RESUMEN

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.

Asunto(s)

Proteínas Bacterianas/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Esterasas/metabolismo , Microbioma Gastrointestinal , Rumen/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Bovinos , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/aislamiento & purificación , Pruebas de Enzimas , Esterasas/genética , Esterasas/aislamiento & purificación , Glucuronatos/biosíntesis , Microbiología Industrial/métodos , Lignina/metabolismo , Metagenoma , Oligosacáridos/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Energía Renovable

RESUMEN

BACKGROUND: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by bgalactosidases. RESULTS: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with b(1 ? 6) and b (1 ? 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. CONCLUSIONS: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.

Asunto(s)

Oligosacáridos/biosíntesis , beta-Galactosidasa/metabolismo , Pantoea , Lactosa/metabolismo , Proteínas Recombinantes , Industria Lechera , Suero Lácteo

RESUMEN

Animal chitosan (Chit-A) is gaining more acceptance in daily activities. It is used in a range of products from food supplements for weight loss to even raw materials for producing nanoparticles and hydrogel drug carriers; however, it has low antioxidant activity. Fungal oligochitosan (OChit-F) was identified as a potential substitute for Chit-A. Cunninghamella elegans is a fungus found in the Brazilian savanna (Caatinga) that produces OligoChit-F, which is a relatively poorly studied compound. In this study, 4 kDa OChit-F with a 76% deacetylation degree was extracted from C. elegans. OChit-F showed antioxidant activity similar to that of Chit-A in only one in vitro test (copper chelation) but exhibited higher activity than that of Chit-A in three other tests (reducing power, hydroxyl radical scavenging, and iron chelation). These results indicate that OChit-F is a better antioxidant than Chit-A. In addition, Chit-A significantly increased the formation of calcium oxalate crystals in vitro, particularly those of the monohydrate (COM) type; however, OChit-F had no effect on this process in vitro. In summary, OChit-F had higher antioxidant activity than Chit-A and did not induce the formation of CaOx crystals. Thus, OChit-F can be used as a Chit-A substitute in applications affected by oxidative stress.

Asunto(s)

Antioxidantes/química , Antioxidantes/farmacología , Quitosano/química , Quitosano/farmacología , Cunninghamella/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos/farmacología , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Oxalato de Calcio/química , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Galactooligosaccharides (GOS) are useful dietary ingredients recognized worldwide as prebiotics. In the present study, we evaluated the ß-galactosidase (ß-gal) activity of a panel of lactic acid bacteria (LAB) in order to select strains for the synthesis of oligosaccharides from lactose (GOS) and lactulose (OsLu) with a potential prebiotic effect. Fifteen strains out of 20 were able to grow on lactose and showed ß-gal activities between 0.03 and 2.06 U mg-1, whereas eleven were able to synthesize GOS. Lactobacillus delbrueckii subsp. bulgaricus CRL450, the strain with the highest ß-gal activity, synthesized a maximum of 41.3% GOS and 21.0% OsLu from lactose and lactulose, respectively, with ß-(1 → 6) and secondary ß-(1 → 3) linkages. When these compounds were tested without purifying, as carbon sources for the development of recognized probiotics and the producer strain, high growth was observed compared to non-prebiotic sugars like glucose and lactose. When the purified oligosaccharides were tested, the bacterial growth decreased, but the microorganisms displayed metabolic activity evidenced by the consumption of carbohydrates and the production of lactic acid. Additionally, the purified oligosaccharides demonstrated a bifidogenic effect. The obtained results support the potential of L. delbrueckii subsp. bulgaricus CRL450 for the production of the prebiotics GOS and OsLu and encourage the optimization of their synthesis for the design of new functional food ingredients.

Asunto(s)

Fermentación , Galactosa/metabolismo , Lactobacillus delbrueckii/metabolismo , Lactosa/metabolismo , Lactulosa/metabolismo , Oligosacáridos/biosíntesis , Prebióticos , Humanos , Probióticos , beta-Galactosidasa/metabolismo

RESUMEN

Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan- magnetic nanoparticles. Immobilization carried out according to a 23-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g L-1, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.

Asunto(s)

Aspergillus/enzimología , Quitosano/química , Enzimas Inmovilizadas/química , Hexosiltransferasas/química , Nanopartículas de Magnetita/química , Oligosacáridos/biosíntesis , Activación Enzimática , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Glutaral , Hexosiltransferasas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Temperatura , Trisacáridos

RESUMEN

Eucalyptus wood is the primary source of fibers to produce paper and cellulose in South American countries. The major by-product generated in the cellulose industry is sawdust derived from chip wood production, which is designated as Eucalyptus by-product (EB). The xylooligosaccharides (XOS) are xylose-based oligomers with proven effects over maintenance and stimulation of beneficial human gut bacteria. This study reported the EB extraction and characterization along with an assessment of hemicellulose hydrolysis using commercial xylanases to produce XOS. Hemicellulose derived from extracted and NaClO2 pretreated (HEEBPT) presented xylan content of 55%, which was similar to 58.5% found in commercial Birchwood hemicellulose (CBH). The enzymatic hydrolysis of HEEBPT and CBH presented 30% as maximum conversion of xylan into XOS without significant difference among the enzymatic extracts evaluated. The XOS production from EB was proven as a technically feasible alternative to recover a value-added product from hemicellulosic fraction generated in the cellulose industry. However, lignin removal with NaClO2 from EB affects the feasibility of an industrial process because they generate toxic compounds in the pretreatment step. Thus, further studies with alternative reagents, such as ionic liquids, are required to asses selectively lignin removal from EB. Graphical Abstract.

Asunto(s)

Endo-1,4-beta Xilanasas/metabolismo , Eucalyptus/metabolismo , Glucuronatos/biosíntesis , Oligosacáridos/biosíntesis , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Hidrólisis

RESUMEN

Chitosan-oligosaccharides (COS) are low-molecular weight chitosan derivatives with interesting clinical applications. The optimization of both COS production and purification is an important step in the design of an efficient production system and for the exploration of new COS applications. Trichoderma harzianum is an innocuous biocontrol agent that represents a novel biotechnological tool due to the production of extracellular enzymes, including those that produce a COS mixture. In this work, we propose different systems for the production of COS using the T. harzianum chitinolitic system. A complete qualitative and quantitative analysis of a partially purified COS mixture were performed. Also, an evaluation of the anticancer and antimicrobial effects of the COS mixture was carried out. Three chitosan variants (colloidal, solid and solution) and two fungus stages (spores and mycelia) were tested for COS production. The best system consisted of the interaction of the solid chitosan and the fungal spores, producing a COS mixture containing species from the monomer to the hexamer, in a concentration range of 7-238 mg/mL, according to chromatographic analysis. The proposed purification method isolated the monomer and the dimer from the COS mixture. Moreover, the COS mixture has an inhibitory effect on the growth of bacteria and changes the morphology of yeasts. As anticancer compounds, COS inhibited the growth of cervical cancer cells at concentration of 4 mg/mL and significantly reduced the survival rate of the cells. In conclusion, T. harzianum proved to be an efficient system for COS mixture production.

Asunto(s)

Quitosano/química , Oligosacáridos/química , Oligosacáridos/farmacología , Trichoderma/metabolismo , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Células HeLa , Humanos , Hidrólisis , Oligosacáridos/biosíntesis

RESUMEN

Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of α-lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since α-lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using α-l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

Asunto(s)

Lacticaseibacillus rhamnosus/enzimología , Oligosacáridos/biosíntesis , alfa-L-Fucosidasa/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Pared Celular/enzimología , Cromatografía Líquida de Alta Presión , Glicósidos/química , Humanos , Espectrometría de Masas , Oligosacáridos/química , Prebióticos , Especificidad por Sustrato , alfa-L-Fucosidasa/metabolismo

