RESUMEN

This study was conducted to evaluate whether adding ß-mannanase alone or in combination with a multi-carbohydrase complex to simple and complex diets could improve diet digestibility, nutrient and energy metabolism, and gut health in weaned pigs. Thirty pigs (7.9 kg ± 0.851 kg) weaned at 28 days were randomly split into a 2 × 3 factorial arrangement, considering a simple (corn and soybean meal-based diet) or complex diet (13% point reduction in inclusion of soybean meal, 5% of whey power, and 2.5% of spray-dried plasma compared to the simple diet) and diet without any addition (control) or the addition of ß-mannanase (BM; 0.300 g/kg of the diet) or ß-mannanase plus a multi-carbohydrase complex blend such as xylanase, ß-glucanase, and arabinofuranosidases (BM + MCC; 0.300 + 0.050 g/kg of the diet) for 17 days post-weaned. Total fecal and urine samples were collected on days 11-17. Fecal samples were collected from all pigs to identify fecal biomarkers using commercial ELISA tests. Blood samples were collected from all pigs at the end of the experimental period to assess serum concentrations of acute-phase proteins. All pigs were euthanized on day 18 for intestinal tissue collection. The simple diet had greater (p < 0.05) protein digestibility and metabolizability coefficients than the complex diet. Greater (p < 0.05) energy digestibility and energy metabolizability coefficients were observed in the BM and BM+ MCC compared to the control diet. On average, BM improved by 64 kcal/kg and BM + MCC improved by 100 kcal/kg of metabolizable energy. Furthermore, the addition of BM and BM + MCC to the diets led to lower fecal moisture and fecal output. Moreover, the BM and BM + MCC diets also reduced fecal calprotectin concentrations by 29 and 46%, respectively, compared to control pigs (p < 0.001). We conclude that simple diets are a suitable alternative to complex diets, without compromising the nutrient digestibility and gut health of post-weaned pigs. The addition of exogenous enzymes improves nutrient and energy utilization, as well as the absorption area, and decreases calprotectin concentrations.

RESUMEN

Cellulose is a major renewable resource for a wide variety of sustainable industrial products. However, for its utilization, finding new efficient enzymes for plant cell wall depolymerization is crucial. In addition to microbial sources, cellulases also exist in plants, however, are less studied. Fleshy fruit ripening includes enzymatic cell wall hydrolysis, leading to tissue softening. Therefore, bilberry (Vaccinium myrtillus L.), which produces small fruits that undergo extensive and rapid softening, was selected to explore cellulases of plant origin. We identified 20 glycoside hydrolase family 9 (GH9) cellulases from a recently sequenced bilberry genome, including four of which showed fruit ripening-specific expression and could be associated with fruit softening based on phylogenetic, transcriptomic and gene expression analyses. These four cellulases were secreted enzymes: two B-types and two C-types with a carbohydrate binding module 49. For functional characterization, these four cellulases were expressed in Pichia pastoris. All recombinant enzymes demonstrated glucanase activity toward cellulose and hemicellulose substrates. Particularly, VmGH9C1 demonstrated high activity and ability to degrade cellulose, xyloglucan, and glucomannan. In addition, all the enzymes retained activity under wide pH (6-10) and temperature ranges (optimum 70 °C), revealing the potential applications of plant GH9 cellulases in the industrial bioprocessing of lignocellulose.

Asunto(s)

Celulasas , Celulosa , Frutas , Celulosa/metabolismo , Celulosa/química , Celulasas/metabolismo , Celulasas/genética , Celulasas/química , Frutas/enzimología , Filogenia , Polimerizacion , Especificidad por Sustrato , Concentración de Iones de Hidrógeno , Temperatura

RESUMEN

Enzymes that degrade ß-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-ß-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%-25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal ß-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-ß-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-ß-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

Asunto(s)

Estabilidad de Enzimas , beta-Glucanos , beta-Glucanos/metabolismo , Cladosporium/enzimología , Cladosporium/genética , Dominios Proteicos , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Endo-1,3(4)-beta-Glucanasa/genética , Endo-1,3(4)-beta-Glucanasa/metabolismo , Endo-1,3(4)-beta-Glucanasa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Clonación Molecular , Temperatura , Saccharomycetales

RESUMEN

ß-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of ß-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of ß-1,6-glucan by ß-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel ß-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-ß-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of ß-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: • ß-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • ß-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported ß-1, 6-glucanase derived from Flavobacterium.

