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Expression and characterization of recombinant L-asparaginase from Pseudomonas fluorescens.

Sindhu, R. and Manonmani, H. K. (2018) Expression and characterization of recombinant L-asparaginase from Pseudomonas fluorescens. Protein Expression and Purification, 143. pp. 83-91. ISSN 1046-5928

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Abstract

l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 °C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified l-asparaginase had a specific activity of 26 U/mg with a Km and Vmax of 0.050 M and 4.032 mol−1min. The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg2+ enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells.

Item Type: Article
Uncontrolled Keywords: L-asparaginase EcoAns PfAns Pseudomonas fluorescens Recombinant enzyme Characterization
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 07 Enzyme Biochemistry
500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology
Divisions: Food Protectants and Infestation Control
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 18 Jan 2018 09:33
Last Modified: 18 Jan 2018 09:33
URI: http://ir.cftri.com/id/eprint/13320

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