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

The gene encoding beta-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 beta-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-beta-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei beta-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 beta-D-galactosidase (51100 U/mg). The activity of P. woesei beta-D-galactosidase was enhanced by thiol compounds, Mg(2+) ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca(2+) ions had no effect.

A proposal to rename the hyperthermophilePyrococcus woeseiasPyrococcus furiosussubsp.woesei

Pyrococcusspecies are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 °C. All species grow heterotrophically and produce H2or, in the presence of elemental sulfur (S°), H2S.Pyrococcus woeseiandP. furiosuswere isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2in the presence of 0.1% maltose and 10–100 µM sodium tungstate in S°-free medium. However,P. woeseishows more extensive autolysis thanP. furiosusin the stationary phase.PyrococcusfuriosusandP. woeseishare three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes ofP. woeseiandP. furiosus.Cloning and sequencing of ISs that were in different contexts inP. woeseiandP. furiosusrevealed that thenapAgene inP. woeseiis disrupted by a type III IS element, whereas inP. furiosus, this gene is intact. A type I IS element, closely linked to thenapAgene, was observed in the same context in bothP. furiosusandP. woeseigenomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to renameP. woeseia subspecies ofP. furiosusbased on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that allP. woeseinucleotide sequences deposited in GenBank to date are > 99% identical toP. furiosussequences.