Investigation of the nutritional and functional roles of a combinational use of xylanase and β-glucanase on intestinal health and growth of nursery pigs

Background Xylanase andβ-glucanase combination(XG)hydrolyzes soluble non-starch polysaccharides that are anti-nutritional compounds.This study aimed to evaluate the effects of increasing levels of XG on intestinal health and growth performance of nursery pigs.Methods Forty pigs(6.5±0.4 kg)were assigned to 5 dietary treatments and fed for 35 d in 3 phases(11,9,and 15 d,respectively).Basal diets mainly included corn,soybean meal,and corn distiller's dried grains with solubles,contained phytase(750 FTU/kg),and were supplemented with 5 levels of XG at(1)0,(2)280 TXU/kg xylanase and 125 TGU/kgβ-glucanase,(3)560 and 250,(4)840 and 375,or(5)1,120 and 500,respectively.Growth performance was measured.On d 35,all pigs were euthanized and jejunal mucosa,jejunal digesta,jejunal tissues,and ileal digesta were collected to determine the effects of increasing XG levels and XG intake on intestinal health.Results Increasing XG intake tended to quadratically decrease(P=0.059)viscosity of jejunal digesta(min:1.74 m Pa·s at 751/335(TXU/TGU)/kg).Increasing levels of XG quadratically decreased(P<0.05)Prevotellaceae(min:0.6%at 630/281(TXU/TGU)/kg)in the jejunal mucosa.Increasing XG intake quadratically increased(P<0.05)Lactobacillaceae(max:40.3%at 608/271(TXU/TGU)/kg)in the jejunal mucosa.Increasing XG intake quadratically decreased(P<0.05)Helicobacteraceae(min:1.6%at 560/250(TXU/TGU)/kg)in the jejunal mucosa.Increasing levels of XG tended to linearly decrease(P=0.073)jejunal Ig G and tended to quadratically increase(P=0.085)jejunal villus height to crypt depth ratio(max:2.62 at 560/250(TXU/TGU)/kg).Increasing XG intake tended to linearly increase the apparent ileal digestibility of dry matter(P=0.087)and ether extract(P=0.065).Increasing XG intake linearly increased(P<0.05)average daily gain.Conclusions A combinational use of xylanase andβ-glucanase would hydrolyze the non-starch polysaccharides fractions,positively modulating the jejunal mucosa-associated microbiota.Increased intake of these enzyme combination possibly reduced digesta viscosity and humoral immune response in the jejunum resulting in improved intestinal structure,and ileal digestibility of nutrients,and finally improving growth of nursery pigs.The beneficial effects were maximized at a combination of 550 to 800 TXU/kg xylanase and 250 to 360 TGU/kgβ-glucanase.

Effects of Plant Extracts and Beauveria bassiana on the Activity of Defense-Related Enzymes in Solanum lycopersicum L.during Interaction with Fusarium oxysporum f.sp.lycopersici

The objective to this work was to evaluate the enzymatic activity in the culture of Solanum lycopersicum L.infected with Fusarium oxysporum after the combined application of Beauveria bassiana and plant extracts.Solanum lycopersicum plantlets were transplanted 15 days after the emergency.Five days after transplanting,Beauveria bassiana spores were applied at a concentration of 1×10^(7)spores mL^(−1)onto soil(along with A.indica(N)and P.auritum(H)leaf extracts)where S.lycopersicum plants were planted.Eight days after transplanting,spores of F.oxysporum strain were applied at a concentration of 1×10^(6)spores mL^(−1)to soil where S.lycopersicum plants were growing.The development of S.lycopersicum plants was monitored for 114 days,whereby a randomized complete block treatment design was used,the roots of the plants were crushed with liquid nitrogen,after which UV/VIS spectrophotometry was used to determine protein concentration,as well as the activity ofβ-1,3-glucanases,peroxidases(POX,EC1.11.1.7),catalase and chitinases.The treatments combining B.bassiana and A.indica and P.auritum extracts,had a significant difference in enzymatic activity levels,thus contributing to better defense mechanisms and a greater protection to S.lycopersicum plants in the presence of F.oxysporum.The application of B.bassiana and plant extracts to the ground mitigated damage caused by F.oxysporum on S.lycopersicum plants.

