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Cloning, Purification, Characterization and Immobilization of L-asparaginase II from E. coli W3110

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Author(s): Magdy M. Youssef | Mohammed A. Al-Omair

Journal: Asian Journal of Biochemistry
ISSN 1815-9923

Volume: 3;
Issue: 6;
Start page: 337;
Date: 2008;
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Keywords: Cloning | expression | purification | immobilization | characterization | glutathione s-transferase

ABSTRACT
asnII) gene from E. coli W3110 into pGEX-2T DNA vector. The L-asparaginase II enzyme (E.C.3.5.1.1) was overexpressed in E. coli BL21(DE3) and purified to homogeneity 238.4 fold by utilizing chromatography technique on DEAE-Sepharose fast flow, Glutathione S sepharose 4B columns and thrombin. SDS-PAGE of the purified enzyme revealed that has Mr of 40 kDa. In addition, we found that the enzyme can be efficiently immobilized in calcium alginate gelatin composites. The free enzyme has an optimum pH at 7.5 but this optimum pH is shifted to 8.5 for the immobilized enzyme. The optimum temperature, for free and immobilized enzyme were 37 and 50C, respectively. The immobilized enzyme retained most of its activity at 60C with high stability compared with the native enzyme when incubated at 60C for 30 min.]]>

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