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1.
PLoS Genet ; 16(9): e1008979, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32877410

RESUMO

The ascomycete Trichoderma reesei is a highly prolific cellulase producer. While XYR1 (Xylanase regulator 1) has been firmly established to be the master activator of cellulase gene expression in T. reesei, its precise transcriptional activation mechanism remains poorly understood. In the present study, TrGAL11, a component of the Mediator tail module, was identified as a putative interacting partner of XYR1. Deletion of Trgal11 markedly impaired the induced expression of most (hemi)cellulase genes, but not that of the major ß-glucosidase encoding genes. This differential involvement of TrGAL11 in the full induction of cellulase genes was reflected by the RNA polymerase II (Pol II) recruitment on their core promoters, indicating that TrGAL11 was required for the efficient transcriptional initiation of the majority of cellulase genes. In addition, we found that TrGAL11 recruitment to cellulase gene promoters largely occurred in an XYR1-dependent manner. Although xyr1 expression was significantly tuned down without TrGAL11, the binding of XYR1 to cellulase gene promoters did not entail TrGAL11. These results indicate that TrGAL11 represents a direct in vivo target of XYR1 and may play a critical role in contributing to Mediator and the following RNA Pol II recruitment to ensure the induced cellulase gene expression.


Assuntos
Celulase/genética , Complexo Mediador/genética , Trichoderma/genética , Celulase/biossíntese , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Expressão Gênica , Complexo Mediador/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica/genética , RNA Polimerase II/genética , Transativadores , Fatores de Transcrição/genética , Trichoderma/metabolismo , Xilanos/metabolismo
2.
PLoS One ; 15(7): e0236518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702033

RESUMO

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.


Assuntos
Celulose/metabolismo , Polissacarídeos/metabolismo , Microbiologia do Solo , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Biomassa , Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Filogenia , Especificidade por Substrato , Thermoanaerobacterium/classificação , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/isolamento & purificação
3.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 920-931, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567275

RESUMO

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²âº or 0.1 mmol/L Cu²âº treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.


Assuntos
Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Microbiologia Industrial , Microbiota , Rúmen , Animais , Ciclização , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Microbiologia Industrial/métodos , Engenharia de Proteínas , Rúmen/enzimologia , Rúmen/microbiologia , Temperatura
4.
Arch Anim Nutr ; 74(4): 309-324, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32441546

RESUMO

The study was conducted to determine effects of a complex of fibre-degrading enzymes (xylanase, cellulase and ß-glucanase) on nutrient digestibility, fibre fermentation and concentrations of short chain fatty acids (SCFA) at different parts of digestive tract in pigs fed different fibre-rich ingredients. A total of 36 barrows fitted with T-cannulas in the distal ileum (initial body weight of 41.1 ± 2.7 kg) were randomly allotted to six dietary treatments with three different high-fibre diets including maize bran (MB), sugar beet pulp (SBP) and soybean hulls (SH) with or without supplementation of fibre-degrading enzymes. Enzyme supplementation improved (p < 0.05) apparent ileal digestibility (AID) of dietary gross energy (GE), crude protein, dry matter (DM), organic matter (OM), total dietary fibre (TDF), neutral detergent fibre (NDF) and apparent total tract digestibility (ATTD) of dietary GE, DM, OM, TDF, insoluble dietary fibre (IDF) when pigs were fed MB, SBP or SH diets. When compared to the SBP and SH diets, the AID of GE, DM, ash, OM and NDF in diet MB was higher (p < 0.05), but the hindgut disappearance and ATTD of nutrients, except for ether extract and crude ash, were lower (p < 0.05). Enzyme supplementation increased acetate and total SCFA concentrations in ileal digesta and faeces of pigs. In conclusion, enzyme addition improved IDF fermentation and SCFA concentration in the whole intestine of pigs, and there was a large variation of digestibility of fibre components among MB, SH and SBP owing to their different fibre composition. Therefore, fibre-degrading enzymes should be applied to fibrous diets to improve efficient production of swine, especially considering low fibre digestibility of fibre-rich ingredients.


