Peptide amidases are enzymes that convert C-terminal amides of amino acids or peptide into corresponding acids. We investigated amidases for making peptide esters from peptide amides that could be used for chemoenzymatic peptide synthesis. The strategy would enable cheaper and greener production of peptides. However, there are limitations for practical use of peptide amidases in this scheme. Amidases prefer to synthesize peptide acids instead of peptide esters in the presence of water and are very sensitive to alcohol when added to the reaction medium. Furthermore, they are not industrially suitable due to moderate thermostability and organic solvent tolerance. In this thesis, we cloned novel peptide amidase from soybean (SbPam). Then we genetically engineered thermostable variants of SbPam and another cloned peptide amidase (Pam). Both Pam and SbPam were improved to withstand higher temperatures and organic solvents in the reaction medium. We demonstrated that peptide amidases could be successfully used for the synthesis of activated peptide methyl esters in organic solvents with minimum amount of water present. The discovery enables us to use peptide amidase to provide activated precursors that can be used in chemoenzymatic peptide synthesis.
|Qualification||Doctor of Philosophy|
|Place of Publication||[Groningen]|
|Publication status||Published - 2018|