Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride

The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion.     

The Elicitation of Ethylene Biosynthesis by a Trichoderma Xylanase Is Not Related to the Cell Wall Degradation Activity of the Enzyme

A [beta]-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.     

The infection of oranges by Trichoderma viride and mixed infection by Trichoderma viride and Penicillium digitatum

Trichoderma viride spores applied in water to apparently uninjured skin of oranges do not cause lesions. Adding orange juice, rind extract, citric acid or orange essential oil did not influence infection. Oranges became infected only when the stem-end cuts or wounds deeper than 6 mm into oil vesicles were inoculated. Sound oranges in contact with decayed oranges did not become infected. Diphenyl-impregnated wrappers reduced infection. A mixed inoculum of T. viride and Penicillium digitatum caused as fast rotting as P. digitatum, which caused faster rotting than T. viride alone. Lesions infected with P. digitatum could become infected by T. viride but those caused by T. viride did not become infected by P. digitatum. T. viride was antagonistic to P. digitatum in vivo and in vitro, possibly because it produces a heat-labile diffusible substance toxic to P. digitatum.     

Sensitivity to an Ethylene Biosynthesis-Inducing Endoxylanase in Nicotiana tabacum L. cv Xanthi Is Controlled by a Single Dominant Gene

The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). Sensitivity to EIX is controlled by a single dominant gene, based on chi-square analysis of segregation ratios.     

Purification and characterization of a chitinase from Trichoderma viride

Usukizyme, a commercial enzyme preparation from Trichoderma viride, showed multiple chitin- degrading activities. One of these was purified to homogeneity by sequential DEAE Sepharose CL-6B, Q-Sepharose FF, and Sephacryl S-100 HR column chromatographies. The purified enzyme showed optimum activity at pH 3.5 and 50 degrees -55 degrees C and was stable in the pH range of 3.5-6.0 and up to 45 degrees C. It showed higher activity toward chitosan-7B, a 62% deacetylated chitosan, as opposed to highly deacetylated chitosan substrates. Products of degradation of a 1% (w/v) solution of partially deacetylated chitin (PC-100) were purified on CM-Sephadex C-25 and analyzed by HPLC, exo-glycosidase digestion, and nitrous acid deamination. The enzyme was unable to split the GlcN-GlcN linkages in the substrate. It produced mainly (GlcNAc)(2) and (GlcNAc)(3) along with mixed oligosaccharides. When subjected to nitrous acid degradation, some of the mixed oligosaccharides produced mainly 2-deoxyglucitol, implying the presence of GlcN at the reducing end of the oligosaccharides.     

pH值对绿色木霉(Trichoderma viride)产纤维素酶的影响

采用微晶纤维素为唯一诱导性碳源,对绿色木霉(Trichoderma viride)在摇瓶发酵过程中控制与不控制pH产纤维素酶进行比较.控制pH时胞外蛋白浓度为0.72 mg/mL比不控制pH时提高43%;FPA、EG、GB和CBH酶活为15.0U/mL,120.0U/mL,1.75U/mL,0.85U/mL分别是不控制pH时的2.1、2.3、11.7和1.7倍.在不同pH下测定纤维素酶液各酶活,表明pH值显著影响纤维素酶各单酶酶活.在pH2.7时,β-葡萄糖苷酶酶活仅为pH4.8时酶活的4%;pH回调试验结果表明β-葡萄糖苷酶对pH敏感,并在催化功能上发生不可逆变化.对纤维素酶液添加分离得到的各单酶,当添加β-葡萄糖苷酶时最多可以提高FPA酶活20%.因此β-葡萄糖苷酶是影响综合酶活的关键酶.通过拉曼光谱检测出β-葡萄糖苷酶在pH5.0有活性状态下,酶蛋白主链结构主要为a-螺旋和无规则卷曲;在pH2.0没有活性状态下,酶蛋白主链结构的无规则卷曲发生较大变化,a-螺旋也受到一定影响.这说明pH对β-葡萄糖苷酶构象的改变是造成其活性变化的主要原因.     

The Ultrastructure of the Fungus Trichoderma viride and Investigation of its Growth on Cellulose

Changes in the ultrastructure of Trichoderma viride during growth in shake cultures on cellobiose and cellulose fibres were examined. Electron micrographs of thin sections of germinating conidia, septate hyphae with ascomycete pores and other cell organelles are presented. Extensive autolysis of hyphae was observed after growth for 20 h on cellobiose. The fungus grew in the lumina and within the walls of cellulose fibres. The hyphae followed the directions of the laminar structure but did not grow across them. The observations indicated that the hyphae penetrated the fibres by causing cracks and by dissolving enzymatically the cellulose.     

Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination.

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.     

The effect of acid pH on the growth kinetics of Trichoderma viride.

Batch cultures of Trichoderma viride have been carried out in a 10 liter stirred fermenter a controlled pH values of 2.5, 2.7, 3.0, and 4.0 and without pH control at a temperature of 28 degrees C. Cell and glucose concentrations and dissolved oxygen values are reported. The yield coefficient was found to be constant at 0.40 kg cells/kg glucose and the maximum specific growth rate was linearly correlated with the hydrogen ion concentration.     

高温对烟叶品质的影响

研究了下列条件下烟叶的成熟和品质的变化.在烟草移栽后第90d进行7d人工高温处理(白天温度为35℃,晚上温度为29℃,光照强度为3×104lx,相对湿度为75%),同期对照的自然条件是3d阴天,日均温为19.7～23.9℃;4d晴天,日均温为25.9～30.8℃,日最强光照强度均在1×1051x以上.结果表明,人工高温处理后,烟叶叶色比对照更绿,成熟期推迟5～7d,没有出现整株烟叶快速变黄的"高温逼熟"现象.对烟叶的细胞结构和化学成分分析,也没有出现"高温逼熟"引起品质变差的变化.因此认为湘南烟区"高温逼熟"导致烟叶品质下降的原因除高温以外,还与强光辐射、"火南风"等因素所造成的危害有关.     

Isolation and characterization of a trypsin-like protease from Trichoderma viride.

A serine endopeptidase with a molecular mass of 25 kDa has been purified from the culture filtrate of Trichoderma viride to electrophoretic homogeneity. The isoelectric point was determined at 7.3. Two carboxyl sites at Arg22 and Lys29 of the oxidized insulin B-chain were cleaved, and peptidyl-p-nitroanilide substrates with Lys or Arg at the P1 position were also hydrolyzed by the enzyme. These results suggest that the specificity of T. viride protease is similar to that of trypsin. However, the hydrolytic activity toward casein of T. viride protease was less than that of porcine trypsin. The amino-terminal sequence of the enzyme protein is similar to that of bovine trypsin. It seems that the trypsin of T. viride is a protease which is promising for the substitution of animal trypsin in the food industry and in medicine at this stage.     

嘧肽霉素影响烟草花叶病毒RNA蓄积量的研究

嘧肽霉素是