From Batch to Continuous Flow Bioprocessing : Use of an Immobilized J-Glutamyl-Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives

J-Glutamyl peptides (J-E-peptides) are raising increasing interest due to their numerous biological properties (e.g., taste enhancement, antioxidant, anticancer, anti-inflammatory and lipid-lowering activity). J-Glutamyl transferases (GGTs, E.C. 2.3.2.2) are naturally involved in the transfer of J-E-moieties from different J-Edonors to both amino acids and peptides, thus producing J-E-derivatives (Scheme). Scheme. GGT-Catalyzed synthesis of J-glutamyl-derivatives (1-4) The GGT from Bacillus subtilis (BsGGT) was covalently immobilized on glyoxylagarose (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 J-E-peptides (1-4) in fully aqueous medium (isolated yields: 19-40%, 5-10 g L-1). BsGGT-GLX-AG was recovered and re-used for five 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., 5-8) and to achieve process intensification. Under the optimized conditions to date, the productivity of the model compound J-E-S-allyl-cysteine (2) was increased by a two-fold factor (7.8 Pmol min-1 g-1) and the impurity profile was improved as well. Further optimization under continuous flow conditions is currently in progress.