Figure 5: PGBD is essential for the optimum activity of LysinB_MF.

A) Domain architecture and comparison of native LysinB_MF and truncated LysinB_MFΔPGBD (EAD). B) The structure of LysinB_MF without the PGBD (LysinB_MFΔPGBD) was built using the native structure as a template. LysinB_MFΔPGBD structure comprises the linker region (in grey) and the EAD (in green). C-D) Comparison of the intact LysinB_MF and LysinB_MFΔPGBD demonstrates a change in electrostatic potential in the active site (indicated with a yellow arrow). E) SDS-PAGE (12%) analysis of purified LysinB_MF and LysinB_MFΔPGBD. Lane 1: protein molecular mass standards. Lane 2: purified recombinant protein LysinB_MF (36.3kDa). Lane 3: LysinB_MFΔPGBD (26 kDa). F) Immunoblot of N-terminal His-tagged LysinB_MF. Lane 1: protein molecular mass standards. Lane 2: N-terminal His-tagged LysinB_MF. G) A white zone indicates Ca²⁺-precipitation by LysinB_MF and LysinB_MFΔPGBD (5 μg each). H) Esterase activity of whole LysinB_MF and LysinB, determined by the PNP release assay (n=6). I) Spot test for intrinsic activity of LysinB_MF and LysinB_MFΔPGBD against M. smegmatis and M. fortuitum. All the assays were performed using 5 μg of each enzyme preparation.