Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
- MeSH
- aktivátory enzymů metabolismus MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fumaráty metabolismus MeSH
- fylogeneze MeSH
- hořčík metabolismus MeSH
- kinetika MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- kyselina jantarová metabolismus MeSH
- metagenom * MeSH
- molekulární sekvence - údaje MeSH
- oxidace-redukce MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- stabilita enzymů MeSH
- sukcinátdehydrogenasa chemie genetika metabolismus MeSH
- teplota MeSH
- vodní organismy MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktivátory enzymů MeSH
- fumaráty MeSH
- hořčík MeSH
- kyselina jantarová MeSH
- rekombinantní proteiny MeSH
- sukcinátdehydrogenasa MeSH
- zinek MeSH
A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLacІ cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn(2+) and Mg(2+). The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k m =0.227 mmol/L, v max= 29.9 U/mg, and k cat/k m=5.44 × 10(4) per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.
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