Pelagic Sargassum events in Jamaica: Provenance, morphotype abundance, and influence of sample processing on biochemical composition of the biomass.

Pelagic Sargassum species have been known for centuries in the Sargasso Sea of the North Atlantic Ocean. In 2011, a new area concentrating high biomass of these brown algae started developing in the Tropical Atlantic Ocean. Since then, massive and recurrent Sargassum influxes have been reported in the Caribbean and off the coast of Western Africa. These Sargassum events have a major negative impact on coastal ecosystems and nearshore marine life, and affect socio-economic sectors, including public health, coastal living, tourism, fisheries, and maritime transport. Despite recent advances in the forecasting of Sargassum events, and elucidation of the seaweed composition, many knowledge gaps remain, including morphotype abundance during Sargassum events, drift of the seaweeds in the months prior to stranding, and influence of sample processing methods on biomass biochemical composition. Using seaweeds harvested on the coasts of Jamaica in summer of 2020, we observed that S. fluitans III was the most abundant morphotype at different times and sampling locations. No clear difference in the geographical origin, or provenance, of the Sargassum mats was observed. The majority of Sargassum backtracked from both north and south of Jamaica experienced ambient temperatures of around 27 °C and salinity in the range of 34-36 psu before stranding. We also showed that cheap (sun) compared to expensive (freeze) drying techniques influence the biochemical composition of biomass. Sun-drying increased the proportion of phenolic compounds, but had a deleterious impact on fucoxanthin content and on the quantities of monosaccharides, except for mannitol. Effects on the content of fucose containing sulfated polysaccharides depended on the method used for their extraction, and limited variation was observed in ash, protein, and fatty acid content within most of the sample locations investigated. These observations are important for the storage and transport of the biomass in the context of its valorisation.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Temporal dynamics and metabolic correlation between lactate-producing and hydrogen-producing bacteria in sugarcane vinasse dark fermentation: The key role of lactate.

This study aimed to better understand the role of different microbial groups and the determining fermentation pathways in a biohydrogen (bioH2)-producing reactor fed with sugarcane vinasse by using next-generation sequencing and principal component analysis (PCA). Both microbial structure and dynamics were characterized. The highest bioH2 production levels were associated with the Thermoanaerobacterium genus, whilst lactate-producing bacteria comprised the dominant genera (e.g. Lactobacillus and Leuconostoc) within the reactor at both stable and unstable bioH2-producing periods. PCA further revealed that the fermentation of lactate played a dual role in the reactor, as both bioH2-producing (acetate+lactate→butyrate+bioH2) and non-bioH2-producing (lactate→propionate+acetate) routes could be observed. Overall, the results suggested that lactate is the primary alternative carbon source in vinasse-fed systems subjected to carbohydrate-shortage conditions.

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification.

Galactanases (endo-ß-1,4-galactanases-EC 3.2.1.89) catalyze the hydrolysis of ß-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-ß-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5-8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4-10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25-45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s(-1) mg(-1) mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE(®)1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

OBJECTIVES: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein. RESULTS: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase. CONCLUSION: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.

Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme.

As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase.

Structural basis for glucose tolerance in GH1 ß-glucosidases.

Product inhibition of ß-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some ß-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal ß-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family ß-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

Molecular and functional characterization of a putative PA28γ proteasome activator orthologue in Schistosoma mansoni.

PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28γ in larval growth/development of the S. mansoni.

In vitro schistosomicidal activity of curcumin against Schistosoma mansoni adult worms.

The in vitro schistosomicidal activity of curcumin (doses ranging from 5 to 100 microM) was carried out against Schistosoma mansoni adult worms. Curcumin (at 50 and 100 microM) caused death of all worms. When tested at the doses of 5 and 20 microM, it decreased the worm viability in comparison with negative (Roswell Memorial Park Institute (RPMI) 1640 medium alone or RPMI 1640 medium with 10% dimethyl sulfoxide) and positive (heat-killed worms at 56 degrees C or praziquantel 10 microM) control groups. All pairs of coupled adult worms were separated into individual male and female by the action of curcumin at the doses of 20 to 100 microM. When tested at 5 and 10 microM, curcumin reduced egg production by 50% in comparison with the positive control group. It is the first time that the schistosomicidal activity has been reported for curcumin.