Site-directed mutagenesis enzyme engineering

Estell, D.A., Graycar, T.P., Wells, J.A. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. /. Biol. Chem. 260 6518-6521, 1985. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

By means of genetic engineering, including cloning and site-directed mutagenesis, it has become possible for modern synthetic chemists to utilize a sufficient amount of isolated enzyme catalysts and to modify the reactivity, stability, or even specificity of enzymes. Therefore, polymerizations catalyzed by isolated enzyme are expected to create a new area of precision polymer syntheses. Furthermore, enzymatic polymerizations have great potential as an environmentally friendly synthetic process of polymeric materials. 

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. 

We have seen how oligodeoxynucleotide site-directed mutagenesis can be used to engineer the primary sequences of proteins, and examples were given of how this powerful technique can be used to engineer enzymes and their protein substrates. 

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. 

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