采用酵母双杂交法筛选PSRPK相关蛋白基因

本课题组在前期研究中,从多头绒泡菌中分离到一个SR蛋白激酶基因psrpk,并将其编码蛋白命名为PSRPK(SR protein kinase ofPhysarum Polycephalum).为分离PSRPK相关蛋白基因以了解PSRPK的功能,构建了转化率为2×106转化子/3μg pGADT7-Rec、密度为5.35×108cells/mL、滴度为2.34×109cfu/mL的多头绒泡菌酵母双杂交AD库.以PSRPK为饵蛋白筛选该文库得到接合率为41.18%的杂交酵母,在SD/-Leu/-Trp/-Ade/-H is培养板上筛选获得1476个杂交克隆,其中,X-gal滤纸显色呈强蓝色的克隆有342个.对大于500 bp的67个克隆测序获得35个cDNA片段,其中编码Plasm in C、branched-chain am inoacid am inotransferase(BCAT)类似蛋白、MSF1类似蛋白、m ixed-linked glucanase precursor类似蛋白、FCY1p类似蛋白和40S ribosomal protein S2类似蛋白等7个cDNA片段在阳性杂交酵母克隆中出现多次,其余仅出现一次.在35个cDNA片段的编码序列中有15个具有同源蛋白,31个编码序列的丝氨酸含量大于或等于6%,符合激酶底物的组成特征.

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Purification and some properties of a β-glucanase from a strain, Trichoderma reesei GXC

glucanase was purified from a solid\|state culture of \%Trichoderma reesei \%on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G\|100 chromatography, and DEAE\|Sephadex A\|50 chromatography. The molecular mass was determined to be 35.21 kilodaltons by sodium dodecyl sulfate\|12.5% polyacrylamide gel electrophoresis. The \%β\%\|glucanase at low pHs was more stable than that at high pHs, and optimum pH was 5.0. The optimum temperature was 60 ℃, and \%β\%\|glucanase was relatively stable at below 40 ℃ for 60 min. The \%K\%\-m of the enzyme on \%β\%\|glucan was 10.86 mg/ml, and the \%V\%\-\{max\} on \%β\%\|glucan was 14286 μmol of glucose equivalents per mg of the pure enzyme per min. The \%β\%\|glucanase activity was significantly inhibited by Fe\+\{3+\} ions, and was reduced in the presence of Cu\+\{2+\} ions, Mn\+\{2+\} ions and Mg\+\{2+\} ions at 5 mmol/L and 10 mmol/L, respectively. The \%β\%\|glucanase activity was stimulated by Co\+\{2+\} ions, Ca\+\{2+\} ions, Zn\+\{2+\} ions, and Fe\+\{2+\} ions at 1 mmol/L and 5 mmol/L, respectively.

Non-starch Polysaccharide Degrading Enzymes in Corn and Wheat-Based Broiler Diets： Dual Activity for Major Substrates

This study consolidates the efficacy of a non-starch polysaccharide （NSP）ase enzyme-concept in corn and wheat-based broiler diets. The NSP building blocks of corn, wheat and soybean meal （SBM, 44% protein） were studied first. Cereal NSP primarily consists of arabinose, xylose and glucose molecules that form arabinoxylan, β-glucan and cellulose polysaccharides. In soybean meal （SBM）, glucose （cellulose） predominates, next to galactose and uronic acids that form “pectins”. Broiler performance progress using in-feed NSPase enzymes depends on the concentration, complexity and solubility of the substrate and the genetic potential of the animal, mainly. A dual NSPase enzyme-concept for cereal and SBM NSP, predominantly being arabinoxylan, β-glucan and cellulose, was developed. Methods for measuring enzyme activities （endo-1,4-β-xylanase （EC 3.2.1.8） and endo-1,3（4）-β-glucanase （EC 3.2.1.6）） were developed （AVEVE Biochem UNITS） and preparations thereof （XG） standardized to meet requirements for most challenging NSP （corn, barely soluble）. Feed intake （FI）, bodyweight gain （BWG） and feed：gain ratio （F：G） were assessed in three zootechnical studies, each using 160 Ross 308 broilers split in two feeding groups with 80 birds/group （10 replicates of eight） for 42 d （starter/grower period）. Respective corn-SBM, wheat-SBM and corn/wheat-SBM diets were used as negative control （NC） or added with the enzyme-concept （XG）. In the total period, XG ameliorated BWG and F：G compared to NC in each study, where BWG increased best in diets with corn and F：G lowered most in diets with wheat. The dual NSPase enzyme-concept offers ingredient flexibility in present setting by enhancing the nutritional content of corn, wheat and SBM, expectedly from cleaving major NSP target molecules. Thereby, broiler professionals increase feed formulation liberty and safety and production result simultaneously.

