Trypsin engineering

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

In addition to structure stabilization and catalytic applications, transition metal-binding sites may be designed and exploited for regulatory purposes. For example, a regulatory metal-binding site has been engineered into the active site of trypsin, where metal binding inhibits proteo-... 

Even though, the immobilization procedure should be engineered to maximum retained activity of immobilized enzyme, it is difficult to measure the amount of active enzyme on the carrier without an active site titration. However, this has been done in the case of immobilized trypsin, although only covalent immobilized. (Daly and Shih, 1982)... 

Kotzia, G. A., Lappa, K. and Labrou, N. E. (2007). Tailoring structure-function properties of L-asparaginase engineering resistance to trypsin cleavage. Biochem J. 404(2), 337-343. 

The seeds of squash plants are rich in a family of trypsin and chymotrypsin inhibitors that are approximately 35 amino acids in size and have been extensively investigated not only for their enzyme inhibitory activity, but also because they are very stable mini-protein scaffolds with applications in protein engineering. The best studied examples are Ecballium elaterium trypsin inhibitor (EETI-II) and Cucurbita maxima trypsin inhibitor (CMTI). Both X-ray and NMR have been used to characterise their structures, which incorporate a cystine-knot motif formed by three conserved disulphide bonds.93 We will describe this motif in more detail in a later section describing the plant cyclotides. 

An alternative hypothesis is that ER retention of Z-a,-anti trypsin results in autophagy, specifically of hepatic mitochondria. The basis for this hypothesis is the increase in autophagosomes in cells engineered for inducible expression of Z-tx,-antitrypsin. The mutant protein, along with the chaperone molecule calnexin, can be found in these autophagosomes by immune electron microscopy. It is postulated that mitochondrial dysfunction results from the damage to the mitochondria in the PIZZ liver, leading to the hepatic injury. 

Engineering members of the serine protease family which structurally consist of two homologous, stacked /1-barrels, Hopfner and coworkers designed a hybrid protease by fusing the N-terminal /1-barrel from trypsin to the C-terminal /1-barrel of factor Xa [74], The location of the fusion point in the linker region between the two /1-barrels was chosen following close inspection of the X-ray structures of trypsin and factor Xa. The resulting hybrid was shown to be fully functional as it hydrolyzed a broader but distinct spectrum of peptide substrates in comparison to the parental enzymes. 

S.Q. Yang, C.S. Craik, Engineering bi-dentate macromolecular inhibitors for trypsin and urokinase-type plasminogen... 

FIGURE 4.13 A MALDI-TOF mass spectrum acquired in reflectron mode showing the tryptic peptides from a digestion of bovine serum albumin. The m/z of the monoisotopic ions are assigned and can be entered into a protein database search engine to match to the theoretical trypsin digest of BSA. 

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. 

---