Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s!1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (a-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a highdensity fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4

Acylpeptide Hydrolase Inhibition as Targeted Strategy to Induce Proteasomal Down-Regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy

Identification and Characterisation of a Novel Acylpeptide Hydrolase from Sulfolobus Solfataricus: Structural and Functional Insights

A novel acylpeptide hydrolase, named APEH-3Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a “true” aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3Ss and apehSs (the gene encoding the previously identified APEHSs from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea

Glycosynthases: new enzymes for oligosaccharide synthesis

The mutation of putative acidrbase and nucleophile of the active sites of retaining glycosyl hydrolases, together with kinetic analysis of the mutants, and stereochemical identification of products lead to useful information for the understanding of the reaction mechanism of these enzymes. This was the preliminary and fundamental step toward the preparation of new enzymatic activities called glycosynthases. Direct exploitation of this information has been possible, leading to the design of four new enzymes for oligosaccharides synthesis. The interest for these biocatalysts rises from the fact that the yield of the reaction can be increased and selectivity can be interpreted as key characteristic of the transfer reaction instead of a balance of hydrolytic and transferring pathways followed either by substrates and products. These new biocatalysts possess different specificities and are promising and useful tools in the construction of oligosaccharide molecules of great biological interest. This short review focused the attention on different glycosynthases obtained from four glycosyl hydrolases highlighting on the preparation and development of these new enzyme