Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile <i>Thermotoga neapolitana</i> and Its Application in Acetyl-CoA Production

doi:10.13523/j.cb.1908013

China Biotechnology

2020, Vol. 40

Issue (3): 72-78 DOI: 10.13523/j.cb.1908013

Orginal Article

Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana and Its Application in Acetyl-CoA Production

LE Yi-lin,FU Yu,NI Li,SUN Jian-zhong(

)

Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Jiangsu 212013, China

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Abstract

Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the synthesis of acetyl-CoA from pyruvate and coenzyme A (CoA) using thiamine pyrophosphate (TPP) as coenzyme. The four subunit-type TnPFOR from T. neapolitana was expressed in Escherichia coli and characterized. The gene of TnPFOR from T. neapolitana was cloned into pET-20b(+). TnPFOR was purified by a heat treatment followed by an ion exchange chromatography. The TnPFOR had an optimal condition for its maximum activity at 90℃ and pH 6.5 and it was indeed thermostable with a half-life of more than 1h at 90℃. The application of TnPFOR to catalyze the conversion of pyruvate into acetyl-CoA was also evaluated. The influence of different reaction conditions (reaction temperature, pyruvate concentrations and reaction time) on the synthesis of acetyl-CoA was discussed. The optimal reaction temperature is 90℃, pyruvate concentrations is 1.5mmol/L and the reaction time is 2min.

Key words： Escherichia coli Recombinant expression Thermophilic enzyme Thermophiles Synthesis of acetyl-CoA

Received: 06 August 2019 Published: 18 April 2020

ZTFLH:

Q946.5

Corresponding Authors: Jian-zhong SUN E-mail: jzsun1002@ujs.edu.cn

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	Yi-lin LE
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	Jian-zhong SUN

Cite this article:

LE Yi-lin,FU Yu,NI Li,SUN Jian-zhong. Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana and Its Application in Acetyl-CoA Production. China Biotechnology, 2020, 40(3): 72-78.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1908013 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/72

Fig.1 Arrangement of the gene clusters for the TnPFOR enzymes

Fig.2 PCR product of TnPFOR (a) and confirmation of 3 recombinant clones (b) (a) 1:PCR product of TnPFOR; M: DNA marker (b) 1-3:Recombinant clones digested by Xba Ⅰ and Hind Ⅲ; M:DNA marker

Fig.3 SDS-PAGE(a)and native-PAGE(b) analysis of the expression and purification of TnPFOR from T. neapolitana (a) M: Protein marker; 1:Total proteins of E. coli containing pET-20b(+); 2:Total proteins of E. coli containing pET-PFOR; 3:Recombinant TnPFOR purified after ion exchange chromatography (b) 1-3:Native-PAGE analysis of TnPFOR

Fig.4 Effects of temperature, pH and O₂ on the activity and enzyme stability of TnPFOR

Fig.5 Detection of reaction products using HPLC (a) Standard sample: 2mmol/L CoA and 1mmol/L acetyl-CoA (b) The reaction with TnPFOR (c) The reaction without TnPFOR

Fig.6 The influence of different reaction conditions on the synthesis of acetyl-CoA (a) The synthesis of acetyl-CoA at various temperatures (b) The synthesis of acetyl-CoA at various pyruvate concentrations (c) The synthesis of acetyl-CoA at various reaction time


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