(A) Acylation activity of biapenem with LdtMt2 and mutants R209E, H336N, S351A, H352N, and C354A was monitored at 292 nm wavelength using UV–visible spectrophotometry. Maximum absorbance spectra of biapenem were found at 292 nm that was used to monitor decrease in biapenem concentration upon acylation with the LdtMt2. The chemical structure of biapenem is shown above the biapenem acylation graph. (B) Rate of acylation of 50 µM biapenem per second with LdtMt2 and mutants. (C) ThermoFluor assays for binding of biapenem with LdtMt2 and mutants R209E, E207A, Y330F, S351A, H336N, and C354A mutants. A change in melting temperature (∆Tm) was plotted at y-axis verses the ligand concentrations at x-axis in GraphPad Prism software. (D) Differential fluorescence (−dF/dT) graphs of ThetrmoFluor assay for LdtMt2 and mutants. The dotted line indicates the Tm, and a red arrow indicates the direction of thermal shift. (E) Molecular dynamic (MD) simulations of LdtMt2 in complex with biapenem. LdtMt2 is represented in cartoon with green colour at 0 ns and cyan colour after running the MD simulations at 40 ns, and biapenem is represented in stick model with pink colour. The red arrow indicates the movement of biapenem to a second position revealed by the MD simulations after 40 ns trajectory.