Asunto(s)

Antifúngicos , Pared Celular , Escherichia coli , Flavobacterium , Glicósido Hidrolasas , Flavobacterium/enzimología , Flavobacterium/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hidrólisis , Antifúngicos/farmacología , Antifúngicos/metabolismo , Pared Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucanos/metabolismo , Concentración de Iones de Hidrógeno , beta-Glucanos/metabolismo , Clonación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Especificidad por Sustrato , Polisacáridos

RESUMEN

ß-1,3-glucanases can degrade ß-1,3-glucoside bonds in ß-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the ß-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-ß-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Asunto(s)

Antifúngicos , Flavobacterium , Glucano 1,3-beta-Glucosidasa , Proteínas Recombinantes , Flavobacterium/genética , Flavobacterium/enzimología , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/metabolismo , Antifúngicos/farmacología , Antifúngicos/química , Glucano 1,3-beta-Glucosidasa/genética , Glucano 1,3-beta-Glucosidasa/química , Glucano 1,3-beta-Glucosidasa/metabolismo , Fusarium/efectos de los fármacos , Fusarium/enzimología , Fusarium/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/genética , Especificidad por Sustrato , Clonación Molecular

RESUMEN

Trichoderma harzianum T4 is a soil fungus that plays an important role in the biological control of plant diseases. The aim of this study was to functionally characterize the ß-1,6-glucanase gene Neg1 in T. harzianum T4 and to investigate the effect of its overexpression on biocontrol traits, especially antagonism against pathogenic fungi. We found that overexpression of Neg1 did not affect growth of T. harzianum but enhanced sporulation of T. harzianum T4 cultures. Generally, spores are closely related to the defense ability of defense fungi and can assist their proliferation and improve their colonization ability. Secondly, overexpression of Neg1 also increased the secretion level of various hydrolytic enzymes and enhanced the antagonistic ability against phytopathogenic fungi of Fusarium spp. The results suggest that Neg1 is a key gene for improving the biocontrol effect of T. harzianum T4, which contributes to a better understanding of the mechanism of action of T. harzianum T4 as a fungal biocontrol agent.

Asunto(s)

Antibiosis , Fusarium , Enfermedades de las Plantas , Esporas Fúngicas , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Fusarium/genética , Fusarium/fisiología , Esporas Fúngicas/crecimiento & desarrollo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/genética , Hypocreales/metabolismo , Control Biológico de Vectores , Agentes de Control Biológico/metabolismo , Trichoderma/genética , Trichoderma/fisiología , Trichoderma/metabolismo

RESUMEN

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Asunto(s)

Pinus , Enfermedades de las Plantas , Tylenchida , Pinus/parasitología , Animales , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/genética , Tylenchida/patogenicidad , Tylenchida/genética , Virulencia , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Interacciones Huésped-Parásitos/genética

RESUMEN

Cross-linking chitosan at room and subzero temperature using a series of diglycidyl ethers of glycols (DEs)-ethylene glycol (EGDE), 1,4-butanediol (BDDE), and poly(ethylene glycol) (PEGDE) has been investigated to demonstrate that DEs can be a more powerful alternative to glutaraldehyde (GA) for fabrication of biocompatible chitosan cryogels with tunable properties. Gelation of chitosan with DEs was significantly slower than with GA, allowing formation of cryogels with larger pores and higher permeability, more suitable for flow-through applications and cell culturing. Increased hydration of the cross-links with increased DE chain length weakened intermolecular hydrogen bonding in chitosan and improved cryogel elasticity. At high cross-linking ratios (DE:chitosan 1:4), the toughness and compressive strength of the cryogels decreased in the order EGDE > BDDE > PEGDE. By varying the DE chain length and concentration, permeable chitosan cryogels with elasticity moduli from 10.4 ± 0.8 to 41 ± 3 kPa, toughness from 2.68 ± 0.5 to 8.3 ± 0.1 kJ/m3, and compressive strength at 75% strain from 11 ± 2 to 33 ± 4 kPa were fabricated. Susceptibility of cryogels to enzymatic hydrolysis was identified as the parameter most sensitive to cross-linking conditions. Weight loss of cryogels increased with increased DE chain length, and degradation rate of PEGDE-cross-linked chitosan decreased 612-fold, when the cross-linker concentration increased 20-fold.

