Production, purification and characterization of asparaginase from bacteria E. Coli (Escherichia coli)
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Date
2018
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UMT Lahore
Abstract
Asparaginase is an enzyme having (E.C. 3.5.1.1.), belongs to amidase group. This enzyme plays an important role in the treatment of tumor and non-Hodgkin’s lymphoma; it can act as anti-tumor agent, also necessary agent of chemotherapeutic agent against acute lymphoblastic leukemia. From last 50 years it has been used for the treatment of Acute Lymphoblastic Leukaemia (ALL), in the form of drugs. This was first identified from guinea pig serum in 1963. L-Asparaginase performs catalytic reaction, and involve in the conversion of asparagine to aspartic acid and ammonia. Both normal and tumor cells needs Asparagine for nutritional condition. Currently the clinical sources of ASNase are Erwinia chrysanthemi and Escherichia coli. The benefit in the use of this enzyme is that, it causes L-asparagine reduction; subsequently these amino acids are not synthesized by tumor cells. In a meanwhile there long term usage can causes allergic reactions, hypersensitivity and anaphylaxis. L-Asparaginase (L-AsnA) is broadly present in many microorganisms. This enzyme is also useful in different clinical research field like food industry, medicine and pharmacologic.
The main objective of this research was isolation, production, purification and characterization of an extracellular L-Asparaginase from Escherichia coli. Production media were performed for asparaginase synthesis. By using the enzyme assay it has been characterized, having the principle of hydrolyzing l-asparagine into l-aspartic acid and ammonia. Enzyme assay proceed by nesslerization method. Crude enzyme of l-asparaginase was moderately purified by ammonium sulfate precipitation (70%) and dialysis. In the purification step, the specific activity of L-Asparaginase from E. coli was increases from 42.2 U/mg to 6.73 U/mg. In ammonium sulfate precipitation step, the total protein increased from 0.38 mg to14.54 mg. After that the last step is characterization where the SDS-PAGE was determined the molecular weight of the purified enzyme. Our thesis work exposed that only a single distinctive protein band of purified L-Asparaginase from E. coli was found the molecular weight is 32 kDa.