Xylanase improves the intestinal barrier function of Nile tilapia(Oreochromis niloticus)fed with soybean(Glycine max)meal

Background Soybean(Glycine max)meal is one of the important protein sources for fish,but the non-starch polysaccharides(NSP)in soybean meal impair the intestinal barrier function.Here we aimed to investigate whether xylanase can alleviate the adverse effects on the gut barrier induced by soybean meal in Nile tilapia and to explore the possible mechanism.Results Nile tilapia(Oreochromis niloticus)(4.09±0.02 g)were fed with two diets including SM(soybean meal)and SMC(soybean meal+3,000 U/kg xylanase)for 8 weeks.We characterized the effects of xylanase on the gut barrier,and the transcriptome analysis was performed to investigate the underlying mechanism.Dietary xylanase improved intestinal morphology and decreased the concentration of lipopolysaccharide(LPS)in serum.The results of transcriptome and Western blotting showed that dietary xylanase up-regulated the expression level of mucin2(MUC2)which may be related to the inhibition of protein kinase RNA-like endoplasmic reticulum kinase(perk)/activating transcription factor 4(atf4)signaling pathways.Microbiome analysis showed that addition of xylanase in soybean meal altered the intestinal microbiota composition and increased the concentration of butyric acid in the gut.Notably,dietary sodium butyrate was supplemented into the soybean meal diet to feed Nile tilapia,and the data verified that sodium butyrate mirrored the beneficial effects of xylanase.Conclusions Collectively,supplementation of xylanase in soybean meal altered the intestinal microbiota composition and increased the content of butyric acid which can repress the perk/atf4 signaling pathway and increase the expression of muc2 to enhance the gut barrier function of Nile tilapia.The present study reveals the mechanism by which xylanase improves the intestinal barrier,and it also provides a theoretical basis for the application of xylanase in aquaculture.

黄色瘤胃球菌木聚糖酶基因xynB的异源表达与酶学性质研究

该试验以黄色瘤胃球菌基因组为模板,通过PCR扩增获得目的基因xynB,将xynB与表达载体PET28a连接,获得重组载体PET28a-xynB。用IPTG对含有重组载体的大肠杆菌BL21(DE3)进行诱导表达,镍离子层析柱纯化后检测其酶学性质。生物信息学分析表明,XynB理论大小为47 kDa,预测等电点为4.49,不含信号肽,含有1个糖苷水解酶11家族结构域和1个碳水化合物结合结构域。酶学性质研究结果表明,该酶的最适pH值为5.0,最适反应温度为40℃,金属离子Mg^(2+)、Na^(+)、K^(+)和Ba^(2+)对木聚糖酶XynB有较好的激活效果。综上可知,黄色瘤胃球菌木聚糖酶XynB属于GH11家族,最适pH值为5.0,最适温度为40℃,酶比活力为62.94 U/mg。

Construction and Identification of Intestine-specific Expression Vector of Xylanase Gene(XynB)

The Aspergillus niger XynB gene and core promoter region of porcine RELMβgene were cloned into pcDNA3.1(-),and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc double tags was constructed.The vector was transfected into human colon cancer cells(HT29)and human liver cancer cells(Bel7402)using liposomes.Fluorescence microscopy revealed that the vector could specifically express green fluorescent protein(GFP)in HT29 cells.RT-PCR and Western Blot were performed on the HT29 cells transfected with the expression vector,and the results showed that the XynB gene was normally transcribed in HT29 cells,and the target protein expression was detected in the cells.

Studies on Mutation Breeding of High-Yielding Xylanase Strains by Low-Energy Ion Beam Implantation

As a new mutagenetic method, low-energy ion implantation has been used widely in many research areas in recent years. In order to obtain some industrial strains with high xylanase yield, the wild type strain Aspergillus niger A3 was mutated by means of nitrogen ions implantation （10 keV, 2.6× 10^14 ～ 1.56 × 10^15 ions/cm^2） and a mutant N212 was isolated subsequently. However, it was found that the initial screening means of the high-yielding xylanase strains such as transparent halos was unfit for first screening. Compared with that of the wild type strain, xylanase production of the mutant N212 was increased from 320 IU/ml to 610 IU/ml, and the optimum fermentation temperature was increased from 28 ℃ to 30 ℃.

