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Marianna Turkiewicz

Publications and source records attributed to Marianna Turkiewicz.

6 recordsLinked to original sources
PubMed2006

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis.

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.

Animals↗
PubMed2005

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.

Bacterial Proteins↗
PubMed2005

Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b.

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 degrees C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 degrees C and during 28 h at 15 degrees C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.

Amino Acid Sequence↗
PubMed2003

A cold-adapted extracellular serine proteinase of the yeast Leucosporidium antarcticum.

An extracellular serine proteinase, lap2, from the psychrophilic antarctic yeast Leucosporidium antarcticum 171 was purified to homogeneity and characterized. The enzyme is a glycoprotein with a molecular mass of 34.4 kDa and an isoelectric point of pH 5.62. The proteinase is halotolerant, and its activity and stability are dependent neither on Ca(2+) nor on other metal ions. Lap2 is a true psychrophilic enzyme because of low optimal temperature (25 degrees C), poor thermal stability, relatively small values of free energy, enthalpy and entropy of activation, and high catalytic efficiency at 0-25 degrees C. The 35 N-terminal amino acid residues of lap2 have homology with subtilases of the proteinase K subfamily (clan SB, family S8, subfamily C). The proteinase lap2 is the first psychrophilic subtilase in this family.

Amino Acid Sequence↗
PubMed2003

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase.

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.

Antarctic Regions↗