Functional and structural characterization of an endo-ß-1,3-glucanase from Euglena gracilis.

Endo-ß-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading ß-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-ß-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml-1,kcat 1.20 s-1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml-1,kcat 0.23 ml mg-1 s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified ß-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml-1, kcat 0.88 ml mg-1 s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the ß-1,3/ß-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 µM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing ß-glucans.

Paramylon and synthesis of its ionic derivatives: Applications as pharmaceutical tablet disintegrants and as colloid flocculants.

Paramylon, a high molecular weight polysaccharide, is a linear and unbranched (1 → 3)-ß-d-glucan. Despite its numerous biological benefits, the poor aqueous solubility of crystalline paramylon is a drawback that has hampered some of its applications. In an effort to make this biomaterial amenable to practical uses, cationic and anionic paramylon derivatives were obtained. The degrees of substitution of both products were determined. The products were characterized by FT-IR spectrocopy, ESI mass spectrometry, 1H, 13C and 1H-13C NMR and SEM microscopy. These techniques confirmed the success of the substitution reactions. 1H NMR analysis was used to develop alternative methods for an approximate estimation of the degree of substitution. 1H-13C HSQC NMR spectra were assigned for both derivatives. New applications of native, cationic and anionic paramylon were found. Native paramylon showed similar performance as pharmaceutical tablet disintegrant than sodium croscarmellose. Cationic paramylon behavior as colloid flocculant was comparable with commercial cationic polyacrylamides. The anionic derivative could eventually be used in the formulation of matrix controlled release systems or as a suspending agent.

Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.