Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Erratum to "Improved fermentation strategies in a bioreactor for enhancing poly(3-hydroxybutyrate) (PHB) production by wild type Cupriavidus necator from fructose" [Heliyon 7 (1) (January 2021) Article e05979].

[This corrects the article DOI: 10.1016/j.heliyon.2021.e05979.].

Improved fermentation strategies in a bioreactor for enhancing poly(3-hydroxybutyrate) (PHB) production by wild type Cupriavidus necator from fructose.

Poly(3-hydroxybutyrate) (PHB) belongs to the family of polyhydroxyalkanoates, biopolymers used for agricultural, industrial, or even medical applications. However, scaling up the production is still an issue due to the myriad of parameters involved in the fermentation processes. The present work seeks, firstly, to scale up poly(3-hydroxybutyrate) (PHB) production by wild type C. necator ATCC 17697 from shaken flasks to a stirred-tank bioreactor with the optimized media and fructose as carbon source. The second purpose is to improve the production of PHB by applying both the batch and fed-batch fermentation strategies in comparison with previous works of wild type C. necator with fructose. Furthermore, thinking of biomedical applications, physicochemical, and cytotoxicity analyses of the produced biopolymer, are presented. Fed-batch fermentation with an exponential feeding strategy enabled us to achieve the highest values of PHB concentration and productivity, 25.7 g/l and 0.43 g/(l h), respectively. The PHB productivity was 3.3 and 7.2 times higher than the one in batch strategy and shaken flask cultures, respectively. DSC, FTIR, 1H, and 13C NMR analysis led to determine that the biopolymer produced by C. necator ATCC 17697 has a molecular structure and characteristics in agreement with the commercial PHB. Additionally, the biopolymer does not induce cytotoxic effects on the NIH/3T3 cell culture. Due to the improved fermentation strategies, PHB concentration resulted in 40 % higher of the already reported one for wild type C. necator using other fed-batch modes and fructose as a carbon source. Thus the produced PHB could be attractive for biomedical applications, which generate a rising interest in polyhydroxyalkanoates during recent years.

Impact of hepatitis B virus genotype F on in vitro diagnosis: detection efficiency of HBsAg from Amerindian subgenotypes F1b and F4.

The influence of the high genetic variability of hepatitis B virus (HBV) on the sensitivity of serological assays has received little attention so far. A major source of variability is related to viral genotypes and subgenotypes. Their possible influence on diagnosis and prophylaxis is poorly known and has mostly been evaluated for genotypes A, B, C and D. Robust data showing the detection efficiency of HBsAg from genotype F is lacking. This study examined the effect of virus-like particles containing HBsAg from genotypes A and F (particularly, F1b and F4) produced in Pichia pastoris in relation to the anti-HBs antibodies used in the immunoassays for in vitro diagnosis and compared it with that exerted by the G145R S-escape mutant. The results showed that HBsAg detection rates for subgenotypes F1b and F4 differed significantly from those obtained for genotype A and that subgenotype F1b had a major impact on the sensitivity of the immunoassays tested. Prediction of the tertiary structure of subgenotypes F1b and F4 revealed changes inside and outside the major hydrophilic region (aa 101-160) of the HBsAg compared to genotype A and the G145R variant. A phosphorylation site (target for protein kinase C) produced by the G145R substitution might prevent recognition by anti-HBs antibodies. In conclusion, the use of different genotypes or variants for diagnosis could improve the rate of detection of HBV infection. The incorporation of a genotype-F-derived HBsAg vaccine in areas where this genotype is endemic should be evaluated, since this might also affect vaccination efficacy.

Pectinase production by Aspergillus giganteus in solid-state fermentation: optimization, scale-up, biochemical characterization and its application in olive-oil extraction.

The application of pectinases in industrial olive-oil processes is restricted by its production cost. Consequently, new fungal strains able to produce higher pectinase titers are required. The aim of this work was to study the capability of Aspergillus giganteus NRRL10 to produce pectinolytic enzymes by SSF and evaluate the application of these in olive-oil extraction. A. giganteus was selected among 12 strains on the basis of high pectinolytic activity and stability. A mixture composed by wheat bran, orange, and lemon peels was selected as the best substrate for enzyme production. Statistical analyses of the experimental design indicated that pH, temperature, and CaCl2 are the main factors that affect the production. Subsequently, different aeration flows were tested in a tray reactor; the highest activity was achieved at 20 L min-1 per kilogram of dry substrate (kgds). Finally, the pectinolytic enzymes from A. giganteus improved the oil yield and rheological characteristics without affecting oil chemical properties.

