Novel and ancestral α-amino acid ester acyltransferases with higher thermostability for dipeptide synthesis

α-Amino acid ester acyltransferase (AET) catalyzes the hydrolysis of Nα-unprotected amino acid esters (donor) and the transfer of the amino acid to an Nα, Cα-unprotected amino acid (acceptors), forming a dipeptide. Previous research had primarily focused on a limited number of dipeptides, specifically Ala-Gln and Asp-Phe. This study aimed to identify novel enzymes with broader specificity and higher thermostability. A sequence similarity network (SSN) was generated, and twenty-one AET-related enzymes were investigated. Initial screenings revealed that two sequences, referred to as SeqO from Myxococcus stipitatus, and Seq6 from Poliangium fumosum exhibited activity with a number of amino acid methyl esters. LC-MS analysis indicated that the observed activity only related to amino ester hydrolysis, but no peptide synthesis activity could be detected. A second approach constituted of a reconstruction of ancestral variants based on the phylogenetic tree calculated from the SSN. Eight ancestral sequences were synthesized and produced. All selected variants were active with most of the amino acid methyl ester donors. The most promising ancestral variants were N166, N256, and N281, which were chosen for further LC/MS-based analysis. The dipeptide Val-Trp, which has potential industrial importance and was not previously investigated, was synthesized with the highest yield by ancestor N281. The ancestral variants also displayed substantially higher thermostability compared to α-amino acid ester acyltransferase from Sphingobacterium siyangensis (SAET), with N166 showing an increase in melting temperature (T_m) of nearly 30°C. Although some dipeptides are synthesized with a lower yield than that obtained with SAET, their increased thermostability makes them better suited for industrial conditions.