Trypsin, immobilization

In addition, it has been shown that other enzymes such as trypsin can be successfully immobilized and used for the conversion of substrates with higher molecular masses [76]. Petro et al. [94] compared the activity of trypsin immobilized on macroporous beads and on monolithic supports. They were able to show that the catalytic activity of trypsin bound to a monolith was much higher and resulted in a much higher throughput. Other enzymes such as invertase [76] and... 

Fig. 8. Effect of linear flow velocity of an L-benzoyl arginine ethylester solution (0.2 mol/1) on the enzymatic activity of trypsin immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads (curve 1) and monolith (curve 2) (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Reactor 50 mm x 8 mm i.d., temperature 25 °C...

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]. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

In addition, the GMA/EDMA copolymer proved to serve as a basic unit for the fabrication of highly permeable bioreactors in capillary format. Trypsin immobilization after epoxide ring opening with diethylamine and attachment of glutaraldehyde is mentioned as the probably most prominent example [64], The immobilization of trypsin was also carried out using another class of reactive monolithic methacrylate polymer, which is based on 2-vinyl-4,4-dimethylazlactone, acrylamide, and ethylene dimethacrylate [65]. In contrast to GMA/EDMA, trypsin can directly be immobilized onto this kind of monolith, as the 2-vinyl-4,4-dimethylazlactone moieties smoothly react with weak nucleophils even at room temperature. 

It was found that cytochrome c, (J-casein, and BS A can be digested by trypsin-immobilized beads in a microchamber on a glass chip. The protein (1 mg/mL) was mobilized by EOF. Although without external heating, Joule heating may assist in the enzymatic digestion reaction of the proteins [116]. 

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. 

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). 

Besides the benefits of scale reduction and trypsin immobilization, microsystem technology has other advantages to offer. For example, Ekstrom et al. [345] described a device that integrated an enzyme microreactor with a sample pretreatment robot and... 

Although a variety of (specific) proteolytic enzymes ate available, tiypsin is applied in most studies. Trypsin cleaves the protein at the C-terminal side of Lys or Arg, unless the next C-terminal amino acid is Pro. Other missed cleavage sites occur as well. Tryptic peptides are often indicated with Tx, where x is the number of the tryptic peptide, counted from the A-terminal side of the protein. The reaction can be performed in various ways homogeneous digestion, with tiypsin immobilized on beads, or by means of a trypsin immobilized enzyme reaction (IMER). 

D.S. Peterson, T. Rohr, F. Svex, J.M.J. Frechet, High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI-TOF and ESI- TOF-MS, J. Proteome Res., 1 (2002) 563. 

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. 

Immobilized trypsin-agarose was prepared as reported elsewhere (6). Trypsin immobilization was carried out in presence of borate buffer (50 mM) containing benzamidine (75 mM) at pH 10 and 25°C. After 72 hours, the trypsin-agarose was reduced with NaBH4 (Img/ml) for 30 minutes. Finally, the immobilized preparation was washed with water and stored at 4 C. The derivative obtained had around 320 units/ml gel using benzoyl-arginine ethyl ester (BAEE) as substrate. One unit of enzyme activity is defined as... 

Rocha, C., Ducso, L., Gonsalves, M. R, Teixeira, J. A. (2005). Spent-grains and zeolites as potencial carriers for trypsin immobilization, 4 Mercosur Congress on Process Systems Engineering Proceedings (CD-ROM), Costa Verde, Brasil. 

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]. 

Open-channel PDMS chip Trypsin Adsorption Poly(vinylidene fluoride) porous membrane with the adsorbed trypsin immobilized on channels of PDMS Cyt-C identified with as little as 0.04 pmol. [89]... 

Adsorption PMMA chip modified with zeolite nanopartides silanol functional groups introduced to PMMA and modified with silicalite-1 nanoparticle Trypsin immobilized using silica-sol-gel matrix (Si—O—Si bridge via tethering to silanol, groups) Cyt-C and BSA digestion at 4 ftl/min for 5 s... 

Entrapment Trypsin immobilized through a Si-0—Si bridge formed by tethering to... 

Open tubular capillary microreactor Trypsin Covalent linking Trypsin immobilized on carboxyl-modified beads digestion of Cyt-C, lactalbumin, enhanced by applying vibration using sinusoid wave from generator and piezoelectric transducer [116]... 

Open tubular capillary microreactor Trypsin Covalent linking Trypsin immobilization by photo-initiated polymerization on a portion of silica capillary digestion performance tested using carbonic anhydrase, Cyt-C, hemoglobin substrates, then subjected to peptide mapping [117]... 

Open tubular capillary microreactor Trypsin Covalent linking IMER in fused silica capillaries integrated with nano-electrospray emitter trypsin immobilized on surface of the inner wall of the fused silica capillary tubing fast on-line digestion and peptide mass mapping of angiotensin II, Cyt-C, haemoglobin, and p-casein [118]... 

Trypsin immobilized onto monolithic capillary colurrm prepared by in situ [125]... 

Monolith capillary microreactor Trypsin S. aureus V-8 protease (Glu-C) Trypsin +Glu-C Covalent linking Porous methacrylate base monoHth prepared by photografting with glycidyl methacrylate trypsin immobilized using carbonyldiimidazole digestions of Cyt-C, apo-myoglobin at flow rates from 0.2 to 1 pl/min, residence times of 0.24-1.4 min of 0.24-1.4 min [129]... 

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