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.

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-IA5

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-glucanasc production which maximized atoptimal temperature of 37℃ and decreased significantly when temperature was over 37℃ . Charge quantity affected β-glucanasc production significantly. Adding oxygen vector N-dodecanc or acetic ether benefited β-glucanasc production, but it depended on the concentration and charge quantity. The results of fractional factorialdesign showed that age and size of inoculum and shaking speed were the key factors affecting β-glueanase production and the cultivation time span to reach the highest β-glucanasc activity. The optimal cultural conditionsfor p-glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82% (v/v)),shaking speed 210 r/rain, 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 p-glucanase, a-amylase and neutral protease produced by B subtills ZJF-1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased signifi-cantly 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.

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

β-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-Sepha-dex A-50 chromatography, rIlae molecular mass was determined to be 35.21 kilodahons 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-glueanase was relatively stable at below 40° for 60min. The Km of the enzyme on β-glucan was 10.86 mg/ml, and the Vmax on β-glucanwas 14286 pmol of glucose equivalents per nag of the pure enzyme per rain. 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 5mmol/L and 10mmol/L, respectively. The β-glucanase activity was stimulated by Co^2+ ions, Ca^2 + ions,Zn^2+ ions, and Fe^2+ ions at 1mmol/L and 5mmol/L, respectively.

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 fermenta-tion 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 NaCl 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 α-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.

Effects of nitrogen ion irradiation on endoglucanase activity and gene mutation of Bacillus subtilis Bac01

Bacillus subtilis Bac01 was mutated by 15 keV N+ ions of 1.5×1016 cm-2. The mutant strain Bac11 with high yield of endoglucanase was isolated using carboxymethylcellulose sodium and congo red indicative plates. It exhibited higher endoglucanase activitiy (381.89IU) than the original strain Bac01 (93.33IU). Two 1,500 bp endoglucanase gene fragments were obtained with PCR amplification from B. subtilis Bac01 and mutant strain Bac11. BLAST comparison result indicated that 10 nucleotides mutated. Bioinformatics methods were used to analyze the two predicted amino acid sequences, and it was found that 5 amino acid residues changed, being all in the cellulose-binding domain of endoglucanase.

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, EGs1 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 °C for the wild-type enzyme to Tm=65.5 °C for the mutant EGs1 and 67.5 °C 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.

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.

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.

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.

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.

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°C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30°C 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.

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.

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.

哈茨木霉β-葡聚糖酶诱导、纯化及对黄瓜幼苗的促生防病作用

在实验室条件下分别以β-葡聚糖、麦麸和黄瓜枯萎病菌(Fusarium oxysporum)细胞壁作为唯一碳源诱导哈茨木霉菌(Trichoderma harzianum)Th-30发酵产生β-葡聚糖酶,采用硫酸铵盐析和琼脂糖凝胶色谱柱层析法对β-葡聚糖酶进行分离纯化,以津研4号黄瓜为试验材料,探究β-葡聚糖酶对黄瓜幼苗抗性生理指标、形态指标的影响及黄瓜常见土传病害的防控作用。结果表明:不同诱导条件下木霉Th-30发酵产β-葡聚糖酶活性差异显著,经β-葡聚糖诱导发酵的粗酶液酶活性最高,发酵72 h时达75.63 U·mL^(-1);纯化的β-葡聚糖酶比活力为783.56 U·mg^(-1),是粗酶液的45.32倍;5倍液和10倍液β-葡聚糖酶可显著提高黄瓜幼苗的根系活力、游离脯氨酸含量、苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)活性以及根冠比和壮苗指数;β-葡聚糖酶10倍液处理对黄瓜立枯病、黄瓜枯萎病、黄瓜疫病、黄瓜猝倒病、黄瓜菌核病的防效为50.36%~80.63%。

哈茨木霉UN⁃2β⁃葡聚糖酶诱导及对水稻纹枯病的抑菌防病作用

【目的】优化哈茨木霉UN⁃2菌株产β⁃葡聚糖酶的培养基组分及发酵参数,提高其产酶能力,研究β⁃葡聚糖酶粗酶液对水稻纹枯病病原菌的拮抗作用及田间防效,阐明哈茨木霉菌株UN⁃2在水稻纹枯病生物防治中的应用潜力。【方法】采用单因素试验考察不同碳源、氮源和金属离子对哈茨木霉菌株UN⁃2产β⁃葡聚糖酶的影响,利用正交试验确定木霉菌株产β⁃葡聚糖酶的最适温度、pH、接种量、瓶装量、摇床转速和发酵时间,优化哈茨木霉菌株UN⁃2产β⁃葡聚糖酶诱导发酵条件;通过体外拮抗试验和田间防效试验研究β⁃葡聚糖酶粗酶液对水稻纹枯病的抑菌防病作用。【结果】哈茨木霉菌株UN⁃2发酵产β⁃葡聚糖酶最佳碳源和氮源分别为麦麸和硫酸铵,金属离子Ca^(2+)和Mg^(2+)对木霉产β⁃葡聚糖酶活性具有明显的促进作用。哈茨木霉UN⁃2菌株以10.0 g/L麦麸、0.5 g/Lβ⁃葡聚糖、4.0 g/L硫酸铵、1.5 mmol/L Ca^(2+)、0.5 mmol/L Mg^(2+)为培养基,在温度32℃、起始pH 6.5、接种量8 mL(10^(6) cfu/mL)、瓶装量30 mL/250 mL、摇床转速160 r/min条件下发酵64 h获得β⁃葡聚糖酶活性最高。木霉菌株UN⁃2产β⁃葡聚糖酶粗酶液对立枯丝核菌和禾谷丝核菌的菌丝生长和菌核萌发具有明显的抑制作用,对由立枯丝核菌引起的水稻纹枯病的田间防效达70.45%,与5%井冈霉素处理相当,β⁃葡聚糖酶粗酶液处理可提高水稻结实率和稻粒充实度。【结论】哈茨木霉菌株UN⁃2在最优发酵条件下产酶活性达97.68 U/mL,β⁃葡聚糖酶粗酶液对水稻纹枯病具有较强的抑菌防病效果,并对水稻植株具有一定的促生作用。

黑曲霉耐热型葡聚糖酶纤维素结合结构域的替换与融合体酶性质

该文旨在通过更换纤维素结合结构域(cellulose binding domain, CBD)的方式探索获得具有纤维素水解能力的耐热型葡聚糖酶的方法。黑曲霉(Aspergillus niger)来源的葡聚糖酶AnEglA6具有优越的耐热性能,在禽类饲料生产中具有应用前景。通过更换不同家族来源的碳水化合物结合结构域(carbohydrate binding module, CBM)编码区,构建了融合体葡聚糖酶基因,分别在巴斯德毕赤酵母(Pichia pastoris)中进行异源表达。AnEglA6催化结构域与分别来源于嗜热菌(Caldicellulosiruptor kristjanssonii)和荧光假单胞菌(Pseudomonas fluorescens)的木聚糖酶的底物结合结构域CBM9、CBM10结合获得的融合体酶AnEg-CBM9和AnEg-CBM10均具有较高的纤维素水解能力,并且底物范围进一步扩大,对微晶纤维素也具有一定的水解能力。AnEg-CBM10热稳定性有显著的提高,在85℃下酶活力半衰期达到40 min左右,能短时耐受90℃高温。融合体酶AnEg-CBM10是一种热稳定性提高、具有纤维素水解能力的新型葡聚糖酶。