Rational mutagenesis

During the past decade, many enzymes have been improved by directed evolution using random mutagenesis, rational protein design, or via a combination of both methods [12, 13]. Early examples were committed to the enhancement of enzyme stability under unusual conditions such as in the presence of organic solvents or towards increased activity. More recently, directed evolution has been used for tlie creation of enantioselective biocatalysts or, even more, for the reversal of enantioselectivity of an enzyme-catalyzed reaction [72, 73]. 

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

Directed evolution methods, as well as rational structure-based mutagenesis approaches, have been successful in broadening the substrate tolerance of aldolases. A common goal is to... 

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

While the results of this work are encouraging, it is clear that the structural definition of mutant proteins of this type is critical to development of rational interpretation of the results if for no other reason than that the structural perturbation introduced is presumably greater than for simple point mutations. Moreover, it would be particularly interesting to compare the functional properties of mutants compared in this manner in assays involving protein-protein reactions relevant to the species of cytochrome c on which the mutagenesis is based. For example, comparison of the activities of wild-type yeast cytochrome c with that of a loop-insertion mutant modelled on a photosynthetic cytochrome c in the reaction with the photosynthetic reaction center could help define the structural elements involved in the cytochrome c binding domain for the reaction center. 

Identification of hot regions empirically or by rational design and subsequent application of cassette mutagenesis. 

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