Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 22, Issue 18, Pages -Publisher
MDPI
DOI: 10.3390/ijms22189894
Keywords
L-asparaginase; hyperthermophile; heterologous expression; biochemical properties; kinetic characteristics; cytotoxic activity
Funding
- RFBR [19-08-01112]
- Russian Federation fundamental research program for the long-term period
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L-asparaginase from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) shows high specific activity and stability, making it a promising candidate for biotechnological applications. TsA also exhibits strong cytotoxic activity against cancer cell lines.
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 Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 degrees 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 mu M/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 degrees 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.
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