Protein engineering directed evolution

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Hamamatsu, N., Aita, T., Nomiya, Y. et al. (2005) Biased mutation-assembling an efficient method for rapid directed evolution through simultaneous mutation accumulation. Protein Engineering Design Selection, 18, 265-271. 

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

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. 

Protein Engineering in Comparison with Directed Evolution Aminotransferases... 

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. 

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