Abstract
A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver–Burk plot, and K m and V max were 0.89 mM and 0.18 U/mg, respectively.
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We wish to thank Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), New Delhi for providing the financial support during the present study.
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Vidya, J., Pandey, A. Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate. Appl Biochem Biotechnol 167, 973–980 (2012). https://doi.org/10.1007/s12010-012-9617-8
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DOI: https://doi.org/10.1007/s12010-012-9617-8