Добірка наукової літератури з теми "Bariey xylanase"

The composition and properties of a wide range of domestic and foreign enzyme preparations (EP), used as additives to feeds of farm animals and poultry, are analyzed. The content of the main active enzymes – endoglucanases (beta-glucanases), cellobiohydrolases and xylanases, leading to biocatalytic destruction of non-starch polysaccharides, which are anti-nutritional factors of feeds and causing their incomplete digestion, is determined. It is shown that, based on the data on the component composition and the level of different types of activity, the studied enzyme preparations can be divided into three groups: a) with high xylanase and low cellulase (endoglucanase and cellobiohydrolase) content, b) high cellulase and low xylanase content, c) containing cellobiohydrolases, endoglucanases and xylanases in a different ratio, but without significant predominant of any of these enzymes. The ability of EP to reduce the viscosity of water-soluble non-starch polysaccharides – xylans and beta-glucans- has been studied. Among the enzyme preparations that have xylanase in their composition and belong to groups b) and c), a number of preparations have been determined which, at the same dosage according to xylanase activity, most effectively reduced the viscosity of the aqueous extract of rye containing xylans (Econase XT 25, Agroxyl Plus, Agroxyl Premium, Rovabio Max AP, Sunzyme). It was shown that the xylanase of precisely these EP is not inhibited by protein inhibitors of rye. At the same dosage for beta-glucanase activity, the viscosity of water-soluble beta-glucans of barley was most effectively reduced by the EP Xybeten CELL, Cellulase, Agroxyl, Agrocell, Axtra XB 201, Rovabio Max AP and Vilzyme. For all studied EP, no inhibitory effect of the barley extract on beta-glucanase activity was found.

ABSTRACT Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and β-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A and E236A). The gene consists of 981 bp, encodes 327 amino acids, and comprises only one catalytic domain that is highly homologous to the glycoside hydrolase family 10 xylanase catalytic domain. Recombinant XynBE18 purified from Escherichia coli BL21(DE3) showed specificity toward oat spelt xylan and birchwood xylan and β-1,3-1,4-glucan (barley β-glucan and lichenin). Homology modeling and molecular dynamic simulation were used to explore structure differences between XynBE18 and the monofunctional xylanase XynE2, which has enzymatic properties similar to those of XynBE18 but does not hydrolyze β-1,3-1,4-glucan. The cleft containing the active site of XynBE18 is larger than that of XynE2, suggesting that XynBE18 is able to bind larger substrates such as barley β-glucan and lichenin. Further molecular docking studies revealed that XynBE18 can accommodate xylan and β-1,3-1,4-glucan, but XynE2 is only accessible to xylan. These results indicate a previously unidentified structure-function relationship for substrate specificities among family 10 xylanases.

<i>Penicillium ramulosum</i> N1 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest activities of xylanases (250 U/ml) and carboxymethyl cellulose (CMCase; 6.5 U/ml) were produced when 1% barley straw was added as a carbon source. The optimum temperature and pH for xylanase activity was 55 and 3.0°C, respectively. The xylanases exhibited strong protease resistance. CMCase revealed maximum activities at pH 3.0 and in the range of 60-70°C. Filter paper activity was optimally active at pH 5.0 and 55°C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that there are four bands of protein with xylanase activity and three bands of proteins with endoglucanase. The results revealed that <i>P. ramulosum</i> N1 is a promising acidophilic and protease-resistant xylanase-producing microorganism that has great potential to be used in animal feed and food industry applications.

The synthesis of barley (Hordeum vulgare L. cv. Himalaya) aleurone xylanase was found to be dependent on both gibberellic acid (GA3) and Ca2+, but inhibited by cycloheximide and cordycepin. Studies using density labeling of barley aleurone layers showed that xylanase was synthetized de novo in response to GA3 and Ca2+. Neither GA3 nor Ca2+ alone induced a large increase in xylanase activity. The concentration of Ca2+ required for maximum xylanase induction was 5 – 40 mM. Xylanase activity was found to develop simultaneously with that of α-amylase in the incubation medium during the first 24 h of incubation with GA3. A critical point with respect to the role of xylanase is the extent of its activity by the time of the initial release of α-amylase. The release of α-amylase into the medium was detectable at 6 h. From 2 to 6% of the cell wall was hydrolysed by xylanase after incubation for 6 h, which was probably sufficient to permit the release of α-amylase. Scanning electron microscopy showed that the purified barley aleurone xylanase hydrolysed the cell walls of barley aleurone layers in the absence of GA3. It is likely that xylanase plays an important role in the release of enzymes from aleurone cells.

