title: Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.
abstract: 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 1.5mM 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 (158.1IU/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.
authors.affiliation: Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization, National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744103, Andaman and Nicobar Islands, India. Electronic address: bmeena79@yahoo.com.
authors.forename: Balakrishnan
authors.initials: B
authors.lastname: Meena
authors.affiliation: Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization, National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744103, Andaman and Nicobar Islands, India. Electronic address: anburajanl@yahoo.co.in.
authors.forename: Lawrance
authors.initials: L
authors.lastname: Anburajan
authors.affiliation: Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization, National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744103, Andaman and Nicobar Islands, India.
authors.forename: Nambali Valsalan
authors.initials: NV
authors.lastname: Vinithkumar
authors.affiliation: Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Government of India, Chennai 600100, Tamil Nadu, India.
authors.forename: Divya
authors.initials: D
authors.lastname: Shridhar
authors.affiliation: Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Government of India, Chennai 600100, Tamil Nadu, India.
authors.forename: Rangamaran Vijaya
authors.initials: RV
authors.lastname: Raghavan
authors.affiliation: Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Government of India, Chennai 600100, Tamil Nadu, India.
authors.forename: Gopal
authors.initials: G
authors.lastname: Dharani
authors.affiliation: Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Government of India, Chennai 600100, Tamil Nadu, India. Electronic address: headmbt@niot.res.in.
authors.forename: Ramalingam
authors.initials: R
authors.lastname: Kirubagaran
mesh_terms: D039903:Actinobacteria / Q000235:genetics*; D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use*; D061249:Batch Cell Culture Techniques; D053898:Biosynthetic Pathways / Q000235:genetics; D003001:Cloning, Molecular; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000627:therapeutic use*; D017422:Sequence Analysis, DNA; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D001215:Asparaginase
keywords: Actinobacteria; Glutaminase-free; Nocardiopsis alba; ansA gene; l-Asparaginase