Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses

Research Areas: Biochemistry & Molecular Biology ; Chemistry ; Polymer ScienceGene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 ◦C. It was stable between pH 5.0–8.0, retaining 85% activity and between 5 ◦C–45 ◦C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley β-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg− 1 s − 1 ) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.info:eu-repo/semantics/publishedVersio

Impact of dietary Chlorella vulgaris and carbohydrate-active enzymes incorporation on plasma metabolites and liver lipid composition of broilers

Research Areas: Veterinary SciencesBackground: Chlorella vulgaris has been proposed as a sustainable green feedstock in poultry nutrition due to its ease of cultivation, minimal environmental impact and balanced nutritional composition. However, the majority of studies documents the use of C. vulgaris as a dietary supplement in broilers instead of a feed ingredient. To the best of our knowledge, no report has shown the effect of a high-level incorporation (>2 % in the diet) of C. vulgaris on plasma metabolites and hepatic lipid composition of broilers. One hundred and twenty Ross 308 male birds were housed in 40 wired-floor cages and randomly distributed by the following experimental diets at 22 days of age (n = 10) during 15 days: (1) a corn-soybean meal based diet (control); (2) based diet with 10% of C. vulgaris; (3) diet 2 supplemented with 0.005% Rovabio® Excel AP; and (4) diet 2 supplemented with 0.01% of a pre-selected four-CAZyme mixture. Results: The inclusion of C. vulgaris at 10% in the diet, regardless of the presence of exogenous CAZymes, changed plasma metabolites but did not compromise broilers growth. Plasma total lipids increased in broilers fed C. vulgaris combined with the two feed CAZymes (p < 0.001) compared with the control diet. Moreover, the supplementation with Rovabio® increased total cholesterol and LDL-cholesterol, while the addition of the four-CAZyme mixture increased triacylglycerols, VLDL-cholesterol and ALP activity. In opposition, HDL-cholesterol levels decreased in broilers fed microalga alone (p = 0.002). Regarding hepatic composition, the inclusion of C. vulgaris in broiler diets, individually or combined with exogenous CAZymes, had a minor effect on fatty acids but improved the n-6/n-3 ratio and total carotenoids. Conclusions: In summary, the inclusion of a high level (10%) of C. vulgaris in broiler´s diet, regardless of the presence of exogenous CAZymes, improved hepatic antioxidant composition and did not impair broiler’s performance. In addition, the feed supplementation with CAZymes increased broilers lipemia. Therefore, dietary C. vulgaris at this incorporation level seems to be safe for animal health and do not compromise performance traits, with no need of CAZymes supplementation.info:eu-repo/semantics/publishedVersio

Immobilization of bacterial feruloyl esterase on mesoporous silica particles and enhancement of synthetic activity by hydrophobic-modified surface

Here, we demonstrated the immobilization of bacterial feruloyl esterase (FAE) from Butyrivibrio sp. XPD2006, Lactobacillus crispatus, Butyrivibrio sp. AE2015, Ruminococcus albus, Cellulosilyticum ruminicola and Clostridium cellulovorans on SBA-15 and their ability to synthesize butyl ferulate (BFA). The BFae2 from Butyrivibrio sp. XPD2006 showed the best catalytic efficiency. High BFA yield was produced when the immobilization of BFae2 took place with a high protein loading and narrow pore sized SBA-15, suggesting alteration of enzyme behavior due to the crowding environment in SBA-15. Grafting of SBA-15 with octyl moieties led to shrinking pore size and resulted in 2.5-fold increment of BFA activity compared to the free enzyme and 70%mol BFA was achieved. The BFae2 encapsulated in hydrophobic-modified SBA-15 endured up to seven reaction cycles while the BFA activity remained above 60%. This is the first report showing the superior performance of hydrophobic-modified surface to entrap FAE to produce fatty phenolic esters

An individual alginate lyase is effective in the disruption of Laminaria digitata recalcitrant cell wall

Research Areas: Science & Technology - Other TopicsIn the present study, 199 pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases were assessed, either alone or in combination, to evaluate their capacity to disrupt Laminaria digitata cell wall, with the consequent release of interesting nutritional compounds. A previously characterized individual alginate lyase, belonging to the family 7 of polysaccharide lyases (PL7) and produced by Saccharophagus degradans, was shown to be the most efcient in the in vitro degradation of L. digitata cell wall. The alginate lyase treatment, compared to the control, released up to 7.11 g/L of reducing sugars (p< 0.001) and 8.59 mmol/100 g dried alga of monosaccharides (p< 0.001), and reduced cell wall fuorescence intensity by 39.1% after staining with Calcofuor White (p= 0.001). The hydrolysis of gel-forming polymer alginate by the alginate lyase treatment could prevent the trapping of fatty acids and release benefcial monounsaturated fatty acids, particularly 18:1c9 (p < 0.001), to the extracellular medium. However, no liberation of proteins (p > 0.170) or pigments (p > 0.070) was observed. Overall, these results show the ability of an individual alginate lyase, from PL7 family, to partially degrade L. digitata cell wall under physiological conditions. Therefore, this CAZyme can potentially improve the bioavailability of L. digitata bioactive compounds for monogastric diets, with further application in feed industry.info:eu-repo/semantics/publishedVersio

