A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 A degrees 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 6x 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 A degrees C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 A degrees 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.