Production in stirred-tank bioreactor of recombinant bovine chymosin B by a high-level expression transformant clone of Pichia pastoris.

An intense screening of Pichia pastoris clones transformed with the gene of bovine chymosin under methanol-inducible AOX1 promoter was performed, obtaining a transformant clone with a higher milk-clotting activity value in comparison with our previous studies. The scaling of recombinant-chymosin production was carried out by a fed-batch strategy in a stirred-tank bioreactor using biodiesel-byproduct crude glycerol as the carbon source and pure methanol for the induction of chymosin expression, achieving a biomass concentration of 158 g DCW/L and a maximum coagulant activity of 192 IMCU/ml after 120 h of methanol induction. Recombinant bovine chymosin was purified from bioreactor-fermentation culture by a procedure including anion-exchange chromatography which allowed obtaining heterologous chymosin with high level of purity and activity; suggesting that this downstream step could be scaled up in a successful manner for chymosin purification. Thermoestability assay permitted to establish that unformulated recombinant chymosin could be stored at 5 °C without decrease of enzyme activity throughout at least 120 days. Finally, reiterative methanol-inductions of recombinant chymosin expression in bioreactor demonstrated that the reutilization of cell biomass overcame the low enzyme productivity usually reached by P. pastoris system.

A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation.

A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m , and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter.

A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01h(-1). By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37°C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.

Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter.

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Antipathogenic properties of Lactobacillus plantarum on Pseudomonas aeruginosa: the potential use of its supernatants in the treatment of infected chronic wounds.

Pathogenic bacteria delay wound healing through several different mechanisms such as persistent production of inflammatory mediators or maintenance of necrotic neutrophils, which release cytolytic enzymes and free oxygen radicals. One of the most frequent pathogens isolated from infections in chronic wounds is Pseudomonas aeruginosa. This bacterium is extremely refractory to therapy and to host immune attack when it forms biofilms. Therefore, antibiotics and antiseptics are becoming useless in the treatment of these infections. In previous works, we demonstrated that Lactobacillus plantarum has an important antipathogenic capacity on P. aeruginosa. The aim of the present work was to elucidate the mechanism involved in the control of growth of P. aeruginosa on different surfaces by L. plantarum. For this purpose, we investigated the effects of L. plantarum supernatants on pathogenic properties of P. aeruginosa, such as adhesion, viability, virulence factors, biofilm formation, and quorum sensing signal expression. L. plantarum supernatants were able to inhibit pathogenic properties of P. aeruginosa by a quorum quenching mechanism. The antipathogenic properties mentioned above, together with the immunomodulatory, tissue repair, and angiogenesis properties in the supernatants of L. plantarum, make them an attractive option in infected chronic wound treatment.

The structure of the agaran sulfate from Acanthophora spicifera (Rhodomelaceae, Ceramiales) and its antiviral activity. Relation between structure and antiviral activity in agarans.

The sulfated agaran isolated by water extraction from the red seaweed, Acanthophora spicifera (Rhodomelaceae, Ceramiales), is made up of A-units highly substituted with sulfate groups on C-2 (28-30%), sulfates on C-2 and 4,6-O-(1'-carboxyethylidene) groups (9-15%), and only the C-2 sulfate groups (5-8%) with small amounts of C-6 sulfate, 6-O-methyl, and nonsubstituted residues. B-units are formed mainly by 3,6-anhydro-alpha-L-galactose (15-16%) and its precursor, alpha-L-galactose 6-sulfate (10-17%), together with lesser amounts of 3,6-anhydro-alpha-L-galactose 2-sulfate, alpha-L-galactose 2,6-disulfate, alpha-L-galactose 2,3,6-tri-sulfate, alpha-L-galactose 2,6-disulfate 3-xylose, 2-O-methyl-alpha-L-galactose, and unsubstituted alpha-L-galactose. Small, but significant quantities of beta-D-xylose were found in all the fractions, together with small amounts to traces of D-glucose. Some of the fractions have high antiviral activity. Attempts to correlate structure and antiviral activity in agarans are presented.