RESUMEN

As an essential component of the fungal cell wall, ß-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel ß-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous ß-1,6-glycosidic bonding polysaccharides. Based on this, ß-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 105/ml conidia with 0.03 µg/µl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure.

RESUMEN

A novel glycoside hydrolase (GH) family 16 multi-domain ß-1,3-1,4-glucanase (FsGlc16A) from Fibrobacter sp. UWP2 was identified, heterogeneously expressed, and its enzymatic properties, protein structure and application potential were characterized. Enzymological characterization showed that FsGlc16A performed the optimal catalytic activity at pH 4.5 and 50 °C with a specific activity of 3263 U/mg. FsGlc16A exhibited the substrate specificity towards oat ß-glucan, barley ß-glucan and lichenan, and in addition, it hydrolyzed oat ß-glucan and lichenan into different ß-glucooligosaccharides with polymerization degrees of 3-4, which further illustrated that it belonged to the endo-type ß-1,3-1,4-glucanase. FsGlc16A was classified in subfamily25 of GH16. A 'PXSSSS' repeats domain was identified at the C-terminus of FsGlc16A, which was distinct from the typical GH family 16 ß-1,3-1,4-glucanases. Removing the 'PXSSSS' repeats domain affected the binding of the substrate to FsGlc16A and reduced the enzyme activity. FsGlc16A displayed good potential for the applications, which hydrolyzed oat bran into ß-glucooligosaccharides, and reduced filtration time (18.89 %) and viscosity (3.64 %) in the saccharification process. This study investigated the enzymatic properties and domain function of FsGlc16A, providing new ideas and insights into the study of ß-1,3-1,4-glucanase.

Asunto(s)

Glucanos , Especificidad por Sustrato , Hidrólisis , Glucanos/química , Glucanos/metabolismo , Concentración de Iones de Hidrógeno , Secuencia de Aminoácidos , Temperatura , Dominios Proteicos , beta-Glucanos/metabolismo , beta-Glucanos/química , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Cinética , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/metabolismo , Endo-1,3(4)-beta-Glucanasa/genética , Clonación Molecular , Filogenia , Estabilidad de Enzimas

RESUMEN

OBJECTIVES: To evaluate the effect of daily use of a multiple-enzyme lozenge on de novo plaque formation, on gingivitis development, and on the oral microbiome composition. METHODS: This trial with two parallel arms included 24 healthy adults allocated to the Active (n = 12) or Placebo (n = 12) group. Subjects consumed one lozenge three times daily for seven days, and no oral hygiene procedures were allowed. Differences in de novo plaque accumulation between a baseline period, and one and seven days of intervention were assessed by the Turesky-modification of the Quigley-and-Hein-Plaque-Index (TM-QHPI). The development of gingivitis after seven days of intervention was assessed by the Gingival Index (GI). Plaque and saliva samples were collected at baseline and after seven days of intervention, and evaluated by 16S rRNA gene sequencing. RESULTS: All subjects completed the study, and no adverse events were reported. After one day, the average TM-QHPI was significantly lower in the Active than in the Placebo group, as compared to baseline (p = 0.012). After 7 days, average TM-QHPI values did not differ significantly between groups (p = 0.37). GI values did not increase during the intervention period, with no difference between groups (p = 0.62). Bacterial richness increased in both plaque and saliva samples over a seven-day oral hygiene-free period, with a statistically significant difference for the saliva samples (p = 0.0495) between groups. CONCLUSIONS: A multiple-enzymes lozenge decreased the build-up of de novo plaque after one day and slowed down the process of species increment in saliva. The lozenge may be an adjunct to regular mechanical plaque removal. CLINICAL SIGNIFICANCE: Dental plaque is the main cause of caries, gingivitis, and periodontitis. The search for therapeutic adjuncts to mechanical plaque removal that have no harmful effects on the oral microbiome is important. Treatment with multiple plaque-matrix degrading enzymes is a promising non-biocidal approach to plaque control.

