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Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae

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Abstract

Non-specific nuclease (NSN) can be applied in industrial downstream processing to remove nucleic acids from crude protein extracts or in cell-sorting systems to degrade nucleic acids derived from lysed cells. PsNuc from the ice-nucleating bacterium Pseudomonas syringae has the ability to decompose double- and single-stranded DNA in linear or circular form and RNA. It is not affected by the presence of metal-ion chelators such as EDTA and tolerates several protease inhibitors and reducing agents. A multiple sequence alignment of PsNuc with closely related enzymes (97–99% identity on the protein level) within the family Pseudomonaceae revealed the presence of only six amino acid residues that are variable in putative NSN from different members of the genus Pseudomonas. Single amino acid variants were produced in recombinant form in Escherichia coli, purified, and characterized. They showed similar activity compared to PsNuc, but a single variant even displayed an improved performance with an activity of > 20,000 U/mg at 35 °C, while amino acid residues S148 and V161 were found to be essential for enzymatic functionality. These results suggest that homologous nucleases from Pseudomonaceae display high activity levels in a metal-ion-independent manner and are therefore of interest for applications in biotechnology.

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Abbreviations

AEBSF:

4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride

dsDNA:

Double-stranded deoxyribonucleic acid

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

NSN:

Non-specific nuclease

PMSF:

Phenylmethylsulfonyl fluoride

RNA:

Ribonucleic acid

ssDNA:

Single-stranded deoxyribonucleic acid

SP:

Signal peptide

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Acknowledgements

This work was funded by Miltenyi Biotec B.V. & Co. KG. The authors thank Jens Helmer (Miltenyi Biotec B.V. & Co. KG) for peptide mass fingerprinting, mass spectrometry analysis, and for the preparation of Supplementary Fig. S1.

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  1. Miltenyi Biotec B.V. & Co. KG, Friedrich-Ebert-Straße 68, 51429, Bergisch Gladbach, Germany

    Sarah Schmitz, Marek Wieczorek, Volker Nölle & Skander Elleuche

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  1. Sarah Schmitz
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  2. Marek Wieczorek
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Correspondence to Skander Elleuche.

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SS, MW, VN, and SE are employees of Miltenyi Biotec B.V. & Co. KG.

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Schmitz, S., Wieczorek, M., Nölle, V. et al. Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae. Mol Biotechnol 62, 67–78 (2020). https://doi.org/10.1007/s12033-019-00229-8

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