Two experiments were conducted to study the response of laying hens to a commercial enzyme preparation (Safizyme XP20) containing 70 000 IU g-1 xylanase. In the first experiment, 270 laying hens (ISA Brown), aged 28 wk, were fed basal diets of wheat (W), wheat-barley (WB) or wheat-barley and wheat bran (WBB) without xylanase, or supplemented with 1400 IU xylanase kg-1 in 3 × 2 factorial arrangement. In the second experiment, 180 ISA Brown laying hens, aged 28 wk, were assigned to 2 × 2 factorial arrangement and fed wheat-based diets containing 2753 or 2653 kcal metabolizable energy (ME) kg-1 without xylanase, or supplemented with 1400 IU xylanase kg-1. Egg production (% hen-day), egg weight, egg mass, feed conversion ratio and changes in body weight were recorded for 12 wk. The effects of xylanase on true metabolizable energy (TME) values of wheat, barley and wheat bran were determined using roosters. Layers fed W-, WB- or WBB-based diets had similar egg mass. The feed conversion ratio of layers fed the WBB-based diet was better (P ≤ 0.05) than that of the other groups. Xylanase improved (P ≤ 0.05) egg mass of layers fed W- or WB-based diets, but it did not affect the performance of hens fed the WBB-based diet. Low dietary ME significantly (P ≤ 0.05) reduced the performance of laying hens. Xylanase supplementation improved (P ≤ 0.05) egg production, egg mass and feed conversion ratio of layers fed the low energy diet. It did improve the feed conversion ratio of layers fed the high-energy diet. Addition of 1400 IU xylanase kg-1 to the low-energy diet was equivalent to an increase of at least 100 kcal ME kg-1. Xylanase increased (P ≤ 0.05) the TME values by 5.2 and 2.44% for wheat and barley, respectively, and did not affect the TME value of wheat bran. Key words: Xylanase, hens, laying, wheat, barley, wheat bran, metabolizable energy.

Abstract One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. Key points • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

Seventy-two steers (289 kg) were offered ad libitum cubed alfalfa hay, cubed timothy hay, or barley silage supplemented with incremental levels of xylanase (IU) and cellulase (FPU), combined in a ratio of 1 IU:0.04 FPU. For alfalfa hay, low and moderate levels (900 to 4733 IU kg−1 DM) increased weight gain by up to 30% (P < 0.10), whereas, for timothy hay, the highest level (12 000 IU kg−1 DM) improved gain (P < 0.10) by 36%. No response to enzymes was observed for barley silage. Fibrolytic enzymes improve weight gain of cattle but optimal enzyme levels depend upon the type of forage. Key words: Beef cattle, forages, enzymes, cellulase, xylanase, carbohydrases

Exogenous enzymes have been used in pig feed to reduce the antinutritional factors in the cereal components of the diets and, as a consequence, improve energy and protein digestibility. The predominant fibre components in barley are soluble β-glucans and insoluble arabinoxylans, both of which are known to have anti-nutritional effects in the pig but through different mechanisms. In wheat both soluble and insoluble arabinoxylans are relevant, for the same reasons (Partridge, 2001). Consequently a product with a combination of β-glucanase and xylanase may be necessary to elicit a performance improvement in pigs fed diets containing both barley and wheat. Furthermore, much work in grower/finisher pigs has been undertaken at single inclusion levels of enzyme product. The object of the current study was to compare the response of growing and finishing pigs fed diets containing barley and wheat supplemented with a β-glucanase and xylanase product included at different levels in the diet.

