gamma-Glutamyltransferase (GGT) catalyzes the transfer of the gamma-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a gamma-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lidloop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-gamma-glutamic acid (gamma-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. colt GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. colt GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-gamma-glutamyl glutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. colt GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. colt and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity.