RESUMEN
This study assessed the effects of ß-mannanase (BM) supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid (SCFA) production in broiler chickens fed diets with different metabolizable energy (ME) levels. A total of 1,296 male 1-d-old Cobb 500 broilers were randomly distributed in a 3â ×â 2 factorial arrangement (3 ME levelsâ ×â 0 or 200 g/ton BM), with 6 replicates per treatment combination. The 3 ME levels were 3,000 (ME1), 2,930 (ME2), and 2,860 (ME3) kcal/kg, respectively, during the 0 to 3 wk-old stages and 3,150 (ME1), 3,080 (ME2), and 3,010 (ME3) kcal/kg, respectively, during the 3 to 6 wk-old stages. Reducing ME levels increased broiler feed intake (Pâ =â 0.036) and decreased average daily gain (ADG, Pâ =â 0.002) during the entire period. While BM supplementation increased ADG (Pâ =â 0.002) and improved the feed conversion ratio (Pâ =â 0.001) during the 0 to 3 wk-old stages, with no effect during the 3 to 6 wk-old stages. Overall, reducing ME levels increased pancreatic lipase (Pâ =â 0.045) and amylase (Pâ =â 0.013) activity and duodenal amylase activity (Pâ =â 0.047). Notably, BM supplementation significantly increased pancreatic lipase activity (Pâ =â 0.015) and increased lipase (Pâ =â 0.029) and amylase (Pâ =â 0.025) activities in the jejunal chyme. Although diet or enzyme supplementation did not affect microbial diversity, significant differences in microbial communities were observed. At the genus level, decreasing ME levels significantly affected the average abundances of Tyzzerella (Pâ =â 0.028), Candidatus_Bacilloplasma (Pâ =â 0.001), Vibrio (Pâ =â 0.005), and Anaerotruncus (Pâ =â 0.026) among groups, whereas BM supplementation reduced the average abundances of Escherichia-Shigella (Pâ =â 0.048) and increased the average abundances of Barnesiella (Pâ =â 0.047), Ruminococcus (Pâ =â 0.020), Alistipes (Pâ =â 0.050), and Lachnospiraceae_unclassified (Pâ =â 0.009). SCFA concentrations strongly depended on bacterial community composition, and BM supplementation increased acetic acid (Pâ =â 0.004), propionic acid (Pâ =â 0.016), and total SCFA concentrations. In conclusion, BM supplementation improved the performance of younger broilers, and both enzyme supplementation and reduced ME levels positively affected digestive enzyme activity and intestinal microflora.
The broiler industry currently faces numerous challenges, especially the significant rise in feedstuff prices, and the economic considerations with regard to feedstuff formulation have become important factors for reducing costs and enhancing benefits. Low-energy diets have been increasingly suggested to control feed costs; however, lower nutrient concentrations may result in reduced performance, thereby increasing attention toward the potential use of feed enzymes. In this study, we have assessed the impacts of ß-Mannanase (BM), the major ß-mannan degrading enzyme, supplementation on poultry health and digestion. Our study involved a total of 1,296 Cobb broilers, randomly distributed into a 3â ×â 2 factorial arrangement to evaluate the effects of BM supplementation on growth, digestive enzyme activity, cecal microbial communities, and SCFA production in broiler chickens fed diets with varying ME levels. Our findings confirmed previous research by showing that BM-supplemented diets are effective in improving performance in younger broilers. In addition, we found that both BM supplementation and reduced ME levels also have positive impacts on digestive enzyme activity and intestinal flora in chickens, which might account for improving production performance. These results will be a crucial refence for the scientific implementation of BM supplementation, which may help achieve better performance and intestinal microecology, resulting in higher economic productivity of broiler farms.
Asunto(s)
Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Ciego , Pollos , Dieta , Suplementos Dietéticos , Ácidos Grasos Volátiles , Microbioma Gastrointestinal , beta-Manosidasa , Animales , Pollos/crecimiento & desarrollo , Pollos/microbiología , Masculino , Dieta/veterinaria , Alimentación Animal/análisis , Suplementos Dietéticos/análisis , beta-Manosidasa/metabolismo , beta-Manosidasa/farmacología , beta-Manosidasa/administración & dosificación , Ciego/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Ácidos Grasos Volátiles/metabolismo , Metabolismo Energético/efectos de los fármacos , Digestión/efectos de los fármacos , Distribución AleatoriaRESUMEN
This study aimed to investigate the nutritional and functional roles of ß-mannanase on the intestinal health and growth of newly weaned pigs fed a typical or low-cost formulated feeds (LCF). Twenty-four newly weaned pigs at 6.2 kgâ ±â 0.4 body weight (BW) were allotted to three dietary treatments based on a randomized complete block design with sex and initial BW as blocks. Three dietary treatments are as follows: Control, typical nursery feeds including animal protein supplements and enzyme-treated soybean meal; LCF with increased amounts of soybean meal, decreased amounts of animal protein supplements, and no enzyme-treated soybean meal; LCF+, low-cost formulated feed with ß-mannanase at 100 g/t, providing 800 thermostable ß-mannanase unit (TMU) per kg of feed. Pigs were fed based on a three-phase feeding program for a total of 37 d. On day 37 of feeding, all pigs were euthanized and the gastrointestinal tract was removed for sample collection to analyze intestinal health parameters, mucosa-associated microbiota, and gene expression of tight junction proteins. Pigs fed LCF increased (Pâ <â 0.05) the relative abundance of Proteobacteria and Helicobacter in the jejunal mucosa, tended to decrease (Pâ =â 0.097; Pâ =â 0.098) the concentration of malondialdehyde (MDA) and the expression of zona occluden 1 (ZO-1) gene in the jejunum, tended to decrease average daily gain (ADG; Pâ =â 0.084) and final BW (Pâ =â 0.090), and decreased (Pâ <â 0.05) average daily feed intake. Pigs fed LCFâ +â tended to decrease (Pâ =â 0.088) digesta viscosity, decreased (Pâ <â 0.05) the relative abundance of Helicobacter, and increased (Pâ <â 0.05) Lactobacillus in the jejunal mucosa compared to LCF. Additionally, LCFâ +â tended to increase final BW (Pâ =â 0.059) and ADG (Pâ =â 0.054), increased (Pâ <â 0.05) gain to feed ratio (G:F), and reduced (Pâ <â 0.05) fecal score compared to LCF. LCF with decreased amounts of animal protein supplements and increased amounts of soybean meal had negative effects on the composition of the mucosa-associated microbiota, intestinal integrity, and growth performance of nursery pigs. Beta-mannanase supplementation to LCF decreased digesta viscosity, increased the relative abundance of potentially health-benefitting microbiota such as Lactobacillus, and improved growth and fecal score, thus reflecting its efficacy in low-cost formulated feeds with increased amounts of soybean meal.
Immediately following weaning, pigs are exposed to various stressors associated with environmental, social, and dietary shifts that can result in depressed growth, high incidence of diarrhea, and increases in mortality. Major plant-based feedstuffs such as soybean meal contain anti-nutritional compounds including nonstarch polysaccharides causing negative impacts on the intestinal health and growth of nursery pigs due to the immaturity of the intestine and intestinal immune system. ß-mannanase hydrolyzes ß-mannans, nonstarch polysaccharides, resulting in elimination of anti-nutritional properties and the release of mannooligosaccharides. The aim of this study was to investigate the nutritional and functional roles of ß-mannanase on intestinal health and growth of newly weaned pigs fed a low-cost formulated feed (LCF). This study showed that increased amounts of soybean meal in LCF increased pathogenic bacteria associated to the jejunal mucosa of nursery pigs, impaired intestinal integrity, and decreased growth. Supplementation of ß-mannanase to LCF decreased digesta viscosity, increased Lactobacillus in the jejunal mucosa, reduced diarrhea, and improved growth performance of nursery pigs.
Asunto(s)
Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Dieta , Destete , beta-Manosidasa , Animales , Alimentación Animal/análisis , beta-Manosidasa/metabolismo , beta-Manosidasa/genética , Dieta/veterinaria , Masculino , Femenino , Porcinos/crecimiento & desarrollo , Porcinos/fisiología , Microbioma Gastrointestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Suplementos Dietéticos/análisis , Distribución AleatoriaRESUMEN
Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.
Asunto(s)
Bacillus licheniformis , Dominio Catalítico , Mutagénesis Sitio-Dirigida , Bacillus licheniformis/enzimología , Bacillus licheniformis/genética , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Manosidasas/química , Manosidasas/genética , Manosidasas/metabolismo , Especificidad por Sustrato , Hidrólisis , Tetrosas/química , Tetrosas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Conformación Proteica , Mananos/química , Mananos/metabolismo , beta-Manosidasa/química , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , OligosacáridosRESUMEN
Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.
Asunto(s)
Aspergillus niger , Galactanos , Mananos , Oligosacáridos , Gomas de Plantas , Prebióticos , beta-Manosidasa , Mananos/química , Mananos/metabolismo , Gomas de Plantas/química , Galactanos/química , Aspergillus niger/enzimología , Oligosacáridos/química , Hidrólisis , beta-Manosidasa/metabolismo , beta-Manosidasa/química , Concentración de Iones de Hidrógeno , Ácidos Grasos Volátiles/metabolismo , Difracción de Rayos X , Temperatura , Lactobacillus/metabolismo , ProbióticosRESUMEN
Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.
Asunto(s)
Escherichia coli , Fermentación , Proteínas Recombinantes , beta-Manosidasa , beta-Manosidasa/metabolismo , beta-Manosidasa/genética , beta-Manosidasa/biosíntesis , beta-Manosidasa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Mananos/metabolismo , Mananos/química , Mananos/biosíntesis , Reactores Biológicos , Concentración de Iones de Hidrógeno , Aerobiosis , Galactanos/metabolismo , Galactanos/biosíntesis , Galactanos/química , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Gomas de Plantas/química , Gomas de Plantas/metabolismo , Actinobacteria/enzimología , Actinobacteria/metabolismo , Actinobacteria/genética , HidrólisisRESUMEN
Currently, the production of manno-oligosaccharides (MOS) from guar gum faces challenges of low oligosaccharide enzymatic hydrolysis yield and complicated steps in separation and purification. In this work, a potential strategy to address these issues was explored. By combining citric acid pretreatment (300 mM, 130 °C, 1 h) with ß-mannanase hydrolysis, an impressive MOS yield of 61.8 % from guar gum (10 %, w/v) was achieved. The key success lay in the optimizing conditions that completely degraded other galactomannans into monosaccharides, which could be easily removable through Saccharomyces cerevisiae fermentation (without additional nutrients). Following ion exchange chromatography for desalination, and concluding with spray drying, 4.57 g of solid MOS with a purity of 90 % was obtained from 10 g of guar gum. This method offers a streamlined and effective pathway for obtaining high-yield and high-purity MOS from guar gum by combining citric acid pretreatment and enzymatic hydrolysis.
Asunto(s)
Ácido Cítrico , Galactanos , Mananos , Oligosacáridos , Gomas de Plantas , beta-Manosidasa , Mananos/química , Gomas de Plantas/química , Galactanos/química , Hidrólisis , Ácido Cítrico/química , Oligosacáridos/química , beta-Manosidasa/metabolismo , Saccharomyces cerevisiae/metabolismo , FermentaciónRESUMEN
In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.
Asunto(s)
Proteínas Bacterianas , Dominio Catalítico , beta-Manosidasa , Animales , Bovinos , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Manosidasa/genética , beta-Manosidasa/química , beta-Manosidasa/metabolismo , Estabilidad de Enzimas , Hidrólisis , Cinética , Mananos/química , Mananos/metabolismo , Rumen/microbiología , Especificidad por SustratoRESUMEN
As an enzyme, ß-mannanase (BM) can be widely used as feed additive to improve the growth performance of animals. This experiment aimed to determine the effect of the addition of BM to low-energy diet on the immune function and intestinal microflora of broiler chickens. In this study, 384 one-day-old Arbor Acres broilers were randomly divided into 3 groups (8 replicates per group): positive control (PC, received a corn-soybean meal basal diet), negative control (NC, received a low-energy diet with Metabolizable Energy (ME) reduced by 50 kcal/kg) and NC + BM group (NC birds + 100 mg/kg BM). All birds were raised for 42 d. The results showed that BM mitigated the damage of immune function in peripheral blood of broilers caused by the decrease of dietary energy level by increasing the Concanavalin A (Con A) index of stimulation (SI) and macrophages phagocytic activity in the peripheral blood of broilers at 42 d (P < 0.05). The analysis of cecum flora showed that the low-energy diet significantly reduced the observed_species index (P < 0.01), Chao1 index and ACE index (P < 0.05), which reduced the abundance and evenness of species in the cecum of broilers at 21 d. It also significantly reduced the relative abundance of Candidatus_Arthromitus and significantly increased the relative abundance of Pseudomonas in the cecum of broilers at 21 d, while also significantly increasing the relative abundance of Monoglobus at 42 d. BM significantly increased the relative abundance of Lachnospiraceae_UCG-001 and Lachnospiraceae_bacterium_615 in the cecum of broilers at 21 d. In addition, BM inhibited microbial Fatty acid degradation by decreasing the activity of glutaryl-CoA dehydrogenase. Collectively, BM could improve intestinal health by enhancing the immune function of broilers, promoting the proliferation of beneficial bacteria and reducing the number of harmful bacteria, regulating intestinal flora, thereby alleviating the adverse effects of lower dietary energy levels.
Asunto(s)
Alimentación Animal , Pollos , Dieta , Microbioma Gastrointestinal , ARN Ribosómico 16S , Distribución Aleatoria , beta-Manosidasa , Animales , Pollos/inmunología , Pollos/microbiología , Alimentación Animal/análisis , Dieta/veterinaria , Microbioma Gastrointestinal/efectos de los fármacos , beta-Manosidasa/metabolismo , beta-Manosidasa/genética , ARN Ribosómico 16S/análisis , Suplementos Dietéticos/análisis , Masculino , Fenómenos Fisiológicos Nutricionales de los Animales/efectos de los fármacos , MetagenómicaRESUMEN
Glycoside hydrolases (GHs) are known to depolymerize polysaccharides into oligo-/mono-saccharides, they are extensively used as additives for both animals feed and our food. Here we reported the characterization of IDSGH5-14(CD), a weakly-acidic mesophilic bifunctional mannanase/glucanase of GH5, originally isolated from sheep rumen microbes. Biochemical characterization studies revealed that IDSGH5-14(CD) exhibited preferential hydrolysis of mannan-like and glucan-like substrates. Interestingly, the enzyme exhibited significantly robust catalytic activity towards branched-substrates compared to linear polysaccharides (P < 0.05). Substrate hydrolysis pattern indicated that IDSGH5-14(CD) predominantly liberated oligosaccharides with a degree of polymerization (DP) of 3-7 as the end products, dramatically distinct from canonical endo-acting enzymes. Comparative modeling revealed that IDSGH5-14(CD) was mainly comprised of a (ß/α)8-barrel-like structure with a spacious catalytic cleft on surface, facilitating the enzyme to target high-DP or branched oligosaccharides. Molecular dynamics (MD) simulations further suggested that the branched-ligand, 64-α-D-galactosyl-mannohexose, was steadily accommodated within the catalytic pocket via a two-sided clamp formed by the aromatic residues. This study first reports a bifunctional GH5 enzyme that predominantly generates high-DP oligosaccharides, preferentially from branched-substrates. This provides novel insights into the catalytic mechanism and molecular underpinnings of polysaccharide depolymerization, with potential implications for feed additive development and high-DP oligosaccharides preparation.
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Rumen , beta-Manosidasa , Animales , Ovinos , Polimerizacion , Rumen/metabolismo , beta-Manosidasa/metabolismo , Oligosacáridos , Polisacáridos , Glicósido Hidrolasas/metabolismo , Especificidad por Sustrato , HidrólisisRESUMEN
This study aimed to produce enzymes (beta (ß)-mannanase using a recombinant Aspergillus sojae AsT3 and inulinase using Aspergillus niger A42) and oligosaccharides (mannooligosaccharides (MOS), fructooligosaccharides (FOS)) using coffee waste, ground coffee, and coffee extract by solid-state fermentation (SSF). Plackett-Burman Design (PBD) was used to create a design for enzyme production with four different parameters (temperature, pH, solid-to-liquid ratio (SLR), and mix with coffee wastes and ground coffee). The highest ß-mannanase and inulinase activities were 71.17 and 564.07 U/mg of protein respectively. Statistical analysis showed that the temperature was statistically significant for the production of both enzymes (P < 0.05). The produced enzymes were utilized in French Pressed coffee extracts to produce oligosaccharides. As a result of the enzymatic hydrolyzation, the highest mannobiose, mannotriose, mannotetraose, and total MOS levels were 109.66, 101.11, 391.02, and 600.64 ppm, respectively. For the FOS production, the maximal 1,1,1-kestopentaose was 38.34 ppm. Consequently, this study demonstrates that a recombinant Aspergillus sojae AsT3 ß-mannanase and Aspergillus niger A42 inulinase produced from coffee wastes and ground coffee can be used in coffee extracts to increase the amount of oligosaccharides in coffee extracts.