RESUMEN

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the nutritional and technological properties of foods. The rhizosphere of inulin-accumulating plants from the Cerrado (Brazilian savanna) harbor fungi capable of synthesizing FOS from sucrose through the transfructosylating activity of ß-fructosyltransferases and/or ß-fructofuranosidases. Here, we investigated the ability of Penicillium janczewskii Zaleski CCIBt 3352, a fungus isolated from the rhizosphere of Chrysolaena obovata (Asteraceae), to produce FOS in a medium supplemented with sucrose concentrations of 30, 100, or 150 g L-1 . Hydrolytic activity on sucrose was observed in culture filtrates; however, at 150 g L-1 sucrose, the accumulation of 8 g L-1 1-kestose (inulin-type FOS) and 7.3 g L-1 neokestose (neolevan-type FOS) was observed, the latter being a type of FOS not commonly produced by filamentous fungi. In addition, minor amounts of four unidentified oligosaccharides, with a high degree of polymerization, were detected. The production of FOS was also observed in enzymatic assays, indicating the presence of extracellular enzymes with transfructosylating activity in the culture filtrates. Our findings demonstrate the feasibility of isolating promising microorganisms, for the production of FOS-synthesizing enzymes, from the rhizosphere of fructan-producing plants of the Brazilian Cerrado.

Asunto(s)

Fructanos/metabolismo , Inulina/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos/química , Penicillium/metabolismo , Asteraceae/microbiología , Brasil , Fructanos/química , Inulina/química , Estructura Molecular , Oligosacáridos/metabolismo , Penicillium/química , Penicillium/crecimiento & desarrollo

RESUMEN

Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.

Asunto(s)

Bacillus subtilis/enzimología , Fructanos/biosíntesis , Hexosiltransferasas/metabolismo , Sacarosa/metabolismo , Catálisis , Disacaridasas/metabolismo , Disacáridos/metabolismo , Fructanos/metabolismo , Hexosiltransferasas/química , Hexosiltransferasas/genética , Cinética , Peso Molecular , Oligosacáridos/biosíntesis , Oligosacáridos/metabolismo , Sacarosa/química , Trisacáridos/metabolismo

RESUMEN

The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.

Asunto(s)

Proteínas Bacterianas/metabolismo , Oligosacáridos/biosíntesis , Thermotoga maritima/enzimología , alfa-L-Fucosidasa/metabolismo , Calcio/metabolismo , Fucosa/análogos & derivados , Sodio/metabolismo

RESUMEN

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70 °C for Rhodotorula mucilaginosa invertase and 4.5 and 50 °C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75 °C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50 °C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3 g/L of nystose by S. cerevisiae invertase and 12.6 g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.

Asunto(s)

Oligosacáridos/biosíntesis , Rhodotorula/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , beta-Fructofuranosidasa/biosíntesis , beta-Fructofuranosidasa/metabolismo , Catálisis , Estabilidad de Enzimas , Etanol/metabolismo , Industria de Alimentos/métodos , Concentración de Iones de Hidrógeno , Hidrólisis , Industrias , Especificidad de la Especie , Sacarosa/metabolismo , Temperatura , beta-Fructofuranosidasa/química

RESUMEN

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.

Asunto(s)

Proteínas Bacterianas/metabolismo , Pseudoalteromonas/enzimología , Glicósido Hidrolasas/metabolismo , Oligosacáridos/biosíntesis , Temperatura , Carbono/metabolismo , Carragenina/biosíntesis , Espectrometría de Masa por Ionización de Electrospray , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Nitrógeno/metabolismo

RESUMEN

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Asunto(s)

Ciclodextrinas/biosíntesis , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Oligosacáridos/biosíntesis , Paenibacillus/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Ciclización , Ciclodextrinas/metabolismo , Glucosiltransferasas/química , Simulación de Dinámica Molecular , Oligosacáridos/metabolismo , Especificidad por Sustrato

