Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy

• Novel glutaminase-free L-asparaginase producing Nocardiopsis alba NIOT-VKMA08 from marine sediments of Andaman and Nicobar Islands
• Cloning and heterologous expression of L-asparaginase biosynthesis gene (ansA) to achieve the stable inducible system that overproduce the glutaminase-free recombinant L-asparaginase
• Purified recombinant enzyme disclosed an elevated level of asparaginase activity (158.1 IU/mL)
• Glutaminase activity was not detected in the recombinant L-asparaginase, which could reduce the probable side effects during leukaemia therapy

l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5 mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1 IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37 °C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.