Screening conditions should simulate the final application as closely as possible. This is especially a challenge when chromophore-free (e.g., aliphatic) substrates are used and no simple and reliable high-throughput method for quantitative analysis of the respective reaction product is available. The screening procedure should also be generally applicable for a certain class of enzymes. Generation of “surrogate substrates” by derivatization with chromogenic groups can have a negative effect on enzyme development by directed evolution, e.g., by causing a change in substrate specificity.
