Immobilization of γ-glutamyl-transferase from bacillus subtilis for the synthesis of biologically active peptide derivatives from batch to continuous flow bioprocessing

gamma-Glutamyl peptides (gamma-E-peptides) are raising increasing interest due to their ability to elicit kokumi taste perception. Moreover, gamma-E-peptides possess a number of biological properties (e.g., antioxidant, anticancer, anti-inflammatory and lipid-lowering activity)[1]. The production of gamma-E-derivatives by conventional peptide chemistry is wearisome due to the extensive use of protective groups. Biocatalysis provides, instead, a more straightforward route: gamma-glutamyl transferases (GGTs, E.C. 2.3.2.2) catalyze the transfer of gamma-E-moieties from glutamine or other gamma-Edonors to both natural and modified amino acids/peptides, thus producing gamma-E-derivatives The GGT from Bacillus subtilis (BsGGT) was covalently immobilized on glyoxyl-agarose (GLX-AG) resulting in high protein immobilization yield and activity recovery (>95%). BsGGT-GLX-AG was shown to be a robust biocatalyst in the batch synthesis of biologically active gamma-E-peptides (1-4) in fully aqueous medium. Compounds 1-4 were obtained in moderate isolated yields (19-40%, 5-10 g L-1) with some contamination of 5 (0-20%). BsGGT-GLX-AG was recovered and re-used for two consecutive reaction cycles; the biocatalyst retained 90% activity under storage conditions (10 months, 4 °C). Based on these results, we moved from batch to continuous flow biotransformation with the aim to control side-reactions (i.e., donor hydrolysis to glutamic acid and autotranspeptidation, 5-7) and to achieve process intensification. gamma-E-S-Allyl-cysteine (2) was used as the acceptor compound for in flow experiments. Residence time, temperature, pressure, concentration and molar ratio donor/acceptor were screened. Under the optimized conditions to date, the productivity was increased by a two-fold factor (7.8 micromol min-1 g -1 ) and the impurity profile was improved as well. Further optimization under continuous flow conditions is currently in progress.