Intracellular L-Asparaginase from <i>Bacillus</i> sp.PG02: Purification, Biochemical Characterization and Evaluation of Optimum pH and Temperature

Abstract

Bacterial L-asparaginases are amidohydrolases that act on Lasparagine and produce L-aspartate and ammonia. These enzymes have been used in treatment of lymphoblastic leukemia. In the present study, a novel strain, Bacillus sp. PG02 was explored for the production of intracellular L-asparaginase enzyme. The nitrogen source for L-asparaginase production was L-asparagine. New intracellular L-asparaginase was purified using ion exchange chromatography and the purity was assessed using SDS-PAGE. Kinetic parameters k m and V max and thermal properties were studied using L-asparagine as the substrate. SDS-PAGE analysis showed apparent molecular weight of approximately 38 kDa. The enzyme was active in a wide pH ranges (5-10) and it was maximally active at pH 7.5. Bacillus PG02 L-asparaginase was optimally active at 40°C. Thermal inactivation studies exhibited t 1/2 of 32.5 min in 37°C. Also T 50 and ∆G of inactivation were measured. The results revealed that the enzyme had appropriate characteristics and thus could be a potential candidate for medical and basic investigations.

Bacterias productoras de L-asparaginasa aisladas de ambientes salinos peruanos

“…The ammonium released from the hydrolysis of Lasparagine was quantified using the Nessler reagent as described by Imada et al (1973) with the modifications described below. The crude extract was obtained from the bacterial isolates cultured in the medium proposed by Qeshmi et al (2015). The reaction was carried out in two stages.…”

“…The method described by Coêlho et al (2016) using azocasein as substrate (Sigma-Aldrich, MO, USA) was employed with the following modifications. The crude enzyme extract was obtained from the bacterial isolates grown in the medium proposed by Qeshmi et al (2015). The enzyme extract (500 µl) was mixed with 500 µl 0,6% w/v azocasein (dissolved in 50 mM Tris-HCl, pH 8.5) and incubated for 30 min at 37 °C.…”