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.

Characterization of dual enzyme resulted from bicistronic expression of two β-glucanases in porcine cells

Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four artificial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only p Bg A and p Egx showed high activity in transfected pig kidney cells. To improve the p H range and p H stability of β-glucanase, the two β-glucanases, p Bg A and p Egx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of p Bg A3 p Eg and p Bg2 Ap Eg showed significantly enlarged p H range and significantly increased p H stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.

Purification and Some Properties of Endo-1,4-β-Glucanases of Trichoderma harzianum UzCF-28

This work aimed at isolation, purification and study of biochemical features of cellulolytic enzymes synthesized by Trichoderma harzianum UzCF-28 strain. Strain UzCF-28 revealed a high cellulolytic activity during submerged cultivation in the liquid culture on modified Mandels nutrient medium, where wheat straw was used as a source of carbon. As a result of purification by precipitation with ammonium sulfate and further ion exchange chromatography, two isoforms of endo- 1,4-β-glucanase-EG II and EG III with molecular weight of 135 and 75 kDa respectively were revealed. The pH optimum for EG I and EG III was 4.5, while for EG II—4.7, irrespective of the applied substrates—either CMC or “Whatman filter” paper. Heating up to 40°C of EG III did not lead to its inactivation, and on the contrary, its activity increased by more than three times comparing to the initial activity of the enzyme, i.e. thermostability of EG III among tested enzymes significantly varied.

The plasmodesmata-associated β-1,3-glucanase gene GhPdBG regulates fiber development in cotton

Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.

Transformation of Compound K from Saponins in Leaves of Panax notoginseng by Immobilized β-Glucanase

Objective To prepare an active anti-tumor component,compound K(C-K),from saponins in leaves of Panax notoginseng(SLPN) using immobilized β-glucanase.Methods Two entrapments,alginate gel-1(Alg 1) and alginate gel-2(Alg 2),were evaluated for their ability to immobilize β-glucanase.The amount and purity of C-K obtained from the transformation process were analyzed by HPLC,and the immobilizing parameters were optimized.Results β-Glucanase can be immobilized and reused with either of the entrapment.However,using Alg 1 resulted in higher enzyme activity than Alg 2.The optimal concentration of the immobilized enzyme was 10%;The optimal crosslinking time was 4–6 h;and the optimal concentration of the crosslinking agent was 6%– 7%.Conclusion Immobilized β-glucanase shows sustained enzyme activity,good ethanol tolerance,and was reusable for the preparation of C-K from SLPN.

PtrCel9A6, an Endo-l,4-β-Glucanase, Is Required for Cell Wall Formation during Xylem Differentiation in Populus

ABSTRACT Endo-l,4-β-glucanases （EGases） are involved in many aspects of plant growth. Our previous study found that an EGase, PtrCel9A6, is specifically expressed in differentiating xylem cells during Populus secondary growth. In this study, the xylem-specific PtrCel9A6 was characterized for its role in xylem differentiation. The EGase is localized on the plasma membrane with catalytic domain toward the outside cell wall, hydrolyzing amorphous cellulose. Suppression of PtrCel9A6 expression caused secondary cell wall defects in xylem cells and significant cellulose reduction in Populus. Heterologous expression of PtrCelgA6 in Arabidopsis enhanced plant growth as well as increased fiber cell length. In addition, introduction of PtrCel9A6 into Arabidopsis resulted in male sterility due to defects in anther dehiscence. Together, these results demonstrate that PtrCel9A6 plays a critical role in remodeling the 1,4-β-glucan chains in the wall matrix and is required for cell wall thickening during Populus xylem differentiation.

