Prokaryotic expression and enzymatic activity of new Delhi metallo-beta-lactamase-4

Abstract

Objective To express the new Delhi metallo-beta-lactamase-4 (NDM-4) by using bioengineering techniques, determine the kinetic parameters of enzyme reaction with its hydrolyzed substrate, and to study the effects of pH and temperature on NDM-4 activity. Methods Clinically separated blaNDM-4 gene was amplified by polymerase chain reaction (PCR) and cloned into pET30a vector to construct a recombinant prokaryotic expression plasmid. The latter was transformed into E. coli BL21 (DE3) . The engineered bacteria strain[E. coli BL21 (DE3) -pET30a-NDM-4]was measured for resistance to various β-lactam antibiotics, and then used to induce NDM-4 expression. The overexpressed recombinant enzyme was purified by Ni2+-NTA column, identified by SDS-PAGE and mass spectrometry, and determined for the kinetic parameters, optimum pH and temperature of enzyme reaction with its hydrolyzed substrates. Results The recombinant plasmid pET30a-NDM-4 was verified by sequencing. The engineered bacteria strains harboring blaNDM-4 gene were resistant to β-lactam antibiotics. The relative molecular mass of the over-expressed recombinant enzyme was about 28 400. After purification, the NDM-4 protein was at a concentration of 0.4 g/L, presenting high hydrolysis activity against almost all β-lactam antibiotics including Carbapenems, particularly at the optimum pH of 7.5, and the optimum temperature of 35 °C. Conclusion NMD-4 with high hydrolysis activity against β-lactam ring was successfully overexpressed and obtained by using a prokaryotic expression system. This provides a material basis for further study of the action mechanisms and enzymatic properties of NMD-4. Key words: New Delhi metallo-beta-lactamase-4; Prokaryotic expression; Enzyme reaction kinetics; Enzyme activity