L-Asparaginase (ASNase) is used in medicine for neoplasms treatment and in food industry for mitigation of acrylamide in high-temperature processed food. In medicine, commercial ASNases have exhibited side effects and l-glutaminase (GLNase) activity affecting the clinical treatment. The aim of this work was to study a novel ASNase from Enterobacter sp. M55 isolated from Maras Salters in Peru, which was purified and biochemically characterised. This ASNase exhibited a Km of 5.71 mM and a Vmax of 0.16 µmol mL–1 min–1, as well as an optimum temperature and pH of 37 °C and 6, respectively. Moreover, a good activity (80 %) was observed at physiological pH. Likewise, the enzyme increased its activity by around 50% in presence of urea, glutathione, and glucose. Whilst in presence of serum compounds, it kept more that 60 % of activity. In addition, this ASNase showed low GLNase activity.