Development of a scalable recombinant system for cyclic beta-1,2-glucans production.

BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.

Gas-Phase Fragmentation of Cyclic Oligosaccharides in Tandem Mass Spectrometry.

Modern mass spectrometry, including electrospray and MALDI, is applied for analysis and structure elucidation of carbohydrates. Cyclic oligosaccharides isolated from different sources (bacteria and plants) have been known for decades and some of them (cyclodextrins and their derivatives) are widely used in drug design, as food additives, in the construction of nanomaterials, etc. The peculiarities of the first- and second-order mass spectra of cyclic oligosaccharides (natural, synthetic and their derivatives and modifications: cyclodextrins, cycloglucans, cyclofructans, cyclooligoglucosamines, etc.) are discussed in this minireview.

Cyclic Glucans Enhance Solubility of Bioavailable Flavonoids.

Diverse flavonoids are abundant in dietary food constituents and possess useful biological activities. However, some flavonoids have limited bioavailability due to their low solubility in water. As an important approach to enhance aqueous solubility, inclusion of hydrophobic guest molecules in hydrophilic hosts such as cyclic glucans has been used. This review summarizes applications of ß-cyclodextrin, synthetic ß-cyclodextrin derivatives, and newly synthesized derivatives of cyclosophoraoses as complexing agents to enhance the bioavailability of flavonoids such as baicalein, kaempferol, and naphthoflavones.

Carboxymethyl cyclosophoraoses as a flexible pH-responsive solubilizer for pindolol.

In the present study, cyclosophoraoses (CyS) (ß-1,2 linked cyclic glucans, with glucopyranose units ranging from 17 to 23) isolated from Rhizobium leguminosarum biovar viciae VF-39 were modified with carboxymethyl (CM) groups, and the pH-sensitive complexation of CM CyS with pindolol was investigated. The solubility of pindolol increased 32-fold by its complexation with 5mM CM CyS at pH 10, whereas it shows no significant change at pH 3. Pindolol, a ß-adrenergic blocking agent, has a hydrophobic nature at non-ionized state, and CM CyS could solubilize efficiently pindolol in a high alkaline solution. The carboxymethylation of flexible CyS allows them to present a more suitable cavity for the hydrophobic pindolol at pH 10, which is differentiated from CM ß-cyclodextrin (ß-CD). It can be interpreted as that the anionic repulsion effectively modulates the flexible and distorted conformation of CyS rather than rigid annular shape of ß-CD. Resultingly, the highly solubilized CM CyS/pindolol complex was characterized by UV-vis, T1 relaxation, ROESY, DOSY NMR spectroscopy, FT-IR spectroscopy, SEM, and molecular modeling studies. The antioxidant activity of pindolol was also improved 260% in the complex compared to free pindolol. The use of flexible host molecules with pH-responsive substituents would be applied in the development of smart systems for sensing or in biomedical fields.

Solubility enhancement of α-naphthoflavone by synthesized hydroxypropyl cyclic-(1→2)-ß-D-glucans (cyclosophoroases).

Rhizobium leguminosarum produces unbranched cyclic ß-1,2-glucans, cyclosophoraoses (Cys). In the present study, Cys were modified with hydroxypropyl groups via a one step chemical derivatization and the complexation ability and solubility enhancement of hydroxypropyl cyclosophoraoses (HP Cys) with α-naphthoflavone (α-NF) were investigated. In the presence of HP Cys, the aqueous solubility of α-NF greatly increased up to 257-fold. Complex formation of HP Cys and α-NF was confirmed by nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR) spectroscopy, and differential scanning calorimetry (DSC). Furthermore, the morphological structure of α-NF with HP Cys was examined using scanning electron microscopy (SEM). A hypothetical model was proposed based on molecular dynamics (MD) simulations and a docking study of α-NF with HP Cys. Our results suggest that HP Cys form complexes with α-NF and can be utilized as a promising solubilizer. This is the first study to identify carbohydrates that can enhance the solubility of α-NF.