Cultivar and Environmental Effects on p-glucanase Activity in Both Barley Grain and Malt and Its Function in β-glucan Degradation

Eight two-rowed barley cultivars were grown at seven locations in the southern winter-barley zone of China. Mean grainβ-glucanase activity ranged from 39.89 U kg-1 for Suyin2 to 49.75 U kg-1 for Xi-umai3 in 8 cultivars grown at 7 locations, and from 38. 74 U kg-1 in Zhengzhou to 57. 96 U kg-1 in Putian among 7 locations on an average of all cultivars. Correspondingly, mean malt β-glucanase activity of 8 cultivars ranged from 313.33 U kg-1 for ZAU3 to 489. 89 U kg-1 for Daner Barley, and of 7 locations from 330.40 U kg-1 in Yancheng to 418. 24 U kg-1 in Putian. There were significant differences among cultivars and locations in maltβ-glucanase activities. The locations showed much larger variation in maltβ-glucanase activities than cultivars. The reduction of total β-glucan content after malting varied in both cultivars and locations, with a mean of 78.31%. The analysis of correlations showed that maltβ-glucan content was significantly positively and negatively correlated with grain β-glucan content and malt β-glucanase activity, respectively, and malt β-glucanase activity was significantly positively correlated with grain β-glucanase activity.

Expression of an Exo-β-glucanase Gene Is Specifically Related to Lily Pollen Germination and Pollen Tube Growth

The present study is expected to provide better understanding of how exo-β-glucanase acts on the biochemo-rheological property of the cell wall, and thus the regulation mechanism of pollen tube growth. A polymerase chain reaction （PCR）-based strategy and cDNA library screening were used to clone an exo-β-glucanase gene （LP-Exol） from Lilium Iongiflorum pollen tubes. The LP-Exol sequence showed a high level of homology to that of reported plant exo-β-glucan hydrolases. Phylogenetic analysis demonstrated that there was close relationship between LP-Exol and other exo-glucan hydrolases in Poacea plants. Northern analysis indicated that LP-Exol transcripts stored in pollen grains at low levels; LP-Exol transcription level enhanced at 2 h after the onset of pollen incubation and remained relatively high during pollen tube elongation. Moreover, the expression of LP-Exol was undetectable in petals, filaments, stigmas, styles, ovaries, leaves, and stems. These results suggest that LP-Exol gene is a late pollen gene, specifically contributing to the regulation of lily pollen germination and pollen tube growth.

Expression Vector Construction and Genetic Transformation of <i>Rosa rugosa β</i>-l,3-Glucanase Gene (<i>RrGlu</i>)

In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.

Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu)

In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.

Cloning and Expression of Extremely Heat-Resistant Endo-β-1,4-Glucanase Gene from Thermotoga maritima in Bacillus subtilis

Gene encoding endo-β-1,4-glucanase(TM1525)is derived from Thermotoga maritima(T.maritima),which has an open reading frame of 825 bp and encodes a 274 amino acid endo-β-1,4-glucanase.This enzyme has the same high temperature resistance as thermophilic bacteria,which is an ideal property for industrial applications.By molecular biological means,TM1525 was cloned into pHT43 vector and introduced into Bacillus subtilis(B.subtilis)WB800N by electroporation.The results showed that the WB800N expression system was successfully constructed,and extracellular expression of the recombinant gene was achieved.Cellulose hydrolyzed activity of the protein was exhibited.

Antifungal Potential of Beauveria bassiana on Solanum lycopersicum L. Infected with Fusarium oxysporum f. sp. lycopersici

The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymesβ-1,3-glucanases and chitinases.Inoculation of Bb 1205 occurred at a concentration of 1×108 conidia·mL−1,which was administered onto the leaves,directly into the soil and via injection.Infection with Fol 17108 occurred with 1×106 spores·mL−1,which were added directly to the soil.Spectrophotometry was used for measuring agronomic parameters,namely activity of chitinases andβ-1,3-glucanases in foliage and roots.When Bb 1205 was added to the soil,the chlorophyll index and aerial part length showed significant differences.In addition,it was determined that root length,fresh weight of foliage,flower,and fruit count increased 82 days after inoculation(dai).Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai(41.8 and 11.6-fold,respectively).Inoculation on the foliage showed a 10-fold increase ofβ-1,3-glucanases in the roots after 82 dpi.As for leaves,a 3.8-fold increase was found when the stem was inoculated.In the different in vivo applications,Bb 1205 activated its defenses by expressing the chitinase enzymes andβ-1,3-glucanase,thus reducing the damage caused by Fol 17108,demonstrating increase plant growth thereafter.

