Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid.

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.

Description of the Wild Strain Rhizobium rosettiformans DSM26376, Reclassified under Peteryoungia rosettiformans comb.nov., for Producing Glucuronan.

Glucuronan is a polysaccharide composed of ß-(1,4)-linked d-glucuronic acids having intrinsic properties and biological activities recoverable in many fields of application. Currently, the description of Sinorhyzobium meliloti M5N1CS mutant bacterial strain as the sole source of glucuronan makes it relevant to the exploration of new microorganisms producing glucuronan. In this study, the Peteryoungia rosettifformans strain (Rhizobia), was identified as a wild producer of an exopolysaccharide (RhrBR46) related to glucuronan. Structural and biochemical features, using colorimetric assays, Fourier infrared spectroscopy, nuclear magnetic resonance, high pressure size exclusion chromatography coupled to multi-angle light laser scattering, and enzymatic assays allowed the characterization of a polyglucuronic acid, having a molecular mass (Mw¯) of 1.85 × 105 Da, and being partially O-acetylated at C-2 and/or C-3 positions. The concentration of Mg2+ ions in the cultivation medium has been shown to impact the structure of RhrBR46, by reducing drastically its Mw¯ (73%) and increasing its DA (10%). Comparative structural analyses between RhrBR46 and the glucuronan from Sinorhyzobium meliloti M5N1CS strain revealed differences in terms of molecular weight, degree of acetylation (DA), and the distribution of acetylation pattern. These structural divergences of RhrBR46 might contribute to singular properties or biological activities of RhrBR46, offering new perspectives of application.

Discovery of a Novel Glucuronan Lyase System in Trichoderma parareesei.

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear ß-(1,4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei. The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. Nuclear magnetic resonance revealed that the monomeric end product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and high-performance anion-exchange chromatography with pulsed amperometric detection analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales species with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Trichoderma parareseei. Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareesei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

A bacterial endo-ß-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family.

Cellouronate is a (1,4)-ß-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (ß-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized ß-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.

Sulfated polysaccharides of the Vietnamese brown alga Sargassum aquifolium (Fucales, Sargassaceae).

A fucoidan preparation named FSA was isolated from the brown alga Sargassum aquifolium collected from the coastal waters of Vietnam. l-Fucose, d-galactose, d-mannose, d-glucuronic acid, d-xylose, and sulfate were found to be the main constituents of FSA. The preparation was fractionated by anion-exchange chromatography on DEAE-Sephacel eluted stepwise with 0.5, 1.0, 1.5, and 2.0 M NaCl to give four fractions differing in monosaccharide composition and degree of sulfation. Their NMR spectra were too complex to be completely interpreted. Fractions 1.0 M and 1.5 M were analyzed by methylation before and after desulfation. In addition, desulfated 1.0 M was fractionated by anion-exchange chromatography into six fractions according to the uronic acid content. They were characterized by methylation and NMR spectral data, and three structurally different polysaccharides were identified. One of them has a core of alternating 2-linked α-d-Manp and 4-linked ß-d-GlcpA residues, about a half of the former bearing single α-l-Fucp or ß-d-Xylp at position 3. The second polymer is a (1 â†’ 3)-ß-d-glucopyranuronan partially substituted with single ß-d-Xylp or single α-l-Fucp at position 4. The third polysaccharide is a xylo(fuco)galactan having a linear core of alternating 4-linked α-d-Gal and 3-linked ß-d-Gal residues. The latter bear single ß-d-Xylp or a short chain of 4-linked ß-d-Xyl, 6-linked ß-d-Gal, and variously linked α-l-Fuc. In FSA, these polysaccharides are sulfated at different positions and devoid of regularity. Fractions of FSA possess anticoagulant, cytotoxic, and antitumor activities, which increase with the degree of sulfation. The most sulfated fraction 2.0 M that contains mainly a sulfated fucogalactan, is about half as active as anticoagulant as the standard low-molecular mass heparin (enoxaparin).