The DiaCoVAb Study in South Italy: Immune Response to SARS-CoV-2 Vaccination in Dialysis Patients.

INTRODUCTION: Since the pandemic of COVID-19 started from December 2019, remarkable numbers of infections and deaths associated with COVID-19 have been recorded worldwide. End-stage kidney disease patients on dialysis are particularly at high risk of infections due to impairments in the innate and adaptive immune systems. Vaccination on dialysis patients (DP) still remains challenging because of the variable response and a low seroconversion rate compared with healthy participants (HP). Therefore, it is urgently necessary to establish a different vaccination strategy for DP, in terms of the dose and administration time. METHODS: Here, we report an observational prospective cohort study in which the immunogenic efficacies of SARS-CoV-2 vaccine BNT162b2 on DP and HP were evaluated by absolute quantification of IgG levels in the blood. RESULTS: DP showed a delayed seroconversion after two vaccine doses, with a low absolute IgG levels compared to HP. While HP reached complete seroconversion within 10 days from the administration of a second dose, only 76% of DP were seropositive. After the booster dose, DP had a strongly improved seroconversion rate as well as antibody levels, reaching 97% seropositivity and 50 times enhancement on antibody levels. DISCUSSION/CONCLUSION: These results prompt to suggest an additional vaccine dose in DP, reducing the interval of time from the second dose. Since limited data are available on immune response in DP overtime after three vaccine doses currently, our study is among the first reports demonstrating the improved seropositivity and IgG levels in DP after the booster vaccine dose.

Butanol production from laccase-pretreated brewer's spent grain.

BACKGROUND: Beer is the most popular alcoholic beverage worldwide. In the manufacture of beer, various by-products and residues are generated, and the most abundant (85% of total by-products) are spent grains. Thanks to its high (hemi)cellulose content (about 50% w/w dry weight), this secondary raw material is attractive for the production of second-generation biofuels as butanol through fermentation processes. RESULTS: This study reports the ability of two laccase preparations from Pleurotus ostreatus to delignify and detoxify milled brewer's spent grains (BSG). Up to 94% of phenols reduction was achieved. Moreover, thanks to the mild conditions of enzymatic pretreatment, the formation of other inhibitory compounds was avoided allowing to apply the sequential enzymatic pretreatment and hydrolysis process (no filtration and washing steps between the two phases). As expected, the high detoxification and delignification yields achieved by laccase pretreatment resulted in great saccharification. As a fact, no loss of carbohydrates was observed thanks to the novel sequential strategy, and thus the totality of polysaccharides was hydrolysed into fermentable sugars. The enzymatic hydrolysate was fermented to acetone-butanol-ethanol (ABE) by Clostridium acetobutilycum obtaining about 12.6 g/L ABE and 7.83 g/L butanol within 190 h. CONCLUSIONS: The applied sequential pretreatment and hydrolysis process resulted to be very effective for the milled BSG, allowing reduction of inhibitory compounds and lignin content with a consequent efficient saccharification. C. acetobutilycum was able to ferment the BSG hydrolysate with ABE yields similar to those obtained by using synthetic media. The proposed strategy reduces the amount of wastewater and the cost of the overall process. Based on the reported results, the potential production of butanol from the fermentation of BSG hydrolysate can be envisaged.

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template.

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N-phenyl-p-phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set-up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less-branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi-ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (∼6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive materials.

Laccase pretreatment for agrofood wastes valorization.

Apple pomace, potato peels, and coffee silverskin are attractive agrofood wastes for the production of biofuels and chemicals, due to their abundance and carbohydrate content. As lignocellulosic biomasses, their conversion is challenged by the presence of lignin that prevents hydrolysis of polysaccharides, hence demanding a pretreatment step. In this work, the effectiveness of Pleurotus ostreatus laccases (with and without mediator) to remove lignin, improving the subsequent saccharification, was assessed. Optimized conditions for sequential protocol were set up for all agrofood wastes reaching delignification and detoxification yields correlated with high saccharification. Especially noteworthy were results for apple pomace and coffee silverskin for which 83% of and 73% saccharification yields were observed, by using laccase and laccase mediator system, respectively. The herein developed sequential protocol, saving soluble sugars and reducing the amount of wastewater, can improve the overall process for obtaining chemicals or fuels from agrofood wastes.

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia.

The lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods-ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-L-arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used in this study.

Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in saccharification of ammonia fibre expansion-pretreated Arundo donax.

This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and ßG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LßX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F.

Exploring the microbiota dynamics related to vegetable biomasses degradation and study of lignocellulose-degrading bacteria for industrial biotechnological application.

The aims of this study were to evaluate the microbial diversity of different lignocellulosic biomasses during degradation under natural conditions and to isolate, select, characterise new well-adapted bacterial strains to detect potentially improved enzyme-producing bacteria. The microbiota of biomass piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra were evaluated by high-throughput sequencing. A highly complex bacterial community was found, composed of ubiquitous bacteria, with the highest representation by the Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes phyla. The abundances of the major and minor taxa retrieved during the process were determined by the selective pressure produced by the lignocellulosic plant species and degradation conditions. Moreover, cellulolytic bacteria were isolated using differential substrates and screened for cellulase, cellobiase, xylanase, pectinase and ligninase activities. Forty strains that showed multienzymatic activity were selected and identified. The highest endo-cellulase activity was seen in Promicromonospora sukumoe CE86 and Isoptericola variabilis CA84, which were able to degrade cellulose, cellobiose and xylan. Sixty-two percent of bacterial strains tested exhibited high extracellular endo-1,4-ß-glucanase activity in liquid media. These approaches show that the microbiota of lignocellulosic biomasses can be considered an important source of bacterial strains to upgrade the feasibility of lignocellulose conversion for the 'greener' technology of second-generation biofuels.

The effect of Pleurotus ostreatus arabinofuranosidase and its evolved variant in lignocellulosic biomasses conversion.

The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer's spent grains (BSG), after chemical or chemical-physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.

Development of an improved variant of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus by directed evolution.

In this study, the α-l-arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α-l-arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a kcat of 7.3 ×1 0¹ ± 0.3 min⁻¹, around 3-fold higher than that of the wild type (2.2 × 10³ ± 0.2 min⁻¹), and a KM (0.54 ± 0.0 6mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-ß-d-glucopyranoside (kcat of 50.85 ± 0.21 min⁻¹ versus 11.0 ± 0.6 min⁻¹) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.

Regulation of cellulase and hemicellulase gene expression in fungi.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.