Anoxic cell rupture of Prevotella bryantii by high-pressure homogenization protects the Na+-translocating NADH:quinone oxidoreductase from oxidative damage.

Abstract:

:Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L-1 culture and a NADH oxidation activity of 0.8 µmol min-1 mg-1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

journal_name

Arch Microbiol

journal_title

Archives of microbiology

authors

Schleicher L,Fritz G,Seifert J,Steuber J

doi

10.1007/s00203-019-01805-x

subject

Has Abstract

pub_date

2020-07-01 00:00:00

pages

1263-1266

issue

5

eissn

0302-8933

issn

1432-072X

pii

10.1007/s00203-019-01805-x

journal_volume

202

pub_type

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