Protein engineering substrate specificity

Specificity for a particular charged substrate can be engineered into an enzyme by replacement of residues within the enzyme-active site to achieve electrostatic complementarity between the enzyme and substrate (75). Protein engineering, when coupled with detailed stmctural information, is a powerful technique that can be used to alter the catalytic activity of an enzyme in a predictable fashion. 

Wells, J.A., et al. Designing substrate specificity by protein engineering of electrostatic interactions. Proc. Natl. Acad. Sci. USA 84 1219-1223, 1987. 

To overcome these limitations, we have developed culture substrates that enable the highly efficient expansion of specific cells in adherent cultures [37, 85-88]. An important characteristic of these substrates is that specifically engineered growth factors are immobilized on the surface. Extensive protein engineering techniques were used to optimize the presentation of growth factors to cells. 

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. 

Wells, J. A., Cunningham, B.C., Graycar, T. P. Estell, D. A. (1987). Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering. Proceedings of the National Academy of Sciences USA, 84, 5167-71. 

Expansion of Substrate Specificity with Protein Engineering and Directed Evolution... 

In order to overcome limitations derived from the protein structure such as lacking stability, substrate or coenzyme specificity, protein engineering methods... 

Protein engineering has been carried out to redesign substrate specificity of lactate dehydrogenase from Bacillus stearothermophilus (Wilks et al., 1988 Wilks et ah, 1990) ... 

Aspartase exhibits incredibly strict substrate specificity and thus is of little use in the preparation of L-aspartic acid analogues. However, a number of L-phenylalanine analogues have been prepared with various PAL enzymes from the yeast strains Rhodotorula graminis, Rhodotorula rubra, Rhodoturula glutinis, and several other sources that have been cloned into E. call.243 241 Future work in this area will likely include protein engineering to design new enzymes that offer a broader substrate specificity such that additional L-phenylalanine analogues could be prepared. 

Yoshida, H., Kojima, K., Witarto, A. B., and Sode, K. (1999). Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase improvement of EDTA tolerance, thermal stability and substrate specificity. Protein Eng., 12, 63-70. 

In spite of their catalytic versatility and their capacity to transform a variety of pollutant compounds, peroxidases are not applied at large scale yet. The challenges that should be solved to use peroxidases for environmental purposes have been recently reviewed [146], Three main protein engineering challenges have been identified (a) the enhancement of operational stability, specifically hydrogen peroxide stability (see Chap. 11) (b) the increase of the enzyme redox potential in order to widen the substrate range (see Chap. 4) (c) the development of heterologous expression and industrial production (see Chap. 12). 

Svensson, B. 1994. Protein engineering in the a-amylase family Catalytic mechanism, substrate specificity and stability. Plant Molec. Biol. 25,141-157. 

The time is ripe for the widespread application of biocatalysis in industrial organic synthesis and according to a recent estimate [113] more than 130 processes have been commercialised. Advances in recombinant DNA techniques have made it, in principle, possible to produce virtually any enzyme for a commercially acceptable price. Advances in protein engineering have made it possible, using techniques such as site directed mutagenesis and in vitro evolution, to manipulate enzymes such that they exhibit the desired substrate specificity, activity, stability, pH profile, etc. [114]. Furthermore, the development of effective immobilisation techniques has paved the way for optimising the performance and recovery and recycling of enzymes. 

---