Asunto(s)

Biopelículas , Índice de Placa Dental , Placa Dental , Gingivitis , Índice Periodontal , Saliva , Humanos , Placa Dental/microbiología , Femenino , Gingivitis/microbiología , Masculino , Biopelículas/efectos de los fármacos , Adulto , Saliva/microbiología , Proyectos Piloto , Adulto Joven , ARN Ribosómico 16S , Microbiota/efectos de los fármacos , Método Doble Ciego , Higiene Bucal , Resultado del Tratamiento , Hidrolasas/uso terapéutico , Persona de Mediana Edad

RESUMEN

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the Km values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the Km value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself.

Asunto(s)

Pared Celular , Flavobacterium , Glucanos , Dominios Proteicos , Flavobacterium/enzimología , Flavobacterium/genética , Flavobacterium/metabolismo , Pared Celular/metabolismo , Glucanos/metabolismo , Hidrólisis , Dominio Catalítico , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/química , Señales de Clasificación de Proteína

RESUMEN

The capacity of combinations of feed enzymes, natural betaine and a probiotic, combined with alternative plant-based ingredients, to totally replace soybean meal (SBM) in a broiler diet was evaluated. Day-old Ross 308 males (2,574) were assigned to 9 treatments (13 pens/treatment, 22 birds/pen) in a completely randomized design. All diets were pelleted and fed ad libitum in 4 phases: starter, grower, finisher 1, finisher 2 (0-10, 10-21, 21-35, and 35-42 d of age, respectively). Treatments included: 1) control diet containing SBM (SBM control), supplemented with phytase (PhyG), at 2,000, 1,500, 1000 and 1,000 FTU/kg in each phase and xylanase (X) at 750 U/kg, [crude protein (CP): 23.5%, 22.0%, 20.2% and 19.3% in each phase]; 2) to 5), alternative (ALT), SBM-free diets, containing the same CP level as the control ("CP high"), supplemented with PhyG as in the control, protease (P, 800 U/kg) and in 2) xylanase (750 U/kg) (ALT+PhyG+P+X), 3) xylanase-ß-glucanase (XB, 1,200 U/kg and 152 U/kg) (Alt+PhyG+P+XB), 4) XB plus betaine (800 g/ton) (ALT+PhyG+P+XB+Bet), and 5) XB plus a probiotic [150,000 colony forming units (CFU)/g] (ALT+PhyG+P+XB+Prob); 6) to 9) as treatments 2) to 5) but with CP reduced by -2.0 to -1.5% points vs. control ('CP low'). Final (d 42) BW and overall (d 0-42) feed conversion ratio (FCR) of birds fed the SBM control exceeded breeder objectives (+3.8% and -1.9%, respectively). Overall FCR was reduced and d 42 BW increased in birds fed "low" vs. "high" CP (P < 0.01). Overall FCR and feed intake were not different in ALT+PhyG+XB+P+Bet and ALT+PhyG+XB+P+Prob vs. the control, whereas final BW was reduced (P < 0.05) in all ALT treatments but close to breeder objectives (98.3%) in ALT+PhyG+XB+P+Prob. Feed costs of this treatment were similar to the control. Total replacement of SBM with alternative plant-based ingredients in a CP-low diet supplemented with hydrolytic enzymes and probiotics can achieve growth performance outcomes close to commercial breeder objectives.