Effects of cellulase and xylanase enzymes mixed with increasing doses of Salix babylonica extract on in vitro rumen gas production kinetics of a mixture of corn silage with concentrate

An in vitro gas production （GP） technique was used to investigate the effects of combining different doses of Salix babylonica extract （SB） with exogenous fibrolytic enzymes （EZ） based on xylanase （X） and cellulase （C）, or their mixture （XC; 1：1 v/v） on in vitro fermentation characteristics of a total mixed ration of corn silage and concentrate mixture （50：50, w/w） as substrate. Four levels of SB （0, 0.6, 1.2 and 1.8 mL g-1 dry matter （DM）） and four supplemental styles of EZ （1 μL g-1 DM; control （no enzymes）, X, C and XC （1：1, v/v） were used in a 4×4 factorial arrangement. In vitro GP （mL g-1 DM） were recorded at 2, 4, 6, 8, 10, 12, 24, 36, 48 and 72 h of incubation. After 72 h, the incubation process was stopped and supernatant pH was determined, and then filtered to determine dry matter degradability （DMD）. Fermentation parameters, such as the 24 h gas yield （GY24）, in vitro organic matter digestibility （OMD）, metabolizable energy （ME）, short chain fatty acid concentrations （SCFA）, and microbial crude protein production （MCP） were also estimated. Results indicated that there was a SBxEZ interaction （P〈0.0001） for the asymptotic gas production （b）, the rate of gas production （c）, GP from 6 to 72 h, GP2 （P=0.0095）, and GP4 （P=0.02）. The SB and different combination of enzymes supplementation influenced （P〈0.001） in vitro GP parameters after 12 h of incubation; the highest doses of SB （i.e., 1.8 mL g-1 DM）, in the absence of any EZ, quadratically increased （P〈0.05） the initial delay before GP begins （L） and GP at different incubation times, with lowering b （quadratic effect, P〈0.0001 ） and c （quadratic effect, P〈0.0001 ; linear effect, P=0.0018）. The GP was the lowest （P〈0.05） when the highest SB level was combined with cellulose. There were SBxEZ interactions （P〈0.001） for OMD, ME, the partitioning factor at 72 h of incubation （PF72）, GY24, SCFA, MCP （P=0.0143）, and pH （P=0.0008）. The OMD, ME, GY24 and SCFA with supplementation of SB extract at 1.8 mL g-1 DM were higher （P〈0.001） than the other treatments, however,PF72 was lower （quadratic effect, P=0.0194） than the other levels. Both C and X had no effect （P〉0.05） on OMD, pH, ME, GY24, SCFA and MP. The combination of SB with EZ increased （P〈0.001） OMD, ME, SCFA, PFz2 and GP24, whereas there was no impact on pH. It could be concluded that addition of SB extract, C, and X effectively improved the in vitro rumen fermentation, and the combination of enzyme with SB extract at the level of 1.2 mL g-1 was more effective than the other treatments.

Mutation-Screening in Xylanase-Producing Strains by Ion Implantation

With ion implantation (N+, energy 10 keV and dosage 1.56×1015 N+cm-2), a high xylanase-producing strain Aspergillus niger N212 was selected. Based on an orthogonal experiment, an optimal fermentation condition was designed for this high-yield strain. The suitable medium was composed of 8% corncob; 1.0% wheat bran; 0.1%TWEEN20; 0.5% (NH4)2SO4; 0.5%NaNO3; 0.5%FeSO4, 7.5 × 10-4; MnSO4·H2O, 2.5 × 10-4; ZnSO4, 2.0 × 10-4; CoCl2, 3.0 × 10-4. At present, under our experiment condition, xylanase activity of Aspergillus niger N212 reached a level of 600 IU/ml, almost increased by 100% in xylanase production and the time of yielding xylanase was largely reduced to 12 h at 28℃.

