Directed enzyme variants

In order to increase the understanding of ThDP-dependent enzymes, the identification of amino acid side chains important for the catalysis of the carboligase reaction in pyruvate decarboxylase from Zymomonas mohilis (E.C. 4.1.1.1) and benzoylformate decarboxylase from Pseudomonasputida (E.C. 4.1.1.7) was a major task. Using site-directed mutagenesis and directed evolution, various enzyme variants were obtained, differing in substrate specificity and enantioselectivity. 

When taking samples for the determination of enantiomeric purity in a kinetic resolution, it is mandatory to choose the proper time window, e. g., when conversion is between 20% and 80%. If the process of directed evolution leads to enzyme variants differing widely in activity, mass screening needs to be time-resolved, i.e., several samples of a given reaction should be taken as a function of time. 

The key to the successful scale-up was the immobilization of the enzyme, which increased stability and reduced the enzyme costs to an acceptable level. The isomerization step is typically carried out in a parallel series of packed-bed reactors, where the enzyme is immobilized on silica or inert cellulose carriers (Figure 5.22). A ton of immobilized xylose isomerase can catalyze the production of 5000 tons of HFCS [35]. Current research is concentrating on developing a more thermostable enzyme variant which would reach the 5 5 45 ratio directly in the reactor column. 

Directed evolution has previously been used to generate enzyme-variants displaying improved properties such as higher activity and stability [11 - 17,19,81,91,92], The work summarized in this chapter shows that the difficult goal of controlling enantios-electivity of enzymes can also be reached by directed evolution [8 - 10]. In doing so, two major challenges had to be dealt with ... 

Fig. 13.3. Correlation of required mechanistic information, required structural information, importance of screening and number of possible enzyme variants in protein engineering by rational protein design and directed evolution.

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