Zymography assisted quick purification, characterization and inhibition analysis of K. pneumoniae alkaline phosphatase by mercury and thiohydroxyal compounds.

In-gel hydrolysis of para-nitrophenyl phosphate (p -NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with K m , V max and k cat values of (0.7 ± 0.02) mM, (80 ± 4.5) μmol min−1 and (39.2 ± 2.2) × 104 s−1 respectively for p -NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO 4 , Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO 4 and 65% activity at 3 mM Hg2+ showing it's PO 4 3− irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp -ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and β-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and βME reduce the C308–C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase. [Display omitted] • Zymography used as a guide for purification, characterization and inhibition analysis of 54 kDa alkaline phosphatase from a Hg2+ tolerant K. pneumonia e. • Zymography reveal that thiohydroxyals reduce it's C308–C358 disulfide bond resulting in an open conformation and loss of activity. • Hg2+ inhibits the enzyme by binding to the surface-accessible metal binding motifs without changing it's conformation. [ABSTRACT FROM AUTHOR]