Assuntos
Celulase/metabolismo , Digestão , Endo-1,3(4)-beta-Glucanase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Íleo/fisiologia , Nutrientes/fisiologia , Sus scrofa/fisiologia , Ração Animal/análise , Animais , Beta vulgaris/química , Celulase/administração & dosagem , Dieta/veterinária , Suplementos Nutricionais/análise , Endo-1,3(4)-beta-Glucanase/administração & dosagem , Endo-1,4-beta-Xilanases/administração & dosagem , Fermentação , Trato Gastrointestinal , Íleo/efeitos dos fármacos , Masculino , Distribuição Aleatória , Soja/química , Zea mays/química
5.
Poult Sci ; 99(5): 2555-2565, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359591

RESUMO

While arabinoxylans (AX), an important dietary fiber fraction of wheat-based broiler diets, are known for exerting antinutritional effects in the gastrointestinal (GI) tract of broilers, the prebiotic potential of arabinoxylan-oligosaccharides (AXOS) is also well-documented. However, inconsistent performance responses as well as the effectiveness of low amounts of AXOS used in diets of previously conducted experiments put into question the classical prebiotic route being the sole mode of action of AXOS. The objective of this study was to investigate the effects of dietary AXOS addition on the rate of AX digestion in the gastrointestinal tract of broilers as a function of broiler age to gain more insight into the mode of action of these oligosaccharides. A feeding trial was performed on 480 one-day-old chicks (Ross 308) receiving a wheat-based diet supplemented with or without 0.50% AXOS, containing no endoxylanases. Digesta samples from ileum and caeca and fecal samples were analyzed for AX content, AX digestibility, intestinal viscosity, and microbial AX-degrading enzyme activities at 6 different ages (day 5, 10, 15, 21, 28, 35). Chicks fed from hatching with 0.50% AXOS demonstrated a higher ileal viscosity (P < 0.05). Also higher levels of AX solubilization and fermentation compared to control birds at 10 D were observed. This was noted by the higher total tract AX digestibility of water-extractable AX (WE-AX) and total AX (TOT-AX) at this age (P < 0.05). Although no significant difference in AX-degrading enzyme activities was observed among the dietary treatments, AXOS supplementation in young broilers was shown to stimulate or "kick-start" dietary AX digestion, thereby speeding up the development of a fiber-fermenting microbiome in the young broiler. This stimulation effect of AXOS could enable greater functional value to be extracted from dietary fiber in broiler feeds.


Assuntos
Galinhas/fisiologia , Digestão , Endo-1,4-beta-Xilanases/metabolismo , Oligossacarídeos/metabolismo , Xilanos/metabolismo , Envelhecimento , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Masculino , Oligossacarídeos/administração & dosagem , Distribuição Aleatória , Xilanos/administração & dosagem
6.
Poult Sci ; 99(4): 2068-2077, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241492

RESUMO

Two independent studies were performed, each with a 3 × 2 factorial arrangement to compare the response in broilers and turkeys to phytase and xylanase supplementation on cecal fermentation and microbial populations. For both studies, 960 Ross 308 and 960 BUT 10 (1-day-old) were allocated to 1 of 6 experimental treatments: (1) control diet, containing the standard dose (100 g/ton) of phytase (STD-Xyl); (2) the control diet with 100 g/ton of xylanase (STD + Xyl); (3) the control diet supplemented on top with 2 fold the standard dose of phytase (200 g/ton), also referred as superdosing (SD-Xyl); (4) the superdosed diet with 100 g/ton of xylanase (SD + Xyl); (5) the control diet supplemented with 5-fold the standard dose of phytase (500 g/ton), also referred as megadosing (MD-Xyl); and (6) the megadosed diet with 100 g/ton of xylanase (MD + Xyl). Each treatment had 8 replicates of 20 animals. Broiler and turkey diets, based on wheat, soybean meal, rapeseed, and barley, and water were available ad libitum. On day 28, the cecal contents from 5 birds per pen were collected. The profile of short-chain fatty acids (SCFA) and microbiome structure (by % guanidine and cytosine [G + C] method) were analyzed. Selected % G + C fractions were used for 16S rDNA sequencing for the identification of bacteria. No treatment effects were noted on SCFA concentrations in either broilers or turkeys. Broilers fed MD diets had greater proportions of unclassified Clostridiales, Mollicutes (RF9) and Faecalibacterium. Xylanase supplementation in broilers resulted in lower proportions of Lactobacillus but increased Mollicutes (RF9), unclassified Ruminococcus, unclassified Clostridiales, and Bifidobacterium. The microbiome in turkeys was unaffected by phytase supplementation, but xylanase supplementation increased the proportions of Lachnospiraceae (Incertae sedis), Lactobacillus, and Bifidobacterium. Supplementation of turkey diets with increasing doses of phytase did not affect the cecal microbiota in contrast to what was observed in broilers. In contrast, xylanase supplementation in both species led to significant changes in the microbial populations, suggesting a positive influence through the provision of oligosaccharides.


