A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Abstract

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon <i>Thermococcus sibiricus</i> (TsA) was expressed in <i>Escherichia coli</i>, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters K_M and V_max for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni²⁺, Cu²⁺, Mg²⁺, Zn²⁺ and Ca²⁺ and EDTA added in concentrations 1 and 10 mmol/L except for Fe³⁺. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.