Extracts from 14 barley silages inhibited endo-1, 4-β-xylanase and α-amylase activities of a ruminant feed enzyme additive from Trichoderma longibrachiatum by 23 to 50% but had little effect on cellulase activity. The inhibitory factor(s) were <10 kDa in size and were stable to autoclaving. These observations may explain why feed enzymes are generally less effective when applied to silages than when applied to dry feeds. Key words: Silage, fibrolytic enzymes, Trichoderma, xylanase, inhibitors

The overall objective of this study was to evaluate the effects of feeding hulless barley and supplementing a xylanase enzyme on production performance and nutrient utilization of lactating dairy cows. In study 1, we evaluated production performance, milk fatty acid composition, and nutrient digestibility in high-producing dairy cows consuming diets containing corn and hulless barley in different proportions as the grain source. We hypothesized that a plausible reduction in production performance would be explained by an altered rumen function, which would be reflected in a reduction of the proportion of de novo fatty acids in milk fat. The inclusion of hulless barley grain as the energy source in diets for lactating dairy cows resulted in similar production performance and nutrient utilization as corn grain. We concluded that hulless barley is as good as corn grain as an energy source and increasing NDF concentration in hulless barley-based diet is not necessary. In study 2, we evaluated production performance, nutrient digestibility, and milk fatty acid composition of high-producing dairy cows consuming diets containing hulled or hulless barley as the grain source. We hypothesized that rumen function is altered when cows are fed low-forage diets containing barley grains, and this altered rumen function would be reflected in lower production performance and a reduction of fatty acids synthesis in the mammary gland. Contrary to our expectations, feeding hulled barley or hulless barely based diets with different forage to concentrate ratios to lactating dairy cows resulted in similar production performance and nutrient utilization. We concluded that both hulled or hulless barley grains are good energy sources for sustaining high milk production and there is no need to increase NDF concentration in diet when using barley grain as the grain source. In study 3, we evaluated the effects of supplementing a xylanase enzyme on production performance and nutrient digestibility of lactating dairy cows fed diets containing corn or sorghum silage as the forage source. We hypothesized that supplementing a xylanase enzyme product in diets containing corn or sorghum silage increases NDF digestibility, and production performance of lactating dairy cows would also be improved due to enhanced fiber digestion. Supplementation of xylanase for 19 d did not affect cow performance and nutrient utilization. Supplementation of xylanase may require a longer period of time to show any response in production performance and nutrient digestibility. We concluded that supplementing xylanase to cows fed corn or sorghum silage-based diets did not improve fiber digestion. But for feeding hulled or hulless barley grains to lactating dairy cows, increased NDF concentration in diets is not necessary and hulless barley is good as corn grain for feeding lactating dairy cows as the grain source.
Ph. D.

Bibliography: leaves 127-166. This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes.

The objective of this thesis was to evaluate the effect of exogenous enzyme supplementation, phytase and xylanase-glucanase, on AME value of barley in poultry and swine. In the first study, 280 broilers were assigned 1 of 8 treatments. Barley inclusion in the diet resulted in decreased (P < 0.05) performance. There was a treatment × phytase × xylanase-glucanase interaction for dry matter retention with birds fed the corn-SBM-barley diet supplemented with phytase and xylanase-glucanase having higher (P < 0.05) DM retention compared to birds fed corn-SBM-based diet with only xylanase-glucanase supplementation. AME and AMEn of corn-SBM-based diets were greater (P < 0.05) than the corn-SBM-barley-based diets. Energy metabolizability and AMEn of barley significantly increased with xylanase-glucanase supplementation. In the second study, 24 pigs (12 pigs/phase) were assigned to 1 of 4 treatments with xylanase-glucanase and phytase. After a 7-d adaption period, urine and feces were quantitatively collected for 5 d. DE of the barley-based diet supplemented with xylanase-glucanase (3,578 kcal/kg) and phytase and xylanase-glucanase in combination (3,617 kcal/kg) were significantly different. Compared to control diets, exogenous enzymes either significantly improved or had a tendency to improve AME and AMEn value of barley in broilers, but not in growing pigs.

Barley (Hordeum vulgare) is a widely grown, valuable cereal crop that is affected by various pests including nematodes. The cereal cyst nematode (CCN) Heterodera avenae is the most widely distributed and damaging species of nematodes to cereal crops grown in temperate regions, including Australia, and is estimated to cause global annual losses of around $160 billion. The use of cultivars resistant to H. avenae is the preferred approach for nematode management and four resistance loci, Rha1, Rha2, Rha3 and Rha4, have been mapped. The Rha4 locus was mapped in the Galleon/Haruna Nijo population to chromosome 5H and since Rha2 and Rha4 provide the most effective resistance against the Australian H. avenae pathotype Ha13 they have been widely used in barley breeding. Despite CCN resistance loci having been mapped in barley and other cereals, no resistance genes have been isolated and characterized. Recently both Rha2 and Rha4 have been fine-mapped and near-diagnostic markers have been developed to provide simple tools for selection. Fine-mapping indicates that approximately 105 genes are linked to the Rha4 locus, including cell wall-related genes predicted to encode (1-4)-β-xylan endohydrolases, also known as xylanases. This thesis reports on experiments that were undertaken to better understand the resistance conferred by Rha4 and to investigate the functions of the xylanases as prime candidate genes. The xylanase genes at the Rha4 locus were cloned and analysed for allelic differences between sequences from the cultivars Sloop (susceptible) and Flagship (Rha4-resistant). Although genes X1 and X2 have been characterised, the X3 gene has not been well described previously. The genomic sequences were used in reciprocal transformation experiments where, under the control of the 35S promoter, the genes from Sloop were transformed into a Flagship background, and vice versa. Tube tests were used to investigate any changes in nematode infection responses, and therefore resistance status, but no significant alterations were detected. However, overexpression of the xylanase genes proved to be detrimental to the overall health of the plant. The xylanases were concomitantly heterologously expressed in Escherichia coli and the X2 protein was characterized in terms of substrate preference and catalytic rate. In more general approaches not directly linked to Rha4 genotype, the natural variation in root polysaccharide amount and distribution, with a focus on (1,3;1,4)-β-glucan and arabinoxylan, was surveyed in a selection of susceptible and resistant barley cultivars. The effect of changes in amount of (1,3;1,4)-β-glucan was also examined through infection of the betaglucanless mutant and transgenic lines carrying either the overexpressed or silenced (1,3;1,4)-β-glucan synthase CslF6 gene. Variable amounts of (1,3;1,4)-β-glucan did not correlate to rates of nematode infection and no clear patterns of polysaccharide profiles could be linked to susceptible or resistant cultivar status. Finally, RNA sequencing was used to profile transcript changes in nematode infected and control Sloop and Flagship roots up to 24 days post inoculation. The patterns of the 105 genes linked to the Rha4 locus were extracted and a set of 27 genes which showed significant fold changes across the time course were profiled. Of these, three strong candidate genes were selected which are differentially expressed in the two cultivars and are involved in biochemical pathways that are feasibly linked to resistance mechanisms. Their putative roles in conferring resistance and how this might be tested were discussed.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2019

