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

ABSTRACT

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.