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2005 | 52 | 4 | 781-787
Article title

Thermostable Pyrococcus woesei β-D-galactosidase - high level expression, purification and biochemical properties

Content
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EN
Abstracts
EN
The gene encoding β-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei β-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-β-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei β-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type β-D-galactosidase (51100 U/mg). The activity of P. woesei β-D-galactosidase was enhanced by thiol compounds, Mg2+ ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca2+ ions had no effect.
Publisher

Year
Volume
52
Issue
4
Pages
781-787
Physical description
Dates
published
2005
received
2005-04-06
revised
2005-06-28
accepted
2005-07-15
(unknown)
2005-11-07
Contributors
  • Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland
author
  • Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland
  • Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland
  • Institute of Technical Biochemistry, Technical University of Łódź, Łódź, Poland
References
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Document Type
Publication order reference
Identifiers
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv52p781kz
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