In this context, transglutaminases are enzymes able to catalyze an acyl transfer reaction between the carboxamide group of glutamine (Q) and the amino group of a lysine residue in the presence of Ca 2+ , resulting in the formation of an isopeptide bond and a release of ammonia. Amongst this family of enzymes, bacterial transglutaminases have raised as attractive targets for bioconjugation, due to their broader substrate specificity, their calcium independency and their smaller molecular weight. Streptomyces mobaraensis transglutaminase (mTG) is one of the most used transglutaminase in the food industry and has been intensively studied, leading to a deep understanding of its mechanism of action. It has been reported that site specific conjugation can be achieved with mTG, upon the insertion of the recognition sequence LLQG. Strop et al. used this sequence to successfully conjugate various payloads to antibodies and investigated the effect of the conjugation site on the ADC stability and pharmacokinetics. However, the heterologous expression of wild type mTG in Escherichia Coli (E. Coli) was shown to be challenging and only mutated enzymes produced in E. Coli were reported. As mTG paved the way for an enzymatic, site specific bioconjugation, another bacterial transglutaminase, the bacillus subtilis transglutaminase (bTG) has also been reported as a potential candidate for site specific conjugation. bTG was reported to recognize the sequence YAHQAHY, which could be inserted in a test protein and fluorescently labelled with dansylcadaverine. The bioconjugation reaction is also not restricted to the natural lysine substrates as the bTG enzyme has been shown to tolerate other types of primary amines. However, the recognition of the glutamine residue by a transglutaminase is more stringent and has been described as dependent on the accessibility of the Q amino acid, which should be located on a flexible region. This enzyme possesses the advantages of being easily produced in E. coli, is active under mild, aqueous conditions and does not require any additional cofactors. Attracted by these convenient features and by the apparent specificity of the bTG transglutaminase, we studied the requirements, biocatalytic potential and scope of this enzyme and report here a different sequence specific site found under peptide mapping experiments for the conjugation of various primary amines (Figure ). Figure . Aim of this work. In (1), peptide mapping experiments allow the identification of the amino acid bearing the dansylcadaverine moiety. After sequence determination, a protein showing no reactivity with the bTG enzyme (2) can be modified with the identified sequence and conjugated (3).