Asunto(s)
Aspergillus , Glicósido Hidrolasas , Oligosacáridos , beta-Manosidasa , beta-Manosidasa/metabolismo , Oligosacáridos/metabolismo , Aspergillus niger , FermentaciónRESUMEN
To understand the influence of family 3 Carbohydrate Binding Module (hereafter CBM3), single (GH5 cellulase; CelB, CelBΔCBM), bi-chimeric [GH26 endo-mannanase (ManB-1601) and GH11 endo-xylanase (XynB); ManB-XynB [1], ManB-XynB-CBM] and tri-chimeric [ManB-XynB-CelB [1], ManB-XynB-CelBΔCBM] enzyme variants (fused or deleted of CBM) were produced and purified to homogeneity. CBM3 did not alter the pH and temperature optima of bi- and tri-chimeric enzymes but improved the pH and temperature stability of ManB in CBM variants of bi-/tri-chimeric enzymes. Truncation of CBM in CelB shifted the pH optimum and increased the melting temperature (Tm 65 â). CBM3 improved both substrate affinity (Km) and catalytic efficiency (kcat/Km) of fused enzymes in tri-chimera and CelB but only Km for bi-chimera. Far-UV CD of CelB and bi- and tri-chimeric enzymes suggested that CBM3 improved the α-helical content and compactness in the native state but did not prevent disintegration of secondary structural contents at acidic pH. Steady-state fluorescence studies suggested that under acidic conditions CBM3 prevented the exposure of hydrophobic patches in bi-chimeric protein but could not avert the opening up of chimeric enzyme structure. Aqueous enzyme assisted treatment of mature coconut kernel using single, bi- and tri-chimeric enzymes led to cracks, peeling and fracturing of the matrix and improved the oil yield by up to 22%.
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beta-Manosidasa , Aceite de Coco , Hidrólisis , beta-Manosidasa/metabolismo , Temperatura , Proteínas Recombinantes de FusiónRESUMEN
A 6-month-old cat of unknown ancestry presented for a neurologic evaluation due to progressive motor impairment. Complete physical and neurologic examinations suggested the disorder was likely to be hereditary, although the signs were not consistent with any previously described inherited disorders in cats. Due to the progression of disease signs including severely impaired motor function and cognitive decline, the cat was euthanized at approximately 10.5 months of age. Whole genome sequence analysis identified a homozygous missense variant c.2506G > A in MANBA that predicts a p.Gly836Arg alteration in the encoded lysosomal enzyme ß -mannosidase. This variant was not present in the whole genome or whole exome sequences of any of the 424 cats represented in the 99 Lives Cat Genome dataset. ß -Mannosidase enzyme activity was undetectable in brain tissue homogenates from the affected cat, whereas α-mannosidase enzyme activities were elevated compared to an unaffected cat. Postmortem examination of brain and retinal tissues revealed massive accumulations of vacuolar inclusions in most cells, similar to those reported in animals of other species with hereditary ß -mannosidosis. Based on these findings, the cat likely suffered from ß -mannosidosis due to the abolition of ß -mannosidase activity associated with the p.Gly836Arg amino acid substitution. p.Gly836 is located in the C-terminal region of the protein and was not previously known to be involved in modulating enzyme activity. In addition to the vacuolar inclusions, some cells in the brain of the affected cat contained inclusions that exhibited lipofuscin-like autofluorescence. Electron microscopic examinations suggested these inclusions formed via an autophagy-like process.
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beta-Manosidosis , Gatos , Animales , beta-Manosidosis/complicaciones , beta-Manosidosis/diagnóstico , beta-Manosidosis/genética , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Mutación MissenseRESUMEN
Seed germination is the complex adaptive trait of higher plants influenced by a large number of genes and environmental factors. Numerous studies have been performed to better understand how germination is controlled by various environmental factors and applied chemicals, such as cyanide. However, still very little is known about the molecular mechanisms of how extrinsic signals regulate seed germination. Our and previous studies found that non-lethal cyanide treatment promotes seed germination, but the regulatory mechanism is unclear. In this study, we found that a low concentration of cyanide pretreatment significantly enhanced the expression of endo-ß-mannanase 5 (MAN5) gene in Arabidopsis thaliana, and the mutation of this gene impaired cyanide-mediated seed germination. In contrast, overexpression of MAN5 gene enhanced Arabidopsis seed germination ability under both normal and salt stress conditions. Further studies showed that the expression of the MAN5 gene was negatively regulated by ABA insensitive 5 (ABI5); In abi5 mutant seeds, the expression of the MAN5 gene was increased and the seed germination rate was accelerated. Additionally, cyanide pretreatment markedly reduced the gene expression of ABI5 in Arabidopsis seeds. Taken together, our data support the involvement of MAN5 as a key gene in cyanide-mediated seed germination and confirm the role of ABI5 as a critical negative factor involved in cyanide-regulated MAN5 gene expression.
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Proteínas de Arabidopsis , Arabidopsis , Germinación , beta-Manosidasa , Ácido Abscísico/farmacología , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , beta-Manosidasa/metabolismo , Cianuros/metabolismo , Germinación/genética , Semillas/metabolismoRESUMEN
An acid stable α-galactosidase was produced and purified from mannolytic fungal strain, Penicillium aculeatum APS1. Enzyme was produced using wheat bran and copra cake moistened with corn steep liquor by solid state fermentation. APS1αgal having molecular weight of 65.4 kDa was purified to electrophoretic homogeneity by three phase partitioning and gel permeation chromatography with high enzyme recovery. APS1αgal was found to be maximally active at 55 °C and pH 4.5, having high stability at acidic pH. Thermal stability and thermal inactivation kinetics of APS1αgal were also studied. APS1αgal was found to effectively hydrolyse oligosaccharides as well as polysaccharides having α-1,6 linked galactose. Abolishment of enzyme activity in N-brommosuccinimide revealed an important role of tryptophan residue in catalysis. APS1αgal had shown outstanding tolerance to NaCl and proteases. MALDI-TOF MS/MS analysis indicated that enzyme is probably a member of family GH27. Synergistic interaction between APS1αgal and ß-mannanase for hydrolysis of galactomannan was very clear and maximum 2.0° of synergy was found under simultaneous mode of action. This study reports a new source of α-galactosidase with biochemical properties suitable for applications in food and feed industries.
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alfa-Galactosidasa , beta-Manosidasa , beta-Manosidasa/metabolismo , Hidrólisis , alfa-Galactosidasa/química , Espectrometría de Masas en Tándem , Concentración de Iones de Hidrógeno , Estabilidad de Enzimas , Especificidad por Sustrato , CinéticaRESUMEN
Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in Escherichia coli. Extracellular enzymes from Bacillus subtilis, including ß-1,4-xylanase, ß-1,4-glucanase, and ß-mannanase constructed with and without their signal peptides and intracellular enzymes from Pseudomonas stutzeri ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in E. coli BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of ß-1,4-xylanase (Xyn), ß-1,4-glucanase (Cel), and ß-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in E. coli BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, E. coli can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.
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Proteínas de Escherichia coli , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , beta-Manosidasa/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Galactomannans are abundant nonstarch polysaccharides in broiler feed ingredients. In broilers, diets with high levels of galactomannans have been associated with innate immune response stimulation, poor zootechnical performance, nutrient and lipid absorption, and excessive digesta viscosity. However, data about its effects on the gut microbiome are scarce. ß-Mannanases are enzymes that can hydrolyze ß-mannans, resulting in better nutrient utilization. In the current study, we have evaluated the effect of guar gum, a source of galactomannans, supplemented to broiler diets, either with or without ß-mannanase supplementation, on the microbiota composition, in an attempt to describe the potential role of the intestinal microbiota in ß-mannanase-induced gut health and performance improvements. One-day-old broiler chickens (n = 756) were randomly divided into 3 treatments: control diet, guar gum-supplemented diet (1.7%), or guar gum-supplemented diet + ß-mannanase (Hemicell 330 g/ton). The zootechnical performance, gut morphometry, ileal and cecal microbiome, and short-chain fatty acid concentrations were evaluated at different time points. The guar gum supplementation decreased the zootechnical performance, and the ß-mannanase supplementation restored performance to control levels. The mannan-rich diet-induced dysbiosis, with marked effects on the cecal microbiota composition. The guar gum-supplemented diet increased the cecal abundance of the genera Lactobacillus, Roseburia, Clostridium sensu stricto 1, and Escherichia-Shigella, and decreased Intestinimonas, Alistipes, Butyricicoccus, and Faecalibacterium. In general, dietary ß-mannanase supplementation restored the main microbial shifts induced by guar gum to levels of the control group. In addition, the ß-mannanase supplementation reduced cecal isobutyric, isovaleric, valeric acid, and branched-chain fatty acid concentrations as compared to the guar gum-supplemented diet group, suggesting improved protein digestion and reduced cecal protein fermentation. In conclusion, a galactomannan-rich diet impairs zootechnical performance in broilers and results in a diet-induced dysbiosis. ß-Mannanase supplementation restored the gut microbiota composition and zootechnical performance to control levels.