RESUMEN

Fructooligosaccharides (FOS) are prebiotics commonly manufactured using fungal fructosyltransferases (FTases) or ß-fructofuranosidases. Several reports have attempted to optimize FOS production by changing operational conditions. Nevertheless, there is a lack of information related to the molecular enzyme-substrate interaction. In this study, we present an in silico evaluation of the interactions between substrates (i.e., glucose, sucrose, GF2, GF3, and GF4) and native FOS-synthesizing enzymes from fungi, with reported FOS production yield. In addition, a molecular dynamic simulation was conducted to assess the stability of these interactions. Six fungal enzymes with reported data of FOS production were selected: sucrose-sucrose 1-fructosyltransferase from A. foetidus (GenBank No. CAA04131); intracellular invertase from A. niger (GenBank No. ABB59679); extracellular invertase from A. niger (GenBank No. ABB59678); ß-fructofuranidase from A. japonicus ATCC 20611 (GenBank No. BAB67771); fructosyltransferase from A. oryzae N74 (GenBank No. ACZ48670); and fructosyltransferase from A. japonicus (PDB ID 3LF7). These enzymes shared an identity between 15 and 96 %, but have a highly conserved folding, and the characteristic FTases domains. Docking results showed that these enzymes also share a similar protein-ligand interaction profile. It was observed that the production yield of total FOS correlated with the sum of affinity energies for GF2, GF3, and GF4. Finally, we present the first molecular dynamic simulation for FOS and fungal FOS-synthesizing enzymes, showing that the protein-ligand interaction does not induce significant changes on the enzyme stability. Overall, these results represent valuable information to continue understanding the FOS synthesis process by fungal FOS-synthesizing enzymes, and they can have a significant impact toward the improvement in their catalytic properties and the synthesis of specific FOS.

Asunto(s)

Aspergillus/enzimología , Simulación por Computador , Oligosacáridos/biosíntesis , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Enlace de Hidrógeno , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

Oligosaccharides are polymers with two to ten monosaccharide residues which have sweetener functions and sensory characteristics, in addition to exerting physiological effects on human health. The ones called nondigestible exhibit a prebiotic behavior being fermented by colonic microflora or stimulating the growth of beneficial bacteria, playing roles in the immune system, protecting against cancer, and preventing cardiovascular and metabolic issues. The global prebiotics market is expected to grow around 12.7% in the next 8 years, so manufacturers are developing new alternatives to obtain sustainable and efficient processes for application on a large scale. Most studied examples of biotechnological processes involve the development of new strategies for fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, and mannanooligosaccharide synthesis. Among these, the use of whole cells in fermentation, synthesis of microbial enzymes (ß-fructofuranosidases, ß-galactosidases, xylanases, and ß-mannanases), and enzymatic process development (permeabilization, immobilization, gene expression) can be highlighted, especially if the production costs are reduced by the use of agro-industrial residues or by-products such as molasses, milk whey, cotton stalks, corncobs, wheat straw, poplar wood, sugarcane bagasse, and copra meal. This review comprises recent studies to demonstrate the potential for biotechnological production of oligosaccharides, and also aspects that need more investigation for future applications in a large scale.

Asunto(s)

Biotecnología/métodos , Industria de Alimentos , Oligosacáridos/genética , Oligosacáridos/metabolismo , Prebióticos , Biotecnología/economía , Colon/microbiología , Productos Lácteos , Fermentación , Glucuronatos/biosíntesis , Glucuronatos/metabolismo , Humanos , Oligosacáridos/biosíntesis , Oligosacáridos/economía , Polisacáridos/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/metabolismo , beta-Manosidasa/biosíntesis , beta-Manosidasa/metabolismo

RESUMEN

The experiments reported in this research paper describe the effects of ß-galactosidase enzyme dose and cheese whey amount, on the maximum concentration and yield of galacto-oligosaccahride (GOS) and reaction time. The experimental plan was based on central composite rotational design (CCRD) and modelled by response surface methodology (RSM). The results indicate that the proposed mathematical models could adequately describe the concentration and yield of GOS and the reaction time within the limits of the factors that are being investigated. The variance analysis shows high values of coefficients of determination (>0·97) while no significant lack of fit was evident. Hence, the models could be employed to select reaction conditions applied in the manufacture of products enriched in bioactive compounds with high value-added.