Characterization of Exo 1, 4-<i>β</i>glucanase produced from <i>Tricoderma Viridi</i>MBL through solid-state bio-processing of orange peel waste

Agro-industrial residues are primarily composed of complex polysaccharides that strengthen the microbial growth for the production of industrially important enzymes like cellulases. In the present study we aimed to characterize the Exo 1, 4-β glucanase that was indigenously produced from Trichoderma viride MBL. T. viride MBL was cultured in the Solid-State medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 412 ± 12 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Exo 1, 4-β glucanase was 4.17-fold purified with specific activity of 642 U/mg in comparison to the crude extract. To confirm its purity and molecular weight, sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS- PAGE) was performed. The enzyme was shown to have a molecular weight of 60 kDa with an optimum pH and temperature of 5 and 50℃, respectively. Lineweaver-Burk reciprocal plot revealed that the kinetic constants Km and Vmax of purified Exo 1, 4-β glucanase were 76 μM and 240 U/mL.

Measurement of β-1,3 Glucanase Activity in Permeabilized Discs of Leaves of Healthy and Scald-Diseased Plants

Leaf discs of five cultivars of sugarcane exhibiting different degree of susceptibility to leaf scald were used to measure β-1,3 glucanase activity before and after experimental infection with Xanthomonas albilineans. Leaf discs were permeabilized with iso-propanol to facilitate the uptake of the enzyme substrate by intact tissues and to improve the enzyme assay. Bacterial infection significantly enhances β-1,3 glucanase activity of sensitive cultivars whereas significantly decreased that of the resistant one. Low concentrations of salicylate increase the hydrolase activity whereas jasmonic acid do not act as an elicitor of the enzyme and β-1,3 glucanase, such as laminarin, significantly inhibits the production of β-1,3 glucanase. Thus, the enzyme must be considered as a sensitivity factor induced by the pathogen.

Effects of dietary barley inclusion and glucanase supplementation on the production performance,egg quality and digestive functions in laying ducks

This study evaluated the effects of barley inclusion and glucanase supplementation on the productive performance and digestive function in laying ducks.The experiment used a randomized design with a 5×2 factorial arrangement of 5 graded levels of barley(0%,15%,30%,45%and 60%)with or without 1.5 g/kgβ-1,3-1,4-glucanase(15,000 U/kg).During the experimental period of 120 d,the weight and total number of eggs within each pen were recorded daily,and egg quality was determined every 4 wk.At the end of the experiment,3 randomly selected ducks within each replicate were sacrificed,then duodenal digesta and jejunal mucosa was collected.Dietary inclusion of barley had no effects on egg production,daily egg mass or FCR,but supplementation with glucanase improved egg production and FCR(P<0.01).Barley did not affect feed intake of laying ducks,but glucanase tended to increase feed intake(P=0.09).Neither barley norβ-glucanase had effects on the egg quality variables,except for yolk color score,which was decreased with increasing barley supplementation.Glucanase,but not barley,increased the activity of chymotrypsin and amylase in duodenal digesta.Barley inclusion affected the activity of alkaline phosphatase and maltase in jejunal mucosa(P<0.05),butβ-glucanase had no effects on the activity of these brush border enzymes.Barley inclusion increased the glucan content in duodenal digesta,but supplementation of glucanase to barley-based diet reduced digesta glucan content and reduced total volatile fatty acids and increased the proportion of acetic acid in cecal contents.The results indicate that,without glucanase,the optimal dietary barley level in the diets of laying ducks is about 13%for maximal production performance;glucanase supplementation of the barley diets improved production perfor-mance,probably through enhancing digestive function.