Generation of a library of carbohydrate-active enzymes for plant biomass deconstruction

Áreas de pesquisa: Biochemistry & Molecular Biology ; ChemistryIn nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sourcesinfo:eu-repo/semantics/publishedVersio

Effect of Dietary Laminaria digitata with Carbohydrases on Broiler Production Performance and Meat Quality, Lipid Profile, and Mineral Composition

Research Areas: Agriculture; Veterinary Sciences; ZoologyABSTRACT - We hypothesized that dietary inclusion of 15% Laminaria digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing animal growth performance. A total of 120 22-day old broilers were fed the following dietary treatments (n = 10) for 14 days: cereal-based diet (control); control diet with 15% L. digitata (LA); LA diet with 0.005% Rovabio® Excel AP (LAR); LA diet with 0.01% alginate lyase (LAE). Final body weight was lower and feed conversion ratio higher with LA diet than with the control. The ileal viscosity increased with LA and LAR diets relative to control but without differences between LAE and control. The pH of thigh meat was higher, and the redness value of breast was lower with LA diet than with control. Meat overall acceptability was positively scored for all treatments. The γ-tocopherol decreased, whereas total chlorophylls and carotenoids increased in meat with alga diets relative to control. The percentage of n-3 polyunsaturated fatty acids (PUFA) and accumulation of bromine and iodine in meat increased with alga diets compared with control. Feeding 15% of L. digitata to broilers impaired growth performance but enhanced meat quality by increasing antioxidant pigments, with beneficial effects on n-3 PUFA and iodineinfo:eu-repo/semantics/publishedVersio

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies

the Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS) grant LA/P/0059/2020. LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Publisher Copyright: © 2022 The Author(s)The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.publishersversionepub_ahead_of_prin

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - An NMR, X-ray crystallography and computational chemistry approach

12 pags, 6 figs, 1 tabThe direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. © 2008 The Authors.The authors would like to thank the research network REQUIMTE (Project Reqmol), as well as the Portuguese Science and Technology Foundation (FCT-MCTES), for financial support through projectPTDC⁄QUI⁄68286⁄2006 and scholarships SFRH⁄BPD⁄27237⁄2006 and SFRH⁄BD⁄31359⁄200

Effect of Dietary Laminaria digitata with Carbohydrases on Broiler Production Performance and Meat Quality, Lipid Profile, and Mineral Composition

This article belongs to the Section Animal Nutrition.Simple Summary: Seaweeds represent promising alternatives to unsustainable conventional feed sources, such as cereals, incorporated in poultry diets. Brown macroalgae (e.g., Laminaria digitata) correspond to the largest cultured algal biomass worldwide and are rich in bioactive polysaccharides, minerals, and antioxidant pigments. However, their utilization as feed ingredients is limited due to the presence of an intricate gel-forming cell wall composed of indigestible carbohydrates, mainly alginate and fucose-containing sulfated polysaccharides. Therefore, supplementation with carbohydrate-active enzymes is required to disrupt the cell wall and allow seaweed nutrients to be digested and absorbed in poultry gut. The present study aimed to evaluate if the dietary inclusion of 15% L. digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing growth performance of broiler chickens. The results show that L. digitata increases antioxidant pigments and n-3 long-chain polyunsaturated fatty acids in meat, thus improving meat nutritional and health values. On the other hand, feeding algae at a high incorporation level impaired growth performance. Feed enzymatic supplementation had only residual effects, although alginate lyase decreased intestinal viscosity caused by dietary L. digitata with potential benefits for broiler digestibility.Abstract: We hypothesized that dietary inclusion of 15% Laminaria digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing animal growth performance. A total of 120 22-day old broilers were fed the following dietary treatments (n = 10) for 14 days: cereal-based diet (control); control diet with 15% L. digitata (LA); LA diet with 0.005% Rovabio® Excel AP (LAR); LA diet with 0.01% alginate lyase (LAE). Final body weight was lower and feed conversion ratio higher with LA diet than with the control. The ileal viscosity increased with LA and LAR diets relative to control but without differences between LAE and control. The pH of thigh meat was higher, and the redness value of breast was lower with LA diet than with control. Meat overall acceptability was positively scored for all treatments. The γ-tocopherol decreased, whereas total chlorophylls and carotenoids increased in meat with alga diets relative to control. The percentage of n-3 polyunsaturated fatty acids (PUFA) and accumulation of bromine and iodine in meat increased with alga diets compared with control. Feeding 15% of L. digitata to broilers impaired growth performance but enhanced meat quality by increasing antioxidant pigments, with beneficial effects on n-3 PUFA and iodine.The study was funded by the Fundação para a Ciência e a Tecnologia, Portugal, through the PTDC/CAL-ZOO/30238/2017 grant, associated with a postdoctoral contract to M.M.C. and a PhD fellowship to D.C. (SFRH/BD/126198/2016), as well as CIISA (UIDB/00276/2020), AL4AnimalS (LA/P/0059/2020), and LEAF (UIDB/04129/2020) grants.info:eu-repo/semantics/publishedVersio