Record Details

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

IR@CSIR-CFTRI

View Archive Info
 
 
Field Value
 
Relation http://ir.cftri.com/13320/
http://dx.doi.org/10.1016/j.pep.2017.09.009
 
Title Expression and characterization of recombinant L-asparaginase from Pseudomonas
fluorescens.
 
Creator Sindhu, R.
Manonmani, H. K.
 
Subject 07 Enzyme Biochemistry
02 Bacteriology
 
Description 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.
 
Date 2018
 
Type Article
PeerReviewed
 
Format pdf
 
Language en
 
Identifier http://ir.cftri.com/13320/1/Protein%20Expression%20and%20Purification%202017.pdf
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