Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation.
The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.
Biochemistry Department, Faculty of Science, Alexandria University, Mohram Byk, Alexandria, Egypt. elbessoumy@hotmail.com
Ashraf A
AA
El-Bessoumy
Mohamed
M
Sarhan
Jehan
J
Mansour
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001215:Asparaginase / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D004795:Enzyme Stability; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013696:Temperature
D000596:Amino Acids; D001426:Bacterial Proteins; D001215:Asparaginase