Engineering Enzymes for Non-natural Environments

Directed evolution has generated a large number of thermostabilized enzymes (see Table 4-3 for examples) there are too many reports for a comprehensive review here. We will discuss only the general picture that arises from those studies the interested reader is referred to a recent review that deals with evolution of enzyme stability in greater detail12t3). 

Creating enzymes that are both more thermostable and more active is particularly exciting for industrial applications. In addition, these studies nicely demonstrate that behaviors of natural enzymes may not necessarily be due to physical limitations intrinsic to proteins themselves, as is often assumed. Instead they reflect what is both relevant to the organism and accessible to natural evolution 184l 

Directed evolution has also been very effective for increasing enzyme activity in organic solvents 14 For example, the serine protease subtilisin can catalyze specific peptide syntheses and transesterification reactions, but organic solvents are required to drive the reaction towards synthesis. Sequential rounds of error-prone PCR and visual screening yielded a subtilisin variant with twelve amino acid substitutions that was 471 times more active than wild-type in 60% dimethylforma-mide (DMF)[145- 22° this enzyme is much more effective for peptide and polymer synthesis. 

High product concentrations are important in organic synthesis but often detrimental to enzymes. Scientist at Celgene reduced product inhibition in transaminases12211 which are valuable for the production of chiral amines or amino acids. A single round of error-prone PCR and screening of 10000 clones revealed mutants with better product tolerance that translated to a four-fold increase in volumetric productivity for a substituted amphetamine. 

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