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 Km and Vmax were 0.89 mM and 0.18 U/mg, respectively
