Abstract
Hydroxynitrile lyase (MeHNL, EC 4.1.2.39) is a useful enzyme for production of optically active cyanohydrin compounds. Production of MeHNL can be increased by substituting rare codons of the natural sequence of cassava (Manihot esculenta) MeHNL. However, most of the MeHNL produced by this method was in an insoluble form in Escherichia coli expression system. In order to increase the productivity of active form of MeHNL, the effects of cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17°C) were 850 times higher than those obtained at the optimum growth temperature of 37°C. The rate of MeHNL production in the present study was calculated as 3,000 U/h. Low-temperature cultivation is very effective in improving the productivity of the active form of MeHNL and has more potential for large-scale production of MeHNL for optically active cyanohydrin production.
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References
Hughes, J., Carvalho, F. J., and Hughes, M. A. (1994) Purification, characterization, and cloning of α-hydroxynitrile lyase from Cassava (Manihot esculenta Crantz). Arch. Biochem. Biophys. 311, 496–502.
Hughes, J., Lakey, J. H., and Hughes, M. A. (1997) Production and characterization of a plant -hydroxynitrile lyase in Escherichia coli. Biotechnol. Bioeng. 53, 332–338.
Forster, S., Roos, J., Effenberger, F., Wajant, H., and Sprauer, A. (1996) The first hydroxynitrile lyase and its application in the synthesis of (S)-cyanohydrins. Angew Chem. Int. Ed. Engl. 35, 437–439.
Hasslacher, M., Schall, M., Hayn, M., Bona, R., Rumbold, K., Luckl, J., Griengl, H., Kohlwein, S. D., and Schwab, H. (1997) High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expr. Purif. 11, 61–71.
Singh, S. M., and Panda, A. K. (2005) Solubilization and refolding of bacterial inclusion body proteins. J. Biosci. Bioeng. 99, 303–310.
Nishihara, K., Kanemori, M., Yanagi, H., and Yura, T. (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66, 884–889.
Baedeker, M., and Schulz, G. E. (1999) Overexpression of a designed 2.2 kb gene of eukaryotic phenylalanine ammonia-lyase in Escherichia coli. FEBS Lett. 457, 57–60.
Hoshino, K., Ito, K., Masubuchi, A., Adachi, M., Asakawa, T., Watanabe, N., Kosaka, T., and Tanaka, Y. (2007) Cloning expression, and characterization of male cynomolgus monkey liver aldehyde oxidase. Biol. Pharm. Bull. 30, 1191–1198.
Semba, H., Ichige, E., Imanaka, T., Atomi, H., and Aoyagi, H. (2008) Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture. Appl. Microbiol. Biotechnol. 79, 563–569.
Takagi, J. S., Ida, N., Tokushige, M., Sakamoto, H., and Shimura, Y. (1985) Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W. Nucleic Acids Res. 13, 2063–2074.
Albrecht, J., Jansen, I., and Kula, M. R. (1993) Improved purification of an (R)-oxynitrilase from Linum usitatissimum (flax) and investigation of the substrate range.Biotechnol. Appl. Biochem. 17, 191–203.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.
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Semba, H., Ichige, E., Imanaka, T., Atomi, H., Aoyagi, H. (2010). Efficient Production of Active Form Recombinant Cassava Hydroxynitrile Lyase Using Escherichia coli in Low-Temperature Culture. In: Fett-Neto, A. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology, vol 643. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-723-5_10
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DOI: https://doi.org/10.1007/978-1-60761-723-5_10
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