Purification and characterization of camel liver L-asparaginase

L-asparaginase from camel liver was isolated and purified by heat denaturation followed by QAE-Sephadex A-50 column chromatography and SP-Sepharose column chromatography. The purified camel liver L-asparaginase had a molecular weight of 180 kDa [consistent with a homotetramer] and a pI value of 8.6. The enzyme was thermostable with relative structure rigidity and an optimum temperature at 65°C. It had a pH optimum at 9.6 and was stable for storage at 4°C in the refrigerator for 7 days

Expression of recombinant escherichia coli L-asparaginase II, purification and characterization

L-Asparaginase [ASNase] is an anti-cancer [[antineoplastic] or [cytotoxic]] chemotherapy drug that is used for the treatment of acute lymphoblastic leukemia [ALL]. An efficient and economical scheme was developed for over expression and rapid purification of the Escherichia coli enzyme. The gene encoding for the Escherichia coli L-asparaginase was PCR-amplified and cloned in pGEX-4Tl expression vector. The recombinant L-asparaginase was purified to homogeneity by affinity chromatography on glutathione Sepharose column. The recombinant enzyme had an apparent MW of 152 kDa and a K[m] value of 12.5 microM for the main physiological substrate L-asparagine. The pI value was 5.6 while the turnover number [catalytic constant] was 1 x 10[2] s[-1] and the K[cat]/K[m] value [specificity constant] was 0.8 x 10[7] M[-1]s[-1]

Studies on the asparaginolytic activity of the brown-pigmented streptomycetes

Twenty-eight brown-pigmented streptomycetes were isolated from different fertile Egyptian soils. Screening was carried out according to their asparaginolytic activity under static culture condition on glycerol- L-asparagine [GA] medium. Results revealed that FS-39 isolate gave the highest asparaginolytic activity. Therefore, it was selected and subjected to complete identification. The cultural, morphological and physiological characteristics of this isolate indicated that it belongs to Streptomyces phaeochromogenes. The effects of nutritional and environmental conditions on the asparaginase activity of Streptomyces phaeochromogenes FS-39 were studied. Data revealed that the maximal, yield of L-asparaginase from this strain can be obtained by growing it on glycerol-L-asparagine yeast extract [GAY] medium containing [w/v] 2.0% Glycerol, 0.2% L-asparagine, 0.1% yeast extract, 0.1% K[2]HPO[4].3H20, 0.0001% FeSO[4].7H[2]0, 0.0001% MnCl[2]. 4H[2]0, 0.0001% ZnSO[4].7H[2]O which was initially adjusted to pH 7.0, inoculated by 2% [v/v] of homogenized spore suspension [containing approximately 3.2x10[7] spores/ml] of 3 days old culture on starch nitrate medimi and incubated at 30°C for 7 days under static culture condition

Purification and preliminary characterization of L-asparaginase from Erwinia aroideae NRRL B-138.

L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) from Erwinia aroideae NRRL B-138 has been purified to apparent homogeneity by ammonium sulphate precipitation, chromatography on sulfopropyl-sephadex C-50 and sephadex G-200 with 22% recovery and 567-fold purification. The enzyme obtained from sulfopropyl-sephadex C-50 was unstable and lost activity within a few hours. Addition of glycerol helped in restoring the activity of the enzyme. The enzyme has an apparent molecular mass of approximately 155 kDa and has four subunits of identical molecular mass of approximately 38 kDa. The K(m) for L-asparagine is 2.8 x 10(-3) M. Enzyme shows optimal activity at 45 degrees C and pH 8.2. Energy of activation as determined from Arrhenius plot was 9.1 kcal/mol. Substrate L-asparagine and analogue L-glutamine, D-asparagine and 6 diazo-5-oxo-L-norleucine provide full protection to the enzyme against thermal denaturation.