Abstract
The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.
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Acknowledgements
We are thankful for the technical excellence and practical support provided by Bente Smith Thorup, Janni Kristensen, and Gunhild Siboska. Kim K. Mortensen was funded by grants from the Danish Natural Science Research Council and Carlsberg (grant nos. 21-03-0465, 21-04-0149 and ANS-1649/40). We also thank the Greenland Homerule for permission to collect samples and the Danish Ministry of Science, Technology, and Innovation for initial funding.
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Sørensen, H.P., Porsgaard, T.K., Kahn, R.A. et al. Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment. Appl Microbiol Biotechnol 70, 548–557 (2006). https://doi.org/10.1007/s00253-005-0153-0
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DOI: https://doi.org/10.1007/s00253-005-0153-0