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Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428
Sanjay Kumar1, V Venkata Dasu, K Pakshirajan
1Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Abstract:
An intracellular glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4 kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, L-asparagine. The activity of L-asparaginase is activated by mono cations and various effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, Km, Vmax and kcat of purified L-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657 mM, 4.45 U μg(-1) and 2.751×10(3) s(-1), respectively. Optimum pH of purified L-asparaginase for the hydrolysis of L-asparagine was in the range of 8.0-10.0, and its optimum temperature was found to be 40 °C. The purified L-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy.
Insights
A novel glutaminase-free L-asparaginase was purified from Pectobacterium carotovorum MTCC 1428. This enzyme shows high specificity for L-asparagine, offering potential for reduced side effects in anti-cancer therapy.
Area of Science:
- Biochemistry
- Enzymology
- Protein Purification
Background:
- L-asparaginase is a crucial enzyme in anti-cancer therapy, particularly for acute lymphoblastic leukemia.
- Controlling glutaminase activity is essential to minimize side effects associated with L-asparaginase treatment.
Purpose of the Study:
- To isolate and characterize an intracellular L-asparaginase from Pectobacterium carotovorum MTCC 1428.
- To assess the enzyme's specificity and kinetic properties, focusing on the absence of glutaminase activity.
Main Methods:
- Isolation and purification of intracellular L-asparaginase.
- Molecular mass determination using MALDI-TOF MS.
- Enzyme activity assays, including substrate specificity, kinetic parameter determination, and analysis of effector/inhibitor effects.
Main Results:
- Homotetrameric L-asparaginase with a molecular mass of 144.4 kDa and pI of ~8.4 was purified.
- The enzyme exhibited high specificity for L-asparagine, with optimal activity at pH 8.0-10.0 and 40 °C.
- Kinetic parameters (Km, Vmax, kcat) were determined, and the enzyme showed activation by monovalent cations and inhibition by divalent cations, with no detectable glutaminase activity.
Conclusions:
- The purified P. carotovorum MTCC 1428 L-asparaginase is a glutaminase-free enzyme with favorable biochemical properties.
- Its high specificity and lack of glutaminase activity suggest potential as a safer therapeutic agent in cancer treatment.