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Mananos , beta-Manosidasa , Animales , Mananos/metabolismo , beta-Manosidasa/metabolismo , Pollos/fisiología , Disbiosis/veterinaria , Dieta/veterinaria , Suplementos Dietéticos , Alimentación Animal/análisisRESUMEN
MAIN CONCLUSION: Blue light exposure delays tomato seed germination by decreasing endosperm-degrading hydrolase activities, a process regulated by CRY1a-dependent signaling and the hormonal balance between ABA and GA. The germination of tomato seeds (Solanum lycopersicum L.) is tightly controlled by an internal hormonal balance, which is also influenced by environmental factors such as light. In this study, we investigated the blue light (BL)-mediated impacts on physiological, biochemical, and molecular processes during the germination of the blue light photoreceptor CRYPTOCHROME 1a loss-of-function mutant (cry1a) and of the hormonal tomato mutants notabilis (not, deficient in ABA) and procera (pro, displaying a GA-constitutive response). Seeds were germinated in a controlled chamber in the dark and under different intensities of continuous BL (ranging from 1 to 25 µmol m-2 s-1). In general, exposure to BL delayed tomato seed germination in a fluency rate-dependent way due to negative impacts on the activities of endosperm-degrading hydrolases, such as endo-ß-mannanase, ß-mannosidase, and α-galactosidase. However, not and pro mutants presented higher germination speed index (GSI) compared to WT despite the BL influence, associated with higher hydrolase activities, especially evident in pro, indicating that the ABA/GA hormonal balance is important to diminish BL inhibition over tomato germination. The cry1a germination percentage was higher than in WT in the dark but its GSI was lower under BL exposure, suggesting that functional CRY1a is required for BL-dependent germination. BL inhibits the expression of GA-biosynthetic genes, and induces GA-deactivating and ABA-biosynthetic genes. The magnitude of the BL influence over the hormone-related transcriptional profile is also dependent upon CRY1a, highlighting the complex interplay between light and hormonal pathways. These results contribute to a better understanding of BL-induced events behind the photoregulation of tomato seed germination.
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Endospermo , Solanum lycopersicum , Endospermo/genética , Endospermo/metabolismo , Solanum lycopersicum/genética , Germinación , Semillas/fisiología , Criptocromos/genética , Criptocromos/metabolismo , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Percepción , Ácido Abscísico/metabolismo , Giberelinas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
A novel glycoside hydrolase family 26 ß-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35-328) and PpMan26A-Δ205 (aa 206-656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The Km and kcat values for konjac gum were 2.13 mg/mL and 416.66 s-1, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3-8. CBM35pp exerted strong binding activity toward mannans but without ß-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 ß-mannanases and expands the potential industrial application of PpMan26A.
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Paenibacillus polymyxa , beta-Manosidasa , beta-Manosidasa/metabolismo , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/metabolismo , Oligosacáridos/metabolismo , Mananos/metabolismo , Especificidad por Sustrato , HidrólisisRESUMEN
Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.
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Cellvibrio , Mananos , Mananos/metabolismo , Galactosa/metabolismo , alfa-Galactosidasa/metabolismo , beta-Manosidasa/química , beta-Manosidasa/metabolismoRESUMEN
The polysaccharide ß-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a ß-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed ß-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for ß-mannan utilization. A conserved GH26 ß-mannanase with endo-activity depolymerized the ß-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial ß-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-ß-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of ß-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom ß-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade ß-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.