ABSTRACT
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
Subject(s)
Animals , Liver , Camelus , Asparaginase/chemistryABSTRACT
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]