Thermostable ethanol tolerant xylanase from a cold-adapted marine species Acinetobacter johnsonii

A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

To optimize culture conditions for xylanase production by solid state fermentation （SSF） using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K2HPO4, 2.5 g/L; KH2PO4, 1.0 g/L; NaCl, 0.1 g/L; （NH4）2SO4, 2.0 g/L, CaCl2-2H2O, 0.005 g/L; MgCl2.6H2O, 0.005 g/L; and FeCI3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity （142.0 ±0.47 U/g DM] was obtained on the 6th day at 30℃ The optimized paddy husk to liquid fermentation medium ratio was 2：9, and the optimized culture temperature was 40℃. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized （224.2 U/g DM） in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production （290.7 U/g DM） was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum （326.5±0.34 U/g DM）. Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Expression and Characterization of a Thermostable Xylanase Gene xynA from a Themophilic Fungus in Pichia pastoris

The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, which coded 194 amino acids. The putative amino acid sequence and DNA sequence of xylanase from T. lanuginosus SY2 （GenBank no.： GU166389） were 98.97 and 99.49% identical to the other T. lanuginosus （GenBank no.： U35436）. A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. The recombinant strain was identified by G418 selection and confirmed by PCR analysis. It was induced by 1.0% methanol at 28°C to express the recombinant xylanase. The results showed that the recombinant xylanase was secreted into extracellular fermentation liquid. The highest enzyme activity of 113.5 IU mL-1 and protein content of 889.7 μg mL-1 were detected for 216 h of induction. The optimal pH value and temperature of the enzyme activity was 5.5 and 65°C, respectively. The xylanase activity retained above 80% from pH value 2.5 to 8.5 for 48 h. The enzyme activity was above 85% at incubation temperature of 55°C.

In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation

Background： The filamentous fungus Talaromyces versatifis is known to improve the metabolizable energy of wheat- based poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-β（1,4）-xylanases. In order to appreciate their in vivo mode of action, the supplementation effect of two of its xylanases, XynD and XynB from families GH10 and GHll respectively, have been evaluated on two different wheat cultivars Caphorn and Isengrain, which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition. Results： Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1, to mimic monogastric metabolism. Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn. For both cultivars, XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates, which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates. Conclusions： The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn, suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy.

Optimization of Fermentation Conditions for Xylanase Production by Aspergillus niger NS-1

In this study, a xylanase-produeing Aspergillus niger strain, NS-1, was screened and isolated from agricultural and forestry wastes. Based on single-fac- tor experiments, the effects of different carbon sources, composite carbon sources, nitrogen sources, calcium carbonate concentrations, initial pH and surfactants on xylanase production by A. niger NS-1 were investigated. The results indicated that the most appropriate carbon source was corncobs ; the best composite carbon source was corncobs ＋ xylan, which was conducive to xylanase secretion; the most suitable nitrogen source was ammonium sulfate. Xylanase activity reached the highest in the medium added with 1.5% calcium carbonate and SDS as a surfactant with an initial pH of 5.0. This study provided the basis for the production of high-activity xylanase.

Effects of Exogenous NSP Enzymes(Xylanase, β-glucanase and Cellulase) on Morphology and Functions of Digestive Tract in Growing Pigs Fed with Paddy-Based Diets

