Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
For optimizing trypsin for peptide and protein synthesis, the oxyanion hole of the enzyme was engineered by rational mutagenesis. From the six mutated trypsin variants, two enzyme hits with an artificial proline mutation either in position 192 or in position 194 could be identified. Model reactions qualify both biocatalysts as efficient coupling enzymes not only for peptide but also for protein semisynthesis.