Protein activity, directed evolution

B. Lingen, J. Grotzinger, D. Kolter, M. R. Kula, M. Pohl, Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein Eng. 2002, 15, 585-593. 

DNA shuffling is a powerful tool for directed evolution of gene products toward desired properties such as enhanced activity [12-15], improved protein folding [16-19], and altered substrate specificity [20-23]. We have shown that DNA shuffling is extremely useful for creating optimized enzymes, e. g., dioxygenases for the efficient degradation of environmental pollutants [24,25]. 

A breakthrough in recombinant DNA technology and protein engineering was achieved by recognizing that the process of natural selection can be harnessed to evolve effective enzymes in artificial circumstances. In this framework of directed evolution , the processes of natural evolution for selecting proteins with the desired properties are accelerated in a test tube. The starting point is an enzyme with a measurable desired activity which still has to be improved. 

L. You and F. H. Arnold, Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylform-amide, Protein Eng. 1996, 9, 77-83. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

All these approaches have been used to alter protein function, to increase the activity or solubility of proteins, or to adapt enzymes for industrial applications. The goal of artificial man-made proteins with tailor-made activities is, however, still far away and none of the currently existing approaches provides the ultimate solution to the directed evolution of proteins. Nevertheless, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. 

Directed evolution as a tool to probe the basis of protein structure, stability, and function is in its infancy, and many fruitful avenues of research remain to be explored. Studies so far have focused on proteins that unfold irreversibly, making detailed thermodynamic analysis impossible. The application of these methods to reversibly folding proteins could provide a wealth of information on the thermodynamic basis of high temperature stability. A small number of studies on natural thermophilic proteins have identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. The combination of directed evolu-... 

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