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Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

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Abstract

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.

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Acknowledgments

This work was supported by National High Technology Research and Development Program of China (“863”) under Grant No. 2007AA02Z227.

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Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, 530004, Guangxi, China

    Xuqin Wei, Xiaolei Meng, Yunlai Chen, Yutuo Wei, Liqin Du & Ribo Huang

  2. National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, 530007, Guangxi, China

    Ribo Huang

  3. Guangxi Biotechnology Research Center, Guangxi Academy of Sciences, 98 Daling Road, Nanning, 530007, Guangxi, China

    Ribo Huang

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  1. Xuqin Wei
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  2. Xiaolei Meng
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  3. Yunlai Chen
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  4. Yutuo Wei
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  5. Liqin Du
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  6. Ribo Huang
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Correspondence to Ribo Huang.

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Xuqin Wei and Xiaolei Meng have contributed equally to this study.

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Wei, X., Meng, X., Chen, Y. et al. Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans . Biotechnol Lett 36, 159–165 (2014). https://doi.org/10.1007/s10529-013-1346-8

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  • DOI: https://doi.org/10.1007/s10529-013-1346-8

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