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Improving the thermostability and stress tolerance of an archaeon hyperthermophilic superoxide dismutase by fusion with a unique N-terminal domain.

ABSTRACT: The superoxide dismutase from the archaeon Sulfolobus solfataricus (SOD Ss ) is a well-studied hyperthermophilic SOD with crystal structure and possible thermostability factors characterized. Previously, we discovered an N-terminal domain (NTD) in a thermophilic SOD from Geobacillus thermodenitrificans NG80-2 which confers heat resistance on homologous mesophilic SODs. The present study therefore aimed to further improve the thermostability and stress tolerance of SOD Ss via fusion with this NTD. The recombinant protein, rSOD Ss , exhibited improved thermophilicity, higher working temperature, improved thermostability, broader pH stability, and enhanced tolerance to inhibitors and organic media than SOD Ss without any alterations in its oligomerization state. These results suggest that the NTD is an excellent candidate for improving stability of both mesophilic and thermophilic SOD from either bacteria or archaea via simple genetic manipulation. Therefore, this study provides a general, feasible and highly useful strategy for generating extremely thermostable SODs for industrial applications.

SUBMITTER: Li M

PROVIDER: S-EPMC4771647 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Improving the thermostability and stress tolerance of an archaeon hyperthermophilic superoxide dismutase by fusion with a unique N-terminal domain.

Li Mingchang M Zhu Lin L Wang Wei W

SpringerPlus 20160301

The superoxide dismutase from the archaeon Sulfolobus solfataricus (SOD Ss ) is a well-studied hyperthermophilic SOD with crystal structure and possible thermostability factors characterized. Previously, we discovered an N-terminal domain (NTD) in a thermophilic SOD from Geobacillus thermodenitrificans NG80-2 which confers heat resistance on homologous mesophilic SODs. The present study therefore aimed to further improve the thermostability and stress tolerance of SOD Ss via fusion with this NTD ...[more]

PMID: 27026935

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Project description:We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.

Dataset Information

Improving the thermostability and stress tolerance of an archaeon hyperthermophilic superoxide dismutase by fusion with a unique N-terminal domain.

Publications

Improving the thermostability and stress tolerance of an archaeon hyperthermophilic superoxide dismutase by fusion with a unique N-terminal domain.

Similar Datasets

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