Abstract
A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the β-glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl-β-D-glucoside were 0.19mM and 0.25mM, respectively.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Appleyard, R.K. (1980) Genetics 39, 440–445.
Clewell, D.B. & Helsinki, D.R. (1970) Biochem. J. 9, 4428–4440.
Coleman, D.C., Arbuthnott, J.P., Pomeroy, H.M. & Birkbeck, T.H. (1986) Microb. Path. 549–564.
Gabelsberger, J., Liebl, W., Schleifer, KH. (1993) FEMS Microb. Letts., 109, 131–137.
Gagdil, N.J., Daginawala, H.F., Chakrabarti, T. & Khanna, P. (1995) Enzyme Microb. Technol. 17, 942–946.
Gallagher, J., Winters, A., Barron, N., McHale, L. & McHale, A.P. (1996) Biotech. Lett. 18, 537–540.
Liebl, W., Gabelsberger, J. & Schleifer, K.H. (1994) Molec. Gen. Genetics 242, 111–115.
Maniatis, T., Fritsch, E. & Sambrook, J. (1982) Molecular Cloning: A laboratory manual. Cold Spring Harbour Laboratory, New York.
Mastromei, G., Hanhart, E., Perito, B. & Polsinelli, M. (1992) J. Biotechnol. 24, 149–157.
McHale, A.P. & Coughlan, M.P. (1981) Biochim. Biophys. Acta. 662, 145–151.
Perezpons, J.A., Cayetano, A., Rebordosa, X., Lloberas, J. & Gausch, A. (1994) Eur. J. Biochem. 223, 557–565.
Petitdemange, E., Tchunden, T., Valles, S., Pirson, H., Ravel, G. & Gay, R. (1992) Biomass Bioenergy 3, 393–402.
Philippidis, G.P., Smith, T.K., Wyman, C.E. (1993) Biotech. Bioeng. 41, 846–853.
Romaniec, M.P.M., Hiskisson, N., Barker, P. & Demain, A.L. (1993) Enzyme Microb. Technol. 15, 393–400.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Winters, A., Gallagher, J., Barron, N. et al. Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli . Biotechnol Lett 18, 1387–1390 (1996). https://doi.org/10.1007/BF00129340
Issue Date:
DOI: https://doi.org/10.1007/BF00129340