Asunto(s)

Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Betaína , Pollos , Dieta , Suplementos Dietéticos , Glycine max , Animales , Alimentación Animal/análisis , Pollos/crecimiento & desarrollo , Pollos/fisiología , Masculino , Dieta/veterinaria , Suplementos Dietéticos/análisis , Betaína/administración & dosificación , Betaína/metabolismo , Glycine max/química , Fenómenos Fisiológicos Nutricionales de los Animales/efectos de los fármacos , Probióticos/administración & dosificación , Distribución Aleatoria , 6-Fitasa/administración & dosificación , 6-Fitasa/metabolismo , Endo-1,4-beta Xilanasas/administración & dosificación , Endo-1,4-beta Xilanasas/metabolismo

RESUMEN

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the efficacy of ROVABIO® ADVANCE (liquid and solid) which contains endo-1,4-beta-xylanase and endo-1,3(4)-beta-glucanase produced with Talaromyces versatilis IMI 378536 and DSM 26702 as a zootechnical feed additive for weaned piglets at the recommended use level of 1800 U xylanase and 1250 U glucanase per kg feed. In a previous assessment, three long-term trials in weaned piglets were submitted. Two of them were considered to support the efficacy of the additive while a third trial was not further considered due to the large number of veterinary treatments applied. A new trial was provided to support the efficacy of the additive, but it did not show a significant improvement of the performance parameters at the minimum recommended use level. Due to the lack of sufficient data, the FEEDAP Panel is not in the position to conclude on the efficacy of the additive for the target species.

RESUMEN

BACKGROUND: Xylanase and ß-glucanase combination (XG) hydrolyzes soluble non-starch polysaccharides that are anti-nutritional compounds. This study aimed to evaluate the effects of increasing levels of XG on intestinal health and growth performance of nursery pigs. METHODS: Forty pigs (6.5 ± 0.4 kg) were assigned to 5 dietary treatments and fed for 35 d in 3 phases (11, 9, and 15 d, respectively). Basal diets mainly included corn, soybean meal, and corn distiller's dried grains with solubles, contained phytase (750 FTU/kg), and were supplemented with 5 levels of XG at (1) 0, (2) 280 TXU/kg xylanase and 125 TGU/kg ß-glucanase, (3) 560 and 250, (4) 840 and 375, or (5) 1,120 and 500, respectively. Growth performance was measured. On d 35, all pigs were euthanized and jejunal mucosa, jejunal digesta, jejunal tissues, and ileal digesta were collected to determine the effects of increasing XG levels and XG intake on intestinal health. RESULTS: Increasing XG intake tended to quadratically decrease (P = 0.059) viscosity of jejunal digesta (min: 1.74 mPa·s at 751/335 (TXU/TGU)/kg). Increasing levels of XG quadratically decreased (P < 0.05) Prevotellaceae (min: 0.6% at 630/281 (TXU/TGU)/kg) in the jejunal mucosa. Increasing XG intake quadratically increased (P < 0.05) Lactobacillaceae (max: 40.3% at 608/271 (TXU/TGU)/kg) in the jejunal mucosa. Increasing XG intake quadratically decreased (P < 0.05) Helicobacteraceae (min: 1.6% at 560/250 (TXU/TGU)/kg) in the jejunal mucosa. Increasing levels of XG tended to linearly decrease (P = 0.073) jejunal IgG and tended to quadratically increase (P = 0.085) jejunal villus height to crypt depth ratio (max: 2.62 at 560/250 (TXU/TGU)/kg). Increasing XG intake tended to linearly increase the apparent ileal digestibility of dry matter (P = 0.087) and ether extract (P = 0.065). Increasing XG intake linearly increased (P < 0.05) average daily gain. CONCLUSIONS: A combinational use of xylanase and ß-glucanase would hydrolyze the non-starch polysaccharides fractions, positively modulating the jejunal mucosa-associated microbiota. Increased intake of these enzyme combination possibly reduced digesta viscosity and humoral immune response in the jejunum resulting in improved intestinal structure, and ileal digestibility of nutrients, and finally improving growth of nursery pigs. The beneficial effects were maximized at a combination of 550 to 800 TXU/kg xylanase and 250 to 360 TGU/kg ß-glucanase.

