Chitosan Sponges for Efficient Accumulation and Controlled Release of C-Phycocyanin.

The paper proposed a new porous material for wound healing based on chitosan and C-phycocyanin (C-PC). In this work, C-PC was extracted from the cyanobacteria Arthrospira platensis biomass and purified through ammonium sulfate precipitation. The obtained C-PC with a purity index (PI) of 3.36 ± 0.24 was loaded into a chitosan sponge from aqueous solutions of various concentrations (250, 500, and 1000 mg/L). According to the FTIR study, chitosan did not form new bonds with C-PC, but acted as a carrier. The encapsulation efficiency value exceeded 90%, and the maximum loading capacity was 172.67 ± 0.47 mg/g. The release of C-PC from the polymer matrix into the saline medium was estimated, and it was found 50% of C-PC was released in the first hour and the maximum concentration was reached in 5-7 h after the sponge immersion. The PI of the released C-PC was 3.79 and 4.43 depending on the concentration of the initial solution.

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/ß hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.