Improving the Heat Resistance ofβ-1,4 Glucanase by Introducing Disulfide Bonds

Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

ABSTRACT： Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation （SSF） as a po- tential feed additive. The results of the first trial showed that there were at least 5 non-starch polysac- charide enzymes： xylanase, 13-ghicanase, pectinase, mannase and carboxy methyl cellulase （CMCase） contained in the compound. Xylanase and fl-glucanase showed good activities at pH 2.5-7.0, which were in the range of 649-1046 U/g and 444-648 U/g, respec- tively. Pectinase showed good activity in acidic solu- tion （pH 2.5-3.0）,which ranged from 195 to 917 U/g. Mannase showed high activity of 235-298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0, which was in the range of 38.2-78.6 U/g. The second trial was aimed to test the stability of the enzymes in gastric liquor （pH 2.6） of finishing pigs and Na2 HPO4-gastric liquor （ pH 5.5 ）.After 6 h incubation at 40℃ in gastric liquor,the re- tained activity of xylanase, 13-glucanase, pectinase, mannase and CMCase was 26.3% ,65.0% ,71.0%, 74.8% and 85.6%, respectively. While after 6 h in- cubation at 40℃ in Na2I-IPO4-gastric liquor, the re- tained activity of xylanase, [3-glucanase, pectinase, mannase and CMCase was 87.9% ,91.1% ,92.3%, 95.0%, and 97.5%, respectively. The third trial was carried out in a jejunum liquor （ pH 5.8,200 mL）, which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran, respectively. After 8 h incubation at 40℃, 18.7% of soybean hull and 20.1% of wheat bran could be degraded to solu- ble saccharide, respectively. Compared with the tradi- tional methods for feed enzyme testing which involve feeding animals for 1-3 months, enzyme assay in this way was relatively convenient.

Steroidal Plant Growth Promoters vs. Phytopathogens, via Enzymatic Regulation;An in <i>Silico</i>Approach

Steroidal plant growth promoters (SPGP) have been continuously studied due to their high activity increasing biomass and resistance to diverse stress factors. In our hands, a new SPGP family of 22-oxocholestanic compounds stands out at a comparative level to brassinosteroids (BSs). The potential activity of new SPGP against phytopathogens was studied through in silico molecular docking, these assays were performed with relevant ensymes of phytopatogens Chitinase B and 1,3-β-Glucanase. Nine Chitinase B inhibitors and two 1,3-β-Glucanase inhibitors were proposed. The launched study analyzed the interactional and spatial level, determining the presence of interactions with key amino acids in receptors in comparison to reference inhibitors. Even more, the AVR4 and ECP6 effectors were also examined. No compound that blocks ECP6 was found;due to, probably, the influence of its highly hydrophilic environment. In the case of AVR4, two SPGP showed a better docking score (DS) than a chitin fragment (endogenous ligand);this fact demonstrates the latent potential of the 22-oxocholestanic derivatives against phytopathogens, with a specific regulation via proliferation inhibition. Moreover, this SPGP does not affect the symbiotic fungi that are beneficial for the natural plant system.

Production of β-1,3-glucanase and chitinase of two biocon-trol agents and their possible modes of action

Pichia membranefaciens Hansen and Candida guilliermondii (Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1, 3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level, being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole car-bon source. P. membranefaciens was able to produce signifi-cantly higher levels of chitinase (exochitinase and endochiti-nase) in vitro than C. guilliermondii grown in Czapeck mini-mal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources

ZmGns,a maize class I β-1,3-glucanase,is induced by biotic stresses and possesses strong antimicrobial activity

Plant b-1,3-glucanases are members of the pathogenesis-related protein 2（PR-2） family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins（spot 842） identified in a recent proteomic comparison between five pairs of closely related maize（Zea mays L.） lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.

Changes in Protein Content and Chitinase and β-1,3- glucanase Activities of Rice with Blast Resistance Induced by Ag-antibiotic 702

[Objective] The paper was to explore the effect of rice blast resistance induced by Ag-antibiotics 702 on protein content and chitlnase and β-1,3-glucanase activities in rice. [ Method] At the fourth leaf stage of Luliangyou 996, 15 μg/mL Ag-antibiotic 702 was sprayed, while Validamycin and distilled wa- ter were sprayed as positive control and negative control, respectively. All treatments were inoculated with spore fluid of Magnaporthe grisea at 48 h post spraying, and the rice inoculated with only distilled water was used as blank control. The enzymes activities （endochitinase, exochitinase and β-1,3-glucanase） and total pro- tein content in rice leaves were determined every 24 h within 168 h post spraying. [ Result] Compared with the blank control, the rice inoculated with spore fluid of M. grisea could significantly increase the total protein content and the activities of β-1,3-glucanase and chitinase. The induction effect of Ag-antibiotics 702 exceeded that of Validamycin treatment. And the changes in activities of β-1,3-glucanase and chitinase had obvious synchronicity. [ Conclusion] Ag-antibiotic 702 can significantly improve the total protein content and the activities of β-1,3-glucanase and chitinase, thus enhancing the resistance to rice blast.

Continuous Saccharification of Laminarin by Immobilized Laminarinase ULam111 Followed by Ethanol Fermentation with a Marine-Derived Yeast

We isolated a novel laminarinase ULam111 from Flavobacterium sp. strain UMI-01. Purified ULam111 showed degradation activity against laminarin with the specific activity of 224 ± 18 U/mg at 30°C and pH 6.0. Its optimum temperature was 50°C, and degradation activities against laminarin were observed at 4°C - 80°C. With a laminarin degradation system, we investigated the preparation and properties of immobilized ULam111 with the use of the 11 types of carriers. The high activity recoveries of immobilized ULam111 were as follows: 19.4% for IB-S60P carrier beads (the non-ionic type), 15.6% for IB-S60S carrier beads (the non-ionic type), 11.9% for IB-150P carrier beads (the covalent type), and 7.1% for IB-C435 carrier beads (the cationic type). With the repeated use of immobilized ULam111, the enzyme activities immobilized on IB-S60S and those on IB-S60P remained at 40% and 30% respectively after the sixth trial. We selected IB-S60S as suitable beads for enzyme immobilization, and we attempted to construct a reactor system with ULam111 immobilized on IB-S60S beads. In this system, 1.2 - 1.9 g/L glucose was repeatedly produced from 30 mg/mL laminarin solutions after 20 hr when the reactor operation was repeated 10 times. We examined ethanol fermentation from the saccharified solutions with a marine-derived yeast (Saccharomyces cerevisiae C-19), and 0.51 - 0.58 g/L bioethanol was produced from the saccharified solution that contained 1.71 - 1.86 g/L of glucose.

Induction of Volatile Organic Compounds in Leaves of Lycopersicon Esculentum by Nd^(3+)

The effects of Nd^3＋ on the quality and quantity of volatile organic compounds （VOCs） in the leaves of Lycopersicon esculentum were studied. The results demonstrate that Nd^3＋ can increase the total amount of VOC by 75% after treatment for 120 h, as compared with the control. Phyto-oxylipins, terpenoids and aromatic compounds were increased by 73%, 38% and 21%, respectively. （E）-2-hexenal, the most abundant constituent is increased by 74%, β- phellandrene and α-caryophyllene in terpenoids,