Cloning, Expression And Rational Design Of Novel Glycerol Dehydratase Homologous Gene

Since 1,3-propanediol, (1,3-PDO) discovered from 19th, the concern has been focused on it for the excellent characteristic in many aspects. Therefore, a lot of multimational corporations have pay high attention to the production and applyment of 1,3-PDO, especially using by biologic way. Glycerol dehydratase, as the rate-limiting enzyme in the metabolism, has becoming the core of research.In 2003, a novel glycerol dehydratase independented the expensive coenzyme B12 have been reported by French scientist. The cost of production will be decreased greatly. The industrializating pace of production 1,3-propanediol by microbiology method have been improveed. Surprisingly, it showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases. Owning to these reasons, people can believe that the catalytic mechanism of the novel glycerol dehydratase is similar even the same as the catalytic mechanism of the PFL. Therefore, the research of the PFL's catalytic mechanism seems more important. The yqhD gene from Escherichia coli, which can substitute 1,3-propanediol dehydrogenase gene, has been found. And the yield of 1,3-propanediol has greatly improved by using the yqhD gene. It is a tremendous impetus that we can find a new gene that can dehydratase the glycerol from Escherichia coli. Due to the homology between novel glycerol dehydratase and pyruvate formate-lyase from Escherichia coli, we attempt to redesign the novel enzyme, which has the activity of glycerol dehydratase based on the gene from Escherichia coli.In this paper, the gene encoding pyruvate formate-lyase from Escherichia coli has been redesigned by site-directed mutagenesis using computer-aided rational design. The useful information is supported for discovering the catalytic mechanism of the novel glycerol dehydratase and redesigning the novel glycerol dehydratase.All possible stabilizing point mutations in the PFL were calculated in silico for determination of the change in folding free energy between wild-type and mutantproteins. Three most stabilizing animo acid residues(Glu336Cys , Ala273Cys, Glu400Ile) in the primary structure of the enzyme have been exchanged by site-directed mutagenesis according to the result of calculation. The temperature, pH optima and kinetic constants of the enzymatic reaction were not significantly altered by the mutations. The effect of temperature on the stability of the wild-type and mutant enzymes was investigated. Compared to the wt enzyme, half lives of the two mutant enzymes were significantly prolonged. The melting temperatures of the Glu336Cys and Glu400Ile enzymes were increased by 3.1 and 2.0°C, respectively, compared with the melting temperature of the wild-type enzyme.The result was obtained that rational design was an effective and efficient approach for researching the protein, especially in the thermostability.