Search Results (2)

This work aimed to study the structural features and mechanisms of thermoinactivation of hyperthermophilic L-asparaginase (L-ASNase) from archaea Thermococcus sibiricus (TsA) in comparison with bacterial L-ASNases from Melioribacter roseus (MrA) and Rhodospirillum rubrum (RrA). The catalytic parameters of L-asparagine hydrolysis under optimal conditions (pH 9) were determined for these enzymes by circular dichroism (CD) spectroscopy. TsA showed the highest activity among the studied L-ASNases (640 IU/mg at 90 °C). Thermo-inactivation kinetics were studied at temperatures close to the enzyme optimum: the first-order inactivation constants were 0.065 min⁻¹ (TsA), 0.011 min⁻¹ (MrA), and 0.026 min⁻¹ (RrA). In contrast to RrA and MrA, aggregation was detected as one of the thermoinactivation mechanisms for TsA. From the analysis of thermograms obtained with CD spectroscopy, the melting temperatures (Tm) for RrA, MrA, and TsA were determined as 50, 69, and 89 °C, respectively. A significant increase in the percentage of β-structures for TsA during heating (from 8 to 16%) indicating aggregation was observed in the interval from 70 to 100 °C. For RrA and MrA this value did not increase. Changes in the tertiary structure of the enzymes during heating were monitored by fluorescence spectroscopy. Thermal inactivation of RrA and MrA were accompanied by changes in the tertiary structure. For TsA, the observed denaturation enthalpy (ΔH) was 346 kJ/mol, which was 1.5–2 times higher than the same values for RrA and MrA. The study of the specific thermoinactivation mechanisms and structural- features in hyperthermophilic enzymes in comparison with mesophilic ones allows us to shed light on the molecular adaptation variants of the enzyme to function at high temperatures. Full article

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases—bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters K_M and V_max for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni²⁺, Mg²⁺ or EDTA, but increased with the addition of Cu²⁺ and Ca²⁺ by 56% and 77%, respectively, and was completely inhibited by Zn²⁺, Fe³⁺ or urea solutions 2–8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade. Full article