Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library.

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

Binding of Mucin by E. coli from Human Gut.

Cells of E. coli isolates from the gut of healthy volunteers (N=5) and patients with Crohn's disease (N=5) and laboratory E. coli strain DH5α bound mucin in vitro in similar amounts ranging from 0.02 to 0.12 mg/mg of bacterial dry weight. Binding was evaluated by the decrease in optical absorption of mucin solution at 214 nm after incubation with bacteria. Detailed analysis of mucin binding by one of isolates showed that during incubation of 0.09 mg/ml bacteria in 0.15 M NaCl containing 0.1 mg/ml mucin at 25oC, maximum binding was reached in 30 min, while in the presence of 14 mM α-methyl mannoside, mucin binding decreased by 46% (p<0.05). Confocal microscopy revealed intensive binding of FITC-labeled mucin to the surface of a small number of bacterial cells. Mucin binding did not significantly affect zeta potential of bacteria and their energetic status assessed by ATP content; at the same time, ATP content in the extracellular environment slightly increased.

[PEGylated recombinant L-asparaginase from Erwinia carotovora: Production, properties, and potential applications].

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

Bioluminescence assay for cell viability.

Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase.

Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His(6)-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K(m)(ATP) and K(m)(LH)(2) values were increased by a factor of ~1.5-1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K(m)(ATP) and K(m)(LH)(2) values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ~2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.