title: Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.
abstract: L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Richi V
authors.initials: RV
authors.lastname: Mahajan
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Vinod
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Vinoth
authors.initials: V
authors.lastname: Rajendran
authors.affiliation: Technology Based Incubator, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Saran
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Prahlad C
authors.initials: PC
authors.lastname: Ghosh
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.
authors.forename: Rajendra Kumar
authors.initials: RK
authors.lastname: Saxena
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D001215:Asparaginase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D016466:CHO Cells; D045744:Cell Line, Tumor; D003412:Cricetulus; D004795:Enzyme Stability; D005972:Glutaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy; D013379:Substrate Specificity
chemical_list: D000970:Antineoplastic Agents; D001215:Asparaginase; D005972:Glutaminase