Ninety Landrace×Jia 35±0. 40 kg weight growing pigs were randomly allotted to three treatments , each of which was replicated three times with ten pigs per replicate. The pigs were reared on either a conventional corn-based diet (control I ) or a paddy-based diet (control I ) or a paddy diet supplemented with 0.2% NSP enzymes (test group). All pigs were given ad libitum access to both feed and water. The results of feeding trial showed that supplementation of NSP enzymes significantly increased ADG by 8.78% (P< 0.05) and decreased F/G by 9. 42% (P<0. 05) over the control group Ⅱ. No significant differences were found in ADG and F/G between control group I and the test group. The digestive trial showed that adding NSP enzymes significantly improved apparent digestibility of CP, EE and CF by 18. 76 (P<0. 01), 16.04 (P <0.05) and 108. 57%(P<0. 05), respectively, compared to control Ⅱ. The activities of proteolytic enzyme and α-amylase in duodenal contents were increased by 99. 07 (P<0. 01) and 18. 41% (P<0. 05) with the addition of NSP enzymes. No significant differences between test and control Ⅱ group were found in activities of the pepsin in the gastric content, the trypsin and lipase in duodenal contents. the disaccharidase and y-glutany transferase (γ-GT) in intestinal mucosa, but there was a tendency towards higher activities associated with the NSP enzymes diet(P>0. 05). The lengths of the villi within the duodenal, jejunal and ileal sections of the small intestine of pigs receiving the NSP enzymes diet were increased by 23. 68 (P<0. 05), 56. 00 (P<0. 01) and 76. 90%(P<0. 01) respectively, relative to the pigs in controlⅡ.

Screening and Statistical Optimization of Physiochemical Parameters for the Production of Xylanases from Agro-Industrial Wastes

Xylanases are mostly produced through submerged fermentation;nonetheless solid-state fermentation has increased profound attention and consideration of scholars having high conversion level biomass to energy conservation. This study depicted the purification of xylanases and their possible utilization in industry. The present study was carried out to examine the culture influence of fungal strain Fomes fomentarius (F. fomentarius) using different agro-industrial residues (wheat straw, rice husk, sugarcane bagasse and siris pods). F. fomentarius showed maximum enzyme production after 72 h of fermentation, when grown on wheat straw in solid state fermentation process while maximum activity showed on pH 6.0 at 30°C. The other parameters optimized by statistical design (RSM) showed maximum xylanase activity (146 ± 8 IU/mL) at 65% moisture content, 4 mL inoculums size, 175 mg Ammonium sulphate, 200 mg Calcium carbonate and 1.4 grams of glucose. Xylanase was salted out at 60% ammonium sulphate concentration and enzyme was further purified by Sephadex G-100 gel filtration chromatography with 2.2 fold increase in activity. The purified xylanase from F. fomentarius had optimum pH 6.0 and 40°C. Xylanase showed higher specificity for oat spelt xylan with kinetic constants Km 1.25 mg/mL and Vmax 54 mM/min. Xylanases have an industrial important enzyme used extensively in food, feed and paper industry.

Immobilization of Commercial Cellulase and Xylanase by Different Methods Using Two Polymeric Supports

Industrial applications require enzymes highly stable and economically viable in terms of reusability. Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes. The immobilization of two commercial enzymes is reported here (cellulase and xylanase) using three chemical methods (adsorption, reticulation, and crosslinking-adsorption) and two polymeric supports (alginate-chitin and chitosan-chitin). The optimal pH for binding was 4.5 for cellulase and 5.0 for xylanase, and the optimal enzyme concentrations were 170 μg/mL and 127.5 μg/mL respectively, being the chitosan and the ideal support. In some cases, a low concentration of crosslinking agent (glutaraldehyde) improved stability of the immobilization process. Biotechnological characterization showed that the reusability of enzymes was the most striking finding, particularly of immobilized cellulase using glutaraldehyde, which after 19 cycles retained 64% activity. These results confirm the economic and biotechnical advantages of enzyme immobilization for a range of industrial applications.

Mutagenesis and Screening of High Yield Xylanase Production Strain of Aspergillus usamii by Microwave Irradiation

A high yield xylanase producing strain, A. usamii L336-23, was screened out from its parent strain A.usamii L336 after microwave irradiation. Its productivity of xylanase activity increased by 35.7% from 21000μ·m1^-1 to 28500μ·m1^-1 and was stable after frequent subcultures and storage for more than two months.The mechanism of microwave mutation was also discussed.

