Non-canonical amino acids (ncAAs) have shown considerable potential in enzyme engineering to improve catalytic activity, modulate enzyme active sites, probe complex enzyme mechanisms, create new to nature enzymes, and enhance thermostability. In this study, we report site-specific incorporation of the photocrosslinking ncAA: p-benzoyl-phenylalanine (pBpA) at the dimeric interface of the transaminase from Vibrio fluvialis (TAVF) and the effect of bulky ncAA incorporation on its functionality has been evaluated. Incorporating the pBpA at selected sites of TAVF led to the destabilization of the functional dimer and improper folding of variants. Two mutants, V31pBpA and K73pBpA, were isolated in the soluble form. The incorporation of bulky pBpA showed detrimental effects on the enzyme activity. Also, we determined the photocrosslinking efficiency of the incorporated pBpA, which showed a cross-linked variant with only 31pBpA highlighting the importance of the precise selection of the incorporation site for ncAA incorporation. Here we demonstrated the ncAA incorporation at a single site of the enzyme and its effects on the enzyme activity.