Assuntos
6-Fitase/metabolismo , Ceco/microbiologia , Galinhas , Endo-1,4-beta-Xilanases/metabolismo , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal , Perus , 6-Fitase/administração & dosagem , Ração Animal/análise , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Galinhas/metabolismo , Galinhas/microbiologia , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Endo-1,4-beta-Xilanases/administração & dosagem , Fermentação , Masculino , Perus/metabolismo , Perus/microbiologia
7.
Sci Rep ; 10(1): 3824, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123244

RESUMO

Application of crude xylanolytic and pectinolytic enzymes in diverse industrial processes make these enzymes commercially valuable and demand their production process to be cost-effective. Out of four different agrowaste biomass, wheat bran (WB) and citrus peel (CP), when amended as fermentation substrates, respectively induced the highest xylanolytic enzymes and pectinolytic enzymes from both, B. safensis M35 and B. altitudinis J208. Further, the simultaneous amendment of WB and CP yielded concurrent production of these cellulase free xylanolytic and pectinolytic enzymes. Hence, the quadratic model was developed using the Central Composite Design of Response Surface Method (CCD-RSM). The model gave the concentration values for WB and CP substrates to be amended in one single production medium for obtaining two optimized predicted response values of xylanase activity and pectinase activity units, which were further practically validated for the xylanase and pectinase production responses from the optimized production medium (OPM). These practically obtained response values from OPM were found to be in accordance with a range of 95% predicted intervals (PI) values. These observations verified the validity of the predicted quadratic model from RSM and suggested that both xylanase and pectinase enzymes can be induced concurrently from both of the bacterial strains.


Assuntos
Bacillus/metabolismo , Biomassa , Biotecnologia/métodos , Endo-1,4-beta-Xilanases/biossíntese , Indústrias , Poligalacturonase/biossíntese , Agricultura , Endo-1,4-beta-Xilanases/metabolismo , Hidrólise , Cinética , Poligalacturonase/metabolismo
8.
Poult Sci ; 99(3): 1528-1539, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32111320

RESUMO

Two experiments were performed, using broilers or turkeys, each utilizing a 3 × 2 factorial arrangement, to compare their response to phytase and xylanase supplementation with growth performance, nutrient digestibility, and ileal phytate degradation as response criteria. For both experiments, 960 Ross 308 or 960 BUT 10 (0-day-old) birds were allocated to 6 treatments: (1) control diet, containing phytase at 500 FTU/kg; (2) the control diet with xylanase (16,000 BXU/kg); (3) the control diet supplemented on top with phytase (1,500 FTU/kg); (4) diet supplemented with 1,500 FTU/kg phytase and xylanase (16,000 BXU/kg); (5) the control diet supplemented with phytase (3,000 FTU/kg); and (6) diet supplemented with 3,000 FTU/kg phytase and xylanase (16,000 BXU/kg). Each treatment had 8 replicates of 20 birds each. Water and diets based on wheat, soybean meal, oilseed rape meal, and barley were available ad libitum. Body weight gain and feed intake were measured from 0 to 28 D, and feed conversion ratio (FCR) corrected for mortality was calculated. Ileal digestibility for dry matter and minerals on day 7 and 28 were analyzed in addition to levels of inositol phosphate esters (InsP6-3) and myo-inositol. Statistical comparisons were performed using ANOVA. Xylanase supplementation improved 28D FCR in broilers and turkeys. Increasing doses of phytase reduced FI and improved FCR only in broilers. In broilers, the age × phytase interaction for phosphorous digestibility showed that increasing phytase dose was more visible on day 7, than on day 28. Mineral digestibility was lower in 28-day-old turkey compared with 7-day-old turkey. InsP6 disappearance increased with increasing phytase levels in both species, with lower levels analyzed in turkeys. InsP6 disappearance was greater in younger turkeys (day 7 compared with day 28). In conclusion, although broilers and turkeys shared several similarities in their growth and nutrient utilization responses, the outcomes of the 2 trials also differed in many aspects. Whether this is because of difference in diets (InsP or Ca level) or differences between species needs further investigation.


Assuntos
6-Fitase/metabolismo , Galinhas/fisiologia , Digestão/efeitos dos fármacos , Endo-1,4-beta-Xilanases/metabolismo , Minerais/metabolismo , Ácido Fítico/metabolismo , Perus/fisiologia , 6-Fitase/administração & dosagem , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Endo-1,4-beta-Xilanases/administração & dosagem , Íleo/metabolismo , Masculino , Perus/crescimento & desenvolvimento
9.
Arch Microbiol ; 202(5): 1077-1084, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32030461

RESUMO

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.


Assuntos
Biocombustíveis/microbiologia , Cellulomonas/isolamento & purificação , Cellulomonas/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Lignina/metabolismo , Composição de Bases/genética , Biomassa , Celulose/metabolismo , Endo-1,4-beta-Xilanases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Panicum/química , Panicum/genética , Panicum/metabolismo , Pectinas/metabolismo , Filogenia , Plantas/metabolismo , Polissacarídeos/metabolismo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
10.
Poult Sci ; 99(2): 992-1000, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32036991

RESUMO

This study was conducted to determine the standardized ileal digestible amino acids (SID AA) and nitrogen-corrected apparent metabolizable energy (AMEn) contents of 6 wheats from different origins in China and incidentally to investigate the effects of exogenous xylanase addition on SID AA and AMEn determination in broiler chicks. A total of 480 chicks were divided into 48 cages of 10 birds each balanced for body weight and fed 8 types of diets in a completely randomized design (6 replicated cages per diet) from 21 to 26 d of age. The individual wheat constituted the only source of crude protein in a semi-purified experimental diet. A nitrogen-free diet was designed to estimate basal endogenous AA loss and determine the SID AA. Titanium oxide (0.3%) was used as an indigestibility marker, and nutrient digestibility and retention were determined by the substitution method. From day 24 to 26, excreta samples were collected for AMEn determination. On day 26, the birds were euthanized, and ileum contents were obtained for AA digestibility determination. Wheat from Gansu had greater (P < 0.05) SID AA contents except Lys, Thr, Phe, and Cys, with a higher (P < 0.001) AMEn (11.83 MJ/kg) than the other wheats. The SID content of mean indispensable amino acids and dispensable amino acids were 87.35% and 88.17%, respectively, and the average AMEn value of 6 wheats was 11.14 MJ/kg. Compared with the diet without xylanase, the added xylanase resulted in higher (P < 0.05) SID contents of Met, Lys, Trp, Arg, Ile, Leu, Val, Gly, Asp, Glu, Pro, and Ala; the SID AA values were raised by 1.96% (mean of all AA); and the AMEn content was significantly increased (+0.87 MJ/kg) (P < 0.05). In conclusion, origins of wheats have significant effects on SID AA and AMEn values which were positively correlated with crude protein content of wheat; exogenous xylanase addition to a wheat-based poultry diet could significantly improve SID AA and AMEn contents for broilers.


Assuntos
Aminoácidos/fisiologia , Digestão , Endo-1,4-beta-Xilanases/metabolismo , Metabolismo Energético , Íleo/fisiologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Endo-1,4-beta-Xilanases/administração & dosagem , Distribuição Aleatória , Triticum/química
11.
Enzyme Microb Technol ; 134: 109484, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044031

RESUMO

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.


Assuntos
Acremonium/enzimologia , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Hidrolases/metabolismo , Acremonium/genética , Endo-1,4-beta-Xilanases/genética , Hidrolases/genética , Especificidade por Substrato
12.
J Agric Food Chem ; 68(10): 3184-3194, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32105462

RESUMO

Enzymatic hydrolysis of xylan represents a promising way to produce xylooligosaccharide (XOS), which is a novel ingredient in functional food. However, the recalcitrance of xylan in natural lignocellulosic biomass entails effective and robust xylanases. In the present study, we reported the isolation of a thermophilic Streptomyces sp. B6 from mushroom compost producing high xylanase activity. Two xylanases of Streptomyces sp. B6 belonging to GH10 (XynST10) and GH11 (XynST11) families were thus identified and biochemically characterized to be robust enzymes with high alkaline- and thermostability. Direct hydrolysis of neutralized viscose fiber production waste using XynST10 and XynST11 showed that while XynST10 produced 23.22 g/L XOS with a degree of polymerization (DP) of 2-4 and 9.27 g/L xylose, XynST11 produced much less xylose (1.19 g/L) and a higher amounts of XOS with a DP = 2-4 (28.29 g/L). Thus, XynST11 holds great potential for the production of XOS from agricultural and industrial waste.


Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Glucuronatos/química , Oligossacarídeos/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glucuronatos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Resíduos Industriais/análise , Oligossacarídeos/metabolismo , Streptomyces/química , Streptomyces/genética , Xilose/química , Xilose/metabolismo
13.
Food Chem ; 315: 126221, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32000077

RESUMO

Xylo- and arabinoxylo-oligosaccharides (XOS and AXOS) are of interest for their prebiotic activity. The production of these oligomers might be accompanied with monosaccharides. The measurement of both oligosaccharides and monosaccharides usually requires two methods. The current work presents an HPAEC-PAD method based on gradient elution of aqueous solvents sodium hydroxide and sodium acetate, in contrast to conventional isocratic elution, for the simultaneous separation of 16 standards of monosaccharides, xylo-oligosaccharides, arabinoxylo-oligosaccharides and uronic acids using CarboPac PA 200 column. The presented method showed a stable baseline and high-resolution separation of the standards. The method showed acceptable accuracy and precision. Limits of Detection and Quantitation (LOD and LOQ) were estimated for all the standards. The method was applied to measure the activity of a commercial endoxylanase on wheat bran; a steady release of xylose monosaccharide was observed. Enzyme action on oligosaccharide standards showed a preference for the larger oligosaccharides.


Assuntos
Monossacarídeos/análise , Oligossacarídeos/análise , Prebióticos/análise , Triticum/química , Arabinose/análise , Arabinose/metabolismo , Cromatografia por Troca Iônica , Fibras na Dieta , Grão Comestível/química , Endo-1,4-beta-Xilanases/metabolismo , Limite de Detecção , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Reprodutibilidade dos Testes , Ácidos Urônicos/química , Xilose/análise , Xilose/metabolismo
14.
Recent Pat Biotechnol ; 14(1): 5-15, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31333132

RESUMO

BACKGROUND: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability. OBJECTIVE: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability. METHODS: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). RESULTS: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket. CONCLUSION: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.


Assuntos
Proteínas de Bactérias , Endo-1,4-beta-Xilanases , Engenharia de Proteínas/métodos , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Fontes Termais/microbiologia , Temperatura Alta , Simulação de Acoplamento Molecular , Mutação
15.
Appl Biochem Biotechnol ; 190(1): 197-217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31325025

RESUMO

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.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Eucalyptus/metabolismo , Glucuronatos/biossíntese , Oligossacarídeos/biossíntese , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Hidrólise
16.
Biosci Biotechnol Biochem ; 84(3): 640-650, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31746676

RESUMO

Corn fibre xylan (CX) shows high resistance to enzymatic hydrolysis due to its densely decorated side chains. To find enzymes capable of hydrolyzing CX, we isolated a bacterial strain (named H2C) from soil, by enrichment culture using non-starch polysaccharides of corn as the sole carbon source. Analysis based on the 16S rRNA sequence placed strain H2C within genus Paenibacillus. Enzymes were purified from supernatant of culture broth of strain H2C based on solubilizing activities toward CX. Four enzymes, Xyn5A, Xyn10B, Xyn11A, and Xyn30A, were successfully identified, which belong to glycoside hydrolase (GH) families, 5, 10, 11, and 30, respectively. Phylogenetic analysis classified Xyn5A in subfamily 35 of GH family 5, a subfamily of unknown function. Their activities toward beechwood xylan and/or wheat arabinoxylan indicated that these enzymes are ß-1,4-xylanases. They showed high solubilizing activities toward a feed material, corn dried distiller's grains with solubles, compared to five previously characterized xylanases.Abbreviations : CX: corn fibre xylan; DDGS: corn dried distiller's grains with solubles.


Assuntos
Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Paenibacillus/enzimologia , Xilanos/metabolismo , Zea mays , Endo-1,4-beta-Xilanases/classificação , Hidrólise , Filogenia , Polissacarídeos/metabolismo
17.
J Biochem ; 167(2): 203-215, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617574

RESUMO

Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on ßα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.


Assuntos
Substituição de Aminoácidos , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Geobacillus stearothermophilus/enzimologia , Temperatura , Endo-1,4-beta-Xilanases/química , Estabilidade Enzimática , Geobacillus stearothermophilus/genética , Ensaios de Triagem em Larga Escala , Especificidade por Substrato
18.
Prep Biochem Biotechnol ; 50(2): 181-190, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31647386

RESUMO

The synergism of cellulase (C), pectinase (P), and xylanase (X) for the saccharification of sweet potato residues (SPR) was investigated. The removal of starch from SPR was easily achieved by using amylase, but the cellulose conversion of de-starched SPR was relatively low, thus dilute H2SO4, NaOH, and H2O2 pretreatment was conducted to improve the enzymatic digestibility. The lignin content of NaOH pretreated SPR was the lowest, whereas H2SO4 pretreatment resulted in the lowest contents of hemicellulose and pectin. The combination of C, P, and X exhibited different sugar production patterns, C-P displayed synergistic action on glucose and galactose production from each type of SPR, C-X also exhibited synergistic effect on glucose production except when H2SO4 pretreated SPR was used, whereas no synergism between P-X on monosaccharide production was observed. The presence of synergism between cellulase and mixed accessory enzymes [C-(PX)] on glucose formation was determined by C-X, and the degree of synergism between C-P and C-(PX) on glucose production had a positive relationship with pectin content. The highest cellulose conversion of 96.2% was obtained from NaOH pretreated SPR using mixed enzymes comprising C, P, and X with the ratio of 8:1:1.


Assuntos
Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Ipomoea batatas/metabolismo , Poligalacturonase/metabolismo , Hidrólise , Ipomoea batatas/química , Ácidos Sulfúricos/metabolismo
19.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140312, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740412

RESUMO

Global economic growth often leads to depletion of raw materials and generation of greenhouse gases, as industry manufactures goods at ever increasing levels to keep up with the demand. The currently implemented production processes mostly rely on non-renewable resources, they suffer from high energy consumption, and generate waste that often has a negative environmental impact. Eco-friendly production methods are therefore intensely searched for. Among them, enzyme-based processes are appealing, because of their high substrate and reaction specificity and the relatively mild operation conditions required by these catalysts. In addition, renewable raw materials that allow sustainable production processes are also widely explored. Marine xylanases, which catalyze the hydrolysis of xylan, the major component of lignocellulose, are promising biocatalysts. Since they are produced by microorganisms that thrive in a wide variety of environmental conditions, the enzymes may be active at widely different ranges of pH, temperature, and salt concentrations. These properties are important for their successful application in various industrial processes, such as production of bioethanol, bleaching of paper and pulp, and in the food and feed sector. The present work gives a brief overview of marine sources of xylanases, their classification and features, and of the potential applications of these marine enzymes, especially in sustainable processes in the scope of circular economy.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Clareadores/química , Clareadores/metabolismo , Endo-1,4-beta-Xilanases/classificação , Concentração de Íons de Hidrogênio , Cinética , Lignina/metabolismo , Probióticos , Energia Renovável , Alga Marinha/enzimologia
20.
Prep Biochem Biotechnol ; 50(1): 91-97, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517567

RESUMO

Xylanases have gained increasing importance due to their diverse applications in the food, paper, and pharmaceutical industries, however, the production of these enzymes currently uses expensive substrates. It has already been estimated that more than 30% of the enzyme production cost originates from the substrate. The present study aimed to optimize the production of extracellular xylanases by the Bacillus sp. TC-DT 13 using solid-state fermentation with agro-industrial residues, with a view at reducing the production cost of these enzymes. All the agro-industrial residues were tested in submerged fermentation to select the best inductor to produce xylanase. Among these residues, wheat bran was selected as the best inducer of xylanase production with 1500 U/mL. Regarding solid-state fermentation, the use of wheat bran as the only fermentation substrate was used and a ratio of 1:4 moisture over a time of 144 hours induced higher amount of xylanase reaching 2943 U/g. The use of carbon and nitrogen sources did not result in the increase in production of xylanolitic enzymes. The use of agro-industrial residues in the solid-state fermentation, besides increasing the production of xylanase, reduces the cost of production and is an environmentally friendly alternative.


Assuntos
Bacillus/enzimologia , Fibras na Dieta/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Microbiologia Industrial/economia , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Temperatura
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