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.

Cloning of Endo-β-Glucanase Ⅲ and Expression in Eerevisiae Fermentum

Objective The aim was to construct bioengineering strains that could degrade the cellulosic solid waste. Method The cDNA of endo-β-glucanase III of Trichoderma vi ride AS313711 was cloned by RT-PCR method. After sequenced, this gene was constructed to expression vector pESP-2, and then the plasmid was transformed into competent cell of cerevisiae fermentum by electric shock, the transformant was then obtained. The enzyme activity of this transformant at the different temperatures and pH was measured by DNS method. Result The length of ORF of EG III was 1 257 bp, encoding 418 amino acids, while the deduced molecular weight was 44.1 × 103 kD. Conclusion The enzyme activity of EG III was the highest when it was at PH 4.9 and tempeture was of 60℃. Then the corresponding enzyme activity was about 100%.

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.

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 characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp. AN545 from Antarctica

A bacterium hydrolyzing carboxymethylcellulose, isolated from Antarctic sea ice, was identified as Pseudoalteromonas sp. based on 16S rDNA gene sequences and named as Pseudoalteromonas sp. AN545. The extracellular endo-1,4-β-glucanase AN-1 was purified successively by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis. AN-1 could hydrolyze caboxymethylcellulose, avicel and β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30℃ and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30℃ for 1 h. Moreover, the specific activity was enhanced by Ca2＋ and Mg2., and inhibited by Cu2＋. The kinetic parameters Michaelis constant （Km） and maximum velocity （Vmax） of AN-1 were 3.96 mg/mL and 6.06×10-2 mg/（min.mL）, respectively.

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.

Enzymatic Transformation of Notoginsenoside Fe by β-Glucanase

Aim An industrial enzyme β-glucanase was used to transfortn notoginsenoside Fe for the first time. Methods Notoginsenoside Fe was isolated from the leave saponin of Panax notoginseng （Burk.） Chen FH. The enzymatically transformed compounds were detected by HPLC and two transformed compounds were identified as 20 （S） -protopanaxadiol-20- O- α-L-arabinofuranosyl （ 1→6 ） - β-gluco- pyranoside, ginsenoside-Mc） and 20（S）-protopanaxadiol-20-O-β-D-glucopyranoside compound-K （C-K） respectively on the basis of their ^1H NMR and ^13 C NMR spectral data. Results Based on the enzymolytic kinetic curve, the transformation rate of notoginsenoside Fe reached 95% after 24 h. Conclusion The enzymatic transformation pathway of notoginsenoside Fe by β-glucanase has been proposed as notoginsenoside Fe→ginsenoside Mc→C-K.

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Enhanced Production of Hybrid Extracellular β-Glucanase by Re-combinant Escherichia coli Using Experimental Design Method

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid ex-tracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those ob-tained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.

Optimization of cultural conditions for thermostable β-1,3-1,4-glucanase production by Bacillus subtilis ZJF-1A5

The optimization of cultural conditions for β glucanase production by Bacillus subtilis ZJF 1A5 was investigated in flask trials. Temperature had great effect on β glucanase production which maximized at optimal temperature of 37℃ and decreased significantly when temperature was over 37℃.Charge quantity affected β glucanase production significantly. Adding oxygen vector N dodecane or acetic ether benefited β glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β glucanase production and the cultivation time span to reach the highest β glucanase activity. The optimal cultural conditions for β glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted by a second order polynomial model. The amount of β glucanase, α amylase and neutral protease produced by B subtilis ZJF 1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.

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.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems

The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.

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 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.

Syntheses and Olfactory Characters of Some Endo-α-Substituted-Bicyclo[2. 2. 2]-Octenyl (or Octanyl) Methanols

Sixteen title compounds were synthesized, twelve of which are new ones. Their structures were determined by 1H NMR, IR and MS, the refractive indices or melting points were measured. Odors of all the title compounds were evaluated and the structure-odor relationship was briefly discussed.

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.