Asunto(s)

Galactosa/metabolismo , Oligosacáridos/biosíntesis , Prebióticos , Queso/análisis , Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Galactosa/análisis , Galactosa/química , Glucosa/análisis , Calor , Cinética , Lactosa/análisis , Modelos Teóricos , Oligosacáridos/análisis , Oligosacáridos/química , Proteína de Suero de Leche/química , Proteína de Suero de Leche/metabolismo , beta-Galactosidasa/metabolismo

RESUMEN

Byproducts from quinoa are not yet well explored sources of hemicellulose or products thereof. In this work, xylan from milled quinoa stalks was retrieved to 66% recovery by akaline extraction using 0.5 M NaOH at 80 °C, followed by ethanol precipitation. The isolated polymer eluted as a single peak in size-exclusion chromatography with a molecular weight of >700 kDa. Analysis by Fourier transform infrared spectroscopy and nuclear magnetic resonance (NMR) combined with acid hydrolysis to monomers showed that the polymer was built of a backbone of ß(1 → 4)-linked xylose residues that were substituted by 4-O-methylglucuronic acids, arabinose, and galactose in an approximate molar ratio of 114:23:5:1. NMR analysis also indicated the presence of α(1 → 5)-linked arabinose substituents in dimeric or oligomeric forms. The main xylooligosaccharides (XOs) produced after hydrolysis of the extracted glucuronoarabinoxylan polymer by thermostable glycoside hydrolases (GHs) from families 10 and 11 were xylobiose and xylotriose, followed by peaks of putative substituted XOs. Quantification of the unsubstituted XOs using standards showed that the highest yield from the soluble glucuronoarabinoxylan fraction was 1.26 g/100 g of xylan fraction, only slightly higher than the yield (1.00 g/100 g of xylan fraction) from the insoluble fraction (p < 0.05). No difference in yield was found between reactions in buffer or water (p > 0.05). This study shows that quinoa stalks represent a novel source of glucuronoarabinoxylan, with a substituent structure that allowed for limited production of XOs by GH10 or GH11 enzymes.

Asunto(s)

Chenopodium quinoa/química , Glucuronatos/biosíntesis , Oligosacáridos/biosíntesis , Xilanos/aislamiento & purificación , Xilosidasas/metabolismo , Arabinosa/química , Conformación de Carbohidratos , Etanol , Galactosa/química , Glucuronatos/química , Glicósido Hidrolasas/metabolismo , Hidrólisis , Peso Molecular , Oligosacáridos/química , Hidróxido de Sodio , Espectroscopía Infrarroja por Transformada de Fourier , Xilanos/química , Xilanos/metabolismo , Xilosa/química

RESUMEN

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by ß-galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL-1 initial lactose concentration and 6 U mL-1 enzyme concentration, obtaining 25.46 ± 0.01 gL-1 yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL-1 of trisaccharides and 13.79 ± 0.21 gL-1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568-1578, 2017.

Asunto(s)

Enzimas Inmovilizadas/química , Galactosa/química , Oligosacáridos/química , beta-Galactosidasa/química , Galactosa/biosíntesis , Glucosa , Cinética , Kluyveromyces/enzimología , Lactosa/química , Oligosacáridos/biosíntesis , Temperatura

RESUMEN

Background: Current commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields. Results: To improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran ß-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced. Conclusions: Our method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.

Asunto(s)

Oligosacáridos/metabolismo , Almidón/metabolismo , Enzimas/metabolismo , Isomaltosa/metabolismo , Oligosacáridos/biosíntesis , Aspergillus niger/enzimología , Temperatura , Bacillus/enzimología , beta-Amilasa/metabolismo , Glicosilación , Licuefacción , alfa-Amilasas/metabolismo , Fermentación , Glucosidasas/metabolismo , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno