Subtilisin protein engineering

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Transition-state stabilization in subtilisin is dissected by protein engineering... 

The subtilisin mutants described here illustrate the power of protein engineering as a tool to allow us to identify the specific roles of side chains in the catalytic mechanisms of enzymes. In Chapter 17 we shall discuss the utility of protein engineering in other contexts, such as design of novel proteins and the elucidation of the energetics of ligand binding to proteins. 

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

MW 27,500) with no cofactors or metal ions reqnirement for its function, it displays Michaelis-Menten kinetics and it is secreted in large amounts by a wide variety of Bacillus species. Subtilisin is also among the most important industrial enzymes due to its use in laundry detergents. Protein engineering strategies for subtilisin have focused on a number of aspects, namely catalysis, substrate specificity, thermal and oxidative stability and pH profile. We will describe briefly each of these aspects. 

Mitchinson, C. and Wells, J.A. (1989) Protein engineering of disulfide bonds in subtilisin BPN. Biochemistry, 28, 4807-4815. 

Siezen, R. J.,de Vos, W. M.,Leunisen, J. A. Dijkstra,B. W. (1991). Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Engineering, 4, 719-37. 

Subtilisin (see also serine proteases) 27, 28, 30, 450-454, 476 prosequence 540 protein engineering specificity of 452-454... 

The production of mutants by site-specific mutagenesis is the basis of the rational and systematic analysis of proteins and their redesign by protein engineering, as described in the next chapter for the tyrosyl-tRNA synthetase and subtilisin. 

The contribution of the triad to catalysis was quantified experimentally by protein engineering experiments on subtilisin.100 This is discussed in detail in Chapter 15, Part 2, section B. Replacement of the equivalent of Asp-102, His-57, and Ser-195 one at a time by alanine reduced the value of cat by factors of 3 X 104, 2 X 106, and 2 X 106, respectively. Converting all three to alanine also decreased activity by 2 X 106. 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtilisin Novo (Genencor, California). Lipase and cellulase types of detergent enzymes have also begun to appear. 

H. Bisgard-Frantzen, S. Carlsen, and J. M. Mikkelsen, Protein engineering of subtilisins to improve stability in detergent formulations, J. Biotechnol. 1993, 28, 55-68. 

Narinx, E., Baise, E., and Gerday, C. (1997). Subtilisin from psychrophilic antarctic bacteria characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Engineering, 10, 1271—1279. 

Bryan PN. Protein engineering of subtilisin. Biochim. Biophys. Acta 2000 1543 203-222. 

Russell, A., Thomas, P. G. and Fersht, A. R. (1987) Electrostatic Effects on the Modification of Charged Groups in the Active Site Cleft of Subtilisin by Protein Engineering, J. Mol. Biol. 193, 803-813. 

Zhao, H. M. and Arnold, F. H. (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Engineering, 12(1), 47-53. 

Bryan, P.N. Protein engineering of subtilisin. Biochimica et Biophysica Acta—Protein Structure and Molecular Enzymology 2000, 1543 (2), 203-222. 

Last but not least, it should be mentioned that a couple of peptidases have industrial importance. In particular, since subtilisins have a broad substrate specificity and are highly stable at neutral and alkaline pH they are of considerable industrial interest as protein-degrading additives to detergents. These reasons combined with their large data base make subtilisins attractive for protein engineering. Extensive engineering studies have been carried out on the Bacillus subtilins and more than 500 site-directed mutants have been produced to alter specific enzyme properties, such as pH profile, thermal stability or substrate specificity (see e. g. references[37 391). 

Importance of Conformational Variability in Protein Engineering of Subtilisin... 

Once enzymes have been modified, they are crystallized and their spatial structure is determined by x-ray crystallography to characterize the structural effects of the specific modifications. Molecular modeling helps to identify which positions in the molecule can be further modified by protein engineering to get a desired effect (specificity and affinity for a substrate, stability at a given pH and temperature). This approach has been used to alter the substrate specificity of subtilisin proteases, to broaden both their pH-activity and pH-stability profiles, and to increase their bleach stability [57]. 

Neumann, B., Zantl, N., Veihelmann, A., Emmanuilidis, K, Pfeffer, K, Heidecke, C-D., Holzmann, B. (1999). Mechanisms of acute inflammatory lung injury induced by abdominal sepsis. Int Immunol, Vol. 11, No. 2, pp. 217-227, ISSN 1460-2377 Nielsen, P.K., B0nsager, B.C., Fukuda, K, Svensson, B. (2004). Barley a-amylase/subtilisin inhibitor structure, biophysics and protein engineering. Biochimica et Biophysica Acta - Proteins Proteomics, Vol. 1696, No. 2, (February 2004), pp. 157-164 ISSN 1570-9639... 

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