Expression, Purification and Biochemical Characterisation of Babesia bigemina Lactate Dehydrogenase

Babesia bigemina, a tick-borne protozoan parasite, is one of the main causative agents of bovine babesiosis, a disease with significant economic impact on the cattle industry. One of the key enzymes involved in the parasite's metabolism is lactate dehydrogenase (LDH), which plays an essential role in the anaerobic glycolytic pathway by catalysing the conversion of pyruvate to lactate. In this study, B. bigemina LDH gene was cloned, expressed in Escherichia coli and subsequently purified using affinity chromatography. The purified enzyme was subjected to biochemical assays to determine its stability, optimal pH, thermostability, and kinetic parameters. Kinetic analyses indicated a Kₘ value of 0.2585 mM for pyruvate and a K_0.5 value of 0.3094 mM for NADH. B. bigemina LDH exhibits typical Michaelis-Menten kinetics for pyruvate, but its behavior towards NADH is similar to that of Cryptosporidium parvum LDH, suggesting that B. bigemina LDH may function as an allosteric enzyme for NADH. Enzyme activity was found to remain stable for up to 9 days at 4 °C without any preservative agent. Biochemical analysis showed that the optimum enzymatic activity occurred at pH 8.5, and the enzyme retained its activity at both 30 °C and 40 °C. These findings provide valuable insights into the functionality of the enzyme and may contribute to the development of therapeutic interventions targeting the glycolytic pathway of B. bigemina.