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease (NFNB), an important disease of barley in Australia and worldwide. This fungus uses proteinaceous toxins to cause necrosis and different isolates of Ptt differ in their ability to cause symptoms on different cultivars of barley. However, little is known about the roles of pathogen growth and individual toxins in symptom development. This project therefore aimed to determine whether there is a relationship between toxin production, fungal growth and virulence in NFNB. Conidial germination, extent of fungal growth and culture filtrate toxicity were compared for six South Australian Ptt isolates with different virulence on the barley cultivar ‘Sloop’. In addition, Ptt toxin production was optimised before identification and selection of virulence-related candidate proteins (VRCPs) for further characterisation. The biological activity of recombinant VRCPs on susceptible and resistant cultivars and VRCPs gene expression during the interaction of Sloop with each isolate were also compared. In general, the more virulent isolates had higher rates of conidial germination (both in vitro and in planta) and fungal development in planta, represented by longer hyphae and more appressoria, compared with less virulent isolates. Similarly, PttGAPDH and its transcript were more abundant during the interaction of barley with more virulent isolates. A proteomics approach was used to identify proteins unique to the more virulent isolate, proteins from bioactive fractions on either susceptible (Sloop) or resistant cultivars (CI9214 and Beecher) and proteins from the intercellular washing fluids (ICWFs) of infected barley. These analyses revealed that Ptt produced proteins between 37 and 150 kDa that have biological activity. Liquid Chromatography-Electrospray Ionisation Ion-Trap Mass Spectrometry (LC-eSI-IT MS), of individual biologically active proteins was used to identify peptides which matched to 17 proteins that belong to three groups of fungal proteins including virulence-related proteins; fungal growth and development proteins; and those with unknown function (hypothetical proteins). However, Ptt toxins were not detected in the ICWF protein profiles suggesting that Ptt toxins were either in trace amounts or might be internalised into the cell. The four VRCPs selected, were identified as hypothetical proteins with unknown function in the Ptt database. Further bioinformatic analysis characterised these VRCPs as an isochorismatase (PttCHFP1), an endo-1, 4-β- xylanase A (PttXyn11A), a glycophosphatidylinositol (GPI)-anchored common in fungal extracellular membrane (CFEM) domain-containing protein (PttGPICFEM) and an unknown proteinaceous secreted (but conserved) hypothetical protein (PttSP1). These VRCPs were heterologously expressed and characterised using gene expression studies. PttXyn11A had strong homology with the well characterised endoxylanases, TrXyn11A from Trichoderma reesei and BcXyn11A from Botrytis cinerea, known to contribute to virulence. A necrosis-inducing region on the surface of the enzyme was also identified in PttXyn11A, suggesting a potential role in necrosis induction. The culture filtrates for more virulent isolates had significantly greater xylanase activity than those from less virulent isolates. Even though heterologously expressed PttXyn11A was toxic to Escherichia coli, xylanase activity was detectable at very low levels and was not enough to cause symptoms in the bioassay. In addition, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and RT-quantitative PCR (RT-qPCR) analysis demonstrated that PttXyn11A was expressed more abundantly by the more virulent isolates compared with the other isolates in culture and during the plant-pathogen interaction. Together, these results suggest that PttXyn11A plays a role in virulence, either through its ability to degrade the plant cell wall to assist fungal growth or through its necrosisinducing ability. PttCHFP1 showed homology to an isochorismatase, an enzyme that has been proposed to have a role in plant defence via inhibition of salicylic acid production. PttSP1 showed homology to a membrane lipoprotein proposed to have a role in fungal development. Bioassay of recombinant PttCHFP1 and PttSP1 induced chlorosis symptoms in the susceptible barley cultivar (Sloop). The cysteine-rich CFEM domain identified in PttGPI-CFEM has been suggested to have an important role in hyphal attachment and fungal networking. However, E. coli was not able to express this gene probably due to its attachment to the plasma membrane and/or cell wall. Analysis of the gene expression profiles for PttCHFP1, PttGPI-CFEM and PttSP1 showed no significant differences between isolates in vitro and in planta suggesting that all isolates regulated the expression of these genes to the essential level possibly required for pathogenesis. This is the first study to identify the relationship between fungal growth and proteinaceous toxin production, characterise individual proteinaceous toxins in the mixture of Ptt culture filtrate and investigate the expression profiles of genes encoding VRCPs during the Ptt-barley interaction. This study therefore provides a better understanding of the Ptt-barley interaction by identifying the potential toxins which might lead to identify the toxin targets and ultimately support the breeding of resistant cultivars of barley.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013

Modification of the barley grain endosperm in germination is fundamental to successful plant growth but also has important ramifications for down-stream industrial uses of barley, particularly in the malting and brewing industries. There are a battery of enzymes that are involved in the modification process but the sites of synthesis and action of only of a few of these have been described in detail and most have only been studied in isolated tissues. The development of a sensitive and robust in situ mRNA hybridization (ISH) procedure, which allows the detection of specific transcripts representing proteins in grain sections is described here. This first required the optimization of grain fixation, embedding and sectioning procedures as well as the adaptation of a staining method using calcofluor white to accurately assess the modification state of each grain prior to processing. Once these technical parameters were established, the non-radioactive ISH protocol was developed to allow the detection of transcripts of (1,3;1,4)-β-glucanases, (1→4)-β-endo-xylanases, limit dextrinase and limit dextrinase inhibitor in grain sections of the two barley cultivars Sloop and Himalaya. The panel of enzymes selected for study covers several aspects of grain modification, such as (1,3;1,4)-β-glucan, xylan and starch breakdown, which are all important for successful grain germination. The successful ISH technique proved sensitive enough to discriminate between the (1,3;1,4)-β-glucanase and xylanase isoenzymes and clearly defined whether the transcipts for these enzymes were synthesized in both a tissue-specific manner and a fixed temporal sequence during grain germination. The use of monoclonal antibodies specific for the two (1,3;1,4)-β-glucanase isoforms in a related immunolabelling procedure, using the same fixed grains, also allowed the patterns of transcript and protein appearance to be correlated. As expected, use of the ISH method showed that the transcripts of the (1,3;1,4)-β-glucanase, xylanase and limit dextrinase inhibitor genes are variously found in the aleurone cells, the starchy endosperm tissue and the scutellum. However, there were also substantial amounts of transcript detected in the tissues of the growing embryo which suggests that these enzymes may also contribute substantially to early seedling development.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2018