Production of Cellulase and Xylanase by Aspergillus terreus KJ829487 Using Cassava Peels as Subtrates

Cassava (Manihot esculenta, Crantz) is one of the most important food plants in West Africa. Its peels are made up of cellulose, hemicellulose and lignin. This lignocellulolytic biomass can be converted using microbial enzymes to fermentable sugars which have wide range of biotechnological relevance in many fermentation processes. The aim of this study is to screen filamentous fungi from decaying cassava peels that are good producers of xylanases and cellulases. Decaying parts of cassava peels were obtained and brought to the laboratory for further work. Fungi were isolated, identified and screened for cellulase and xylanase production. Isolate with highest frequency of occurrence and enzyme production was identified using phenotypic and molecular method. Optimisation of growth conditions for enzymes production was monitored using the DNSA method, also saccharification of cassava peel were carried out using the enzymes obtained from the isolate. Aspergillus terreus KJ829487 was the predominant fungus. It produces cellulases and xylanases optimally at 40°C, pH 6 and 8, utilising carboxymethylcellulose (CMC) or xylose and yeast extracts as its carbon and nitrogen sources respectively. Saccharification of the peels yielded 584 mg/L glucose, 78 mg/L xylose and 66 mg/L rhamnose. Aspergillus terreus KJ829487 obtained from cassava peels have the ability to produce high concentration cellulases and xylanases which effectively hydrolysed the lignocelluloses’ biomass to fermentable sugars.

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosaccharides with long chains from xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increase in the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19. The addition of D-glucose to the culture of the mutant strain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but not xylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharides with long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized. The hydrolyzates generated by the purified xylanase contained xylobiose, xylotriose, xylotetraose, and xylopentaose, but not xylose.

Effect of Xylanase on mRNA Expression of Cationic Amino Acid Transporters in Intestines of Broilers

480 healthy 1-day-old male yellow-feathered chickens were selected and assigned randomly into groups A and B,each having 6 pens with 40 birds per pen.The birds in group A were fed with wheatbased diet and group B with wheat-based diet supplemented with xylanase(1.2×l0~4 U/kg diet).On day 16,two birds per replication with average live weight were selected and sacrificed.Tissue samples of jejunum and ileum were collected to detect mRNA expression of cationic amino acid transporters using RT-PCR.The results showed that xylanase significantly increased the abundance of mRNA for rBAT and CAT4 in the intestines of broilers fed with wheat-based diets(P<0.05)and had a tendency to increase the mRNA expression of y^+LAT2 and CAT1 in jejunum(P>0.05),y^+LAT2,CAT1 and CAT4 in ileum(P>0.05).The treatment had no effect on the expression of rBAT mRNA in ileum(P>0.05).

Study on Screening and Cultivation Conditions of Xylanase-Producing Alkalophilic Bacterial

An xylanase producting alkalophilic Bacillus NT-9 was obtaind by the screening method of transparent zone on the selective medium, and the effects of carbon source and nitrogen source on xylanase production were studied. The medium composed of xylose 1.5%, (NH 4) 2SO 4 0.25%, K 2HPO 4 0.1%, MgSO 457H 2O 0.02%, with the initial pH of 10, was suggested to be optimal for the enzyme production in this study. When cultivatied at 37 ℃ for 72 h, the enzyme activity elaborated by the strain may reach as high as 10.5 U/mL. The xylanase produced by Bacillus NT-9 was a constituent enzyme.

Effects of Xylanase and Protease Supplementation on Nutrient Digestibility in Diets Based on Peas and Wheat Fed to Weaned Pigs

This experiment was conducted to determine the effects of a commercial enzyme mixture providing xylanase and protease activities on the digestibility of dietary nutrients when added to diets based on peas and wheat and fed to 9. 5 to 15 kg pigs. Pigs were weaned at 18 to 21 days of age and fitted with a simple T-cannula at the distal ileum,6 to 7 days after weaning. After 5 to 7 days for adaptation to their diets, ileal digesta, and feces were collected. Enzyme supplementation had no effect on the apparent ileal or fecal digestibility of any dietary nutrient. There was a numerical improvement （ P 〉 0.05 ） in the ileal digestibility of fiber. In conclusion, xylanase and protease appeared to be ineffective in improving nutrient digestibility when supplemented in diets based on peas and wheat fed to weaned pigs.