Expression and characterization of recombinant L-asparaginase from Pseudomonas fluorescens.
IR@CFTRI: CSIR-Central Food Technological Research Institute, Mysore
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 |
|