Abstract
Pyrococcus woesei (DSM 3773) β-galactosidase gene amplified by polymerase chain reaction was cloned intoKpnI andHindIII binding sites of pET-30LIC expression plasmid. The obtained pGal2 (6785 bp) transcription vector was then transferred toEscherichia coli B121 (DE3) cells. High identity (99.9%) of DNA sequences suggests that β-galactosidases fromP. woesei andPyrococcus furiosus are closely related. This enzyme fromE. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85°C. The crude β-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted fromE. coli cells and has maximal activity at pH 5.4 and temperature of 93°C. Isolated enzyme is active at temperatures up to 110°C and the activity loss after 4h of incubation at 85 and 93°C did not exceed 11 and 15% of the initial value, respectively.
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Dąbrowski, S., Maciuńska, J. & Synowiecki, J. Cloning and nucleotide sequence of the thermostable β-galactosidase gene fromPyrococcus woesei inEscherichia coli and some properties of the isolated enzyme. Mol Biotechnol 10, 217–222 (1998). https://doi.org/10.1007/BF02740841
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DOI: https://doi.org/10.1007/BF02740841