HX-MS is very low about 10 amino acid residues on average, depending on the cleavage sites obtained with pepsin. One way to easily improve the resolution was already shown at the early stage 

Example of two overlapping peptides obtained after proteolysis of myoglobin with pepsin. 

By calculating the difference in deuterium incorporation between these two peptides, it is possible to obtain the deuterium incorporation of region AQGAMTKA. In few lucky cases, it was even possible to obtain the information for just one amide [22], even though a recent article has shown a limitation of this approach caused by variable back exchange [23]. If this resolution improvement can be obtained with pepsin, it can be more frequently met by using a second protease showing a specificity different from the one of pepsin. 

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Commonly Employed Matrices for Immobilization of Proteases and Hydrolases by Adsorption for Use in Nonaqueous Enzymology ... 

Silva, C.J.S.M., Zhang, Q., Shen, J., Cavaco-Paulo, A., 2006. Immobilization of proteases with a water soluble-insoluble reversible polymer for the treatment of wool. Enzyme Microb. Technol. 39, 634-640. 

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... 

R. B. Van Breemen, M. G. Bartlett, Y. Tsou, C. Culver, H. Swaisgood, S. E. Unger, Degradation of Peptide Drugs by Immobilized Digestive Proteases , Drug Metab. Dispos. 1991, 19, 683-690. 

Existing uses of proteases in foods have been discussed in the foregoing section. Expanding such applications in the future depends upon our ability to control both the processes themselves and their costs. The development of continuous reactors utilizing free or immobilized enzymes will address each of these constraints. Furthermore, our understanding of the chemical basis for the various functional properties of proteins must be expanded... 

Because of the large number of proteases it inhibits, WT (wild type) eco has become useful as a biochemical tool. As a selective inhibitor of serine proteases, eco has been used in cell culture assays to probe enzymatic activity as well as to titrate the percentage of catalytically active molecules of a previously uncharacterized serine protease [8]. Immobilized eco has been used for the direct purification of trypsinogen [9]. Finally, eco selectively identified a novel protease implicated in prostate cancer, membrane type serine protease I (MT-SPl) [10]. 

Immobilization of Enzymes. Enzymes (carboxypeptidase A, B, and Y, chymotrypsin, thermolysin, trypsin, and V -protease), obtained from Sigma were applied directly for immobilization. About 20 mg of each enzyme was dissolved into 0.1N phosphate buffer pH 7.0, and placed into a 10 x 75 mm test tube with 1 g of succinylamidopropyl glass beads. After degassing, 0.02 umole of l-ethyl-3-(3-dimethylamlnopropyl)-carbodilmide (EDC) (Sigma Chemical Co.) was added to the tube which then was sealed with paraffin and rotated at 4°C overnight for simultaneous activation/immobilization. 

A molecule of AMG contains four identical polypeptide chains, present as a pair of dimer subunits. The dimers are covalently linked by disulfide bonds, whereas the pairs are held together by noncovalent bonds. The dimer is the active unit one molecule of AMG can therefore bind up to two protease molecules. Each monomer contains both the thiol ester mentioned previously and a bait site, both of which are essential for inhibitory activity. After the bait site reacts with a protease, the thiol ester is activated and may form covalent bonds with lysine residues on the protease, thus immobilizing it. However, covalent binding is not essential for steric capture and inhibition of proteases. ... 

After immobihzation, the resistance toward protease increases. The reason may be an increase of steric access hindrance to the immobilized protease due to the three-dimensional obstacles of the carrier. If we take aminoacylase for example, under the action of trypsin, the activity of the solution enzyme was only 23%, the activity of the enzyme immobilized by DEAE-cellulose was 33% and the activity of the enzyme immobilized by DEAE-dextran was 87%. Under the action of protease, Pronase P, the activity of the solution enzyme, the enzyme immobilized by DEAE-ceUulose and the enzyme immobilized by DEAE-dextran was 48%, 53%, 88%, respectively. Immobilized protease can generally avoid digestion damaging itself. 

Chae, H.J. et al, Optimization of protease immobilization by covalent binding using glutaraldehyde, Appl. Biochem. BiotechnoL, 73,195, 1998. 

The same methods are also used to immobilize whole cells. Immobilization of cells avoids the tedious isolation and puriHcation of enzymes. The activity of the immobilized cells can be reduced by toxicity during immobilization and by hindered diffusion of the substrate. But the immobilized cells may also be more active than the free cells as well, since complete or incomplete destruction of the cell walls removes certain proteases from the cell which would otherwise degrade and thereby deactivate enzymes. For example, E. coli bacteria encapsulated in poly(acrylamide) gels are used commercially to convert sodium fumarate to L-aspartic acid. 

Modification of Polysaccharides and Oligosaccharides and Uses of Modified Polysaccharides and Oligosaccharides Introduction.—Derivatives of polysaccharides continue to be used widely as matrices for the immobilization of enzymes. Reviews have dealt with the chemistry of enzyme immobilization. -The use of immobilized proteases as... 

Similar to Figure 1, the palmitoyl (C-16) group has also been enzymatically grafted onto HEC. The reaction entails the incubation of HEC, vinyl palmitate, and a suitable enzyme in N,N-dimethylacetamide (DMAc) at 50°C. Several enzymes were attempted satisfactory results were obtained with Pseudomonas fluorescens lipase (Amano P-30), Pseudomonas capecia lipase (Lipase PS), or Alcalase immobilized alkaline protease from Novozymes A/S as a catalyst. Note that in this case a polar aprotic solvent (DMAc) is used, and one of the reactants, vinyl palmitate, has a fricile leaving group (vinyl alcohol which leaves as acetaldehyde). 

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