RESUMEN

All living organisms produce only one enantiomer, so we found that all natural compounds are presented in enantiomerically pure form. Asymmetric synthesis is highly spread in medicinal chemistry because enantiomerically pure drugs are highly applicable. This study initially demonstrated the feasibility of a good idea for the asymmetric synthesis of α-alkylated carbonyl compounds with high enantiomeric purity ranging from 91 to 94% using different quinazolinone derivatives. The structure of all compounds was confirmed via elemental analysis and different spectroscopic data and the enantioselectivity was determined via HPLC using silica gel column. The synthesized compounds' mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-beta-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed that compound 6a has a broad biocidal activity and this in-vitro study was in line with the in-silico results. Overall, the formulated compound 6a can be employed as antimicrobial agent without any toxicity with high bioavailability in medical applications.

Asunto(s)

Antiinfecciosos , Simulación del Acoplamiento Molecular , Antiinfecciosos/farmacología , Antiinfecciosos/química , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacocinética , Estereoisomerismo , Pruebas de Sensibilidad Microbiana , Alquilación

RESUMEN

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Asunto(s)

Bacillus , Proteínas Bacterianas , Estabilidad de Enzimas , Escherichia coli , Proteínas Recombinantes , Bacillus/enzimología , Bacillus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Clonación Molecular , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/biosíntesis , Expresión Génica , Temperatura , Especificidad por Sustrato

RESUMEN

The melon (Cucumis melo L.) is a globally cherished and economically significant crop. The grafting technique has been widely used in the vegetative propagation of melon to promote environmental tolerance and disease resistance. However, mechanisms governing graft healing and potential incompatibilities in melons following the grafting process remain unknown. To uncover the molecular mechanism of healing of grafted melon seedlings, melon wild type (Control) and TRV-CmGH9B3 lines were obtained and grafted onto the squash rootstocks (C. moschata). Anatomical differences indicated that the healing process of the TRV-CmGH9B3 plants was slower than that of the control. A total of 335 significantly differentially expressed genes (DEGs) were detected between two transcriptomes. Most of these DEGs were down-regulated in TRV-CmGH9B3 grafted seedlings. GO and KEGG analysis showed that many metabolic, physiological, and hormonal responses were involved in graft healing, including metabolic processes, plant hormone signaling, plant MAPK pathway, and sucrose starch pathway. During the healing process of TRV-CmGH9B3 grafted seedlings, gene synthesis related to hormone signal transduction (auxin, cytokinin, gibberellin, brassinolide) was delayed. At the same time, it was found that most of the DEGs related to the sucrose pathway were down-regulated in TRV-CmGH9B3 grafted seedlings. The results showed that sugar was also involved in the healing process of melon grafted onto squash. These results deepened our understanding of the molecular mechanism of GH9B3, a key gene of ß-1, 4-glucanase. It also provided a reference for elucidating the gene mechanism and function analysis of CmGH9B3 in the process of graft union healing.

Asunto(s)

Cucumis melo , Cucurbita , Cucurbitaceae , Cucumis melo/genética , Cucumis melo/metabolismo , Perfilación de la Expresión Génica , Cucurbita/genética , Cucurbita/metabolismo , Cucurbitaceae/genética , Sacarosa/metabolismo

RESUMEN

BACKGROUND: Degradation via enzymatic processes for the production of valuable ß-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific ß-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6. RESULTS: Crystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical ß-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel's network structure, enhancing the hydrolysis of curdlan by specific ß-1,3-glucanases. CONCLUSIONS: This study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical ß-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with ß-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.

RESUMEN

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Asunto(s)

Celulasa , Celulosa , Animales , Celulosa/metabolismo , Celulasa/genética , Celulasa/metabolismo , Peces/microbiología , Intestinos/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Celulasas/metabolismo , Celulasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo