Purification and characterization of L-asparaginase from erwinia carotovora

An L-asparaginase from Erwinia carotovora was successively purified by ammonium sulfate precipitation, DEAE-cellulose and Sephacryl S-200 columns. The specific activity of the L-asparaginase was increased approximately 55-fold, from 15.5 to 852 U/mg proteins. SDS-PAGE showed that the purified L-asparaginase was homogeneous and the molecular weight was about 115 kDa. The isoelectric point [pI] of the enzyme was about 5.9. Characterization of the enzyme exhibited optimum pH and temperature of 8.4 and 40°C, respectively. The purified enzyme is able to prolong its thermal stability up to 50°C. A Lineweaver-Burk analysis showed a K[m] value of 0.154 mM and V[max] of 41.67 U. The purified enzyme was rich in glycine, alanine, glutamic acid and aspartic acid

Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters.

L-Asparaginase shows antileukemic activity and is generally administered in the body in combination with other anticancer drugs like pyrimidine derivatives. In the present study, L-asparaginase was purified from a bacteria Erwinia carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyl-4-phenyl-2-thioxo, 1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) was studied on the kinetic parameters Km and Vmax of the enzyme using L-asparagine as substrate. The enzyme had optimum activity at pH 8.6 and temperature 35 degrees C, both in the absence and presence of pyrimidine derivative and substrate saturation concentration at 6 mg/ml. For the enzymatic reaction in the absence and presence (1 to 3 mg/ml) of dihydropyrimidine derivative, Km values were 7.14, 5.26, 4.0, and 5.22 M, and Vmax values were 0.05, 0.035, 0.027 and 0.021 mg/ml/min, respectively. The kinetic values suggested that activity of enzyme was enhanced in the presence of dihydropyrimidine derivative.

Fermentative production and isolation of L-asparaginase from Erwinia carotovora, EC-113.

L-Asparaginase, an enzyme-drug used for the treatment of acute lymphoblastic leukemia was isolated from Erwinia carotovora. The effects of different carbon and nitrogen sources on the fermentative production of the enzyme were studied. Lactose, monosodium glutamate, corn steep liquor, tryptone and yeast extract showed significant stimulation of the production. When L-asparagine (0.2%), a substrate of the enzyme was added to a fermentation medium, a mutant strain EC-113 exhibited 6 times higher production indicating a distinct induction. The enzyme was extracted from the cells and purified about 30 fold to apparent homogeneity employing polyacrylamide gel electrophoresis. The methods used in sequence were DEAE cellulose chromatography, sephadex G-200 gel filtration, hydroxylapatite ion-exchange and affinity chromatography on sepharose CL-6B. The recovery of enzyme was 60%. The purified enzyme showed optimal pH at 8.0 and optimal temperature at 50 degrees C. The Km value of purified enzyme was 1.8 x 10(-5) M. LD50 of purified enzyme in mice by intravenous route was 4,80,000 IU/Kg and repeated treatment at 20,000 IU/Kg by intravenous route did not elicit bone marrow depression or damage to intestinal mucosa. The plasma half life was 14-24 hours and clearance time was 4-5 hours. Purified enzyme shows significant antitumor activity on experimental animal models.