Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity
Language English Country United States Media print-electronic
Document type Journal Article
PubMed
30361879
DOI
10.1007/s12223-018-0656-6
PII: 10.1007/s12223-018-0656-6
Knihovny.cz E-resources
- MeSH
- Asparaginase isolation & purification metabolism MeSH
- Glutamine metabolism MeSH
- Glutaminase metabolism MeSH
- Kinetics MeSH
- Cloning, Molecular MeSH
- Hydrogen-Ion Concentration MeSH
- Half-Life MeSH
- Pyrobaculum enzymology genetics MeSH
- Recombinant Proteins genetics metabolism MeSH
- Enzyme Stability MeSH
- Substrate Specificity MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Asparaginase MeSH
- Glutamine MeSH
- Glutaminase MeSH
- Recombinant Proteins MeSH
The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 μmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.
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