Cloning、expression and characterization of pyruvate formate-lyase in Escherichia coli

China Biotechnology

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Cloning、expression and characterization of pyruvate formate-lyase in Escherichia coli

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Abstract Pyruvate formate-lyase(PFL) is one of the key enzymes in the metabolic pathway of anaerobic or facultative anaerobic microorganism. In order to further study the function of PFL, the pfl gene was amplified by PCR using the E. coli JM109 genomic DNA, and the amplified fraction was ligated into the vector pMD18-T for DNA sequencing. The identified pfl gene was cloned into the expression vector pET-22b and the recombinant expression vector was induced and expressed in E. coli BL21(DE3). A new protein band appeared by analysis of SDS-PAGE compared to the control, whose molecular weight was 85kD. The PFL with 6×His-Tag was purified by metal affinity chromatography. The characterization of purified PFL was carried out. The result showed that the temperature optimum and pH optimum were 35℃ and 7.5 , respectively. The Km value of the enzyme was 2.3mmol, and the value of melting temperature was 49.9℃.

Key words： cloning expression enzymatic properties pyruvate formate-lyase

Received: 06 April 2006 Published: 25 August 2006

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. Cloning、expression and characterization of pyruvate formate-lyase in Escherichia coli. China Biotechnology, 2006, 26(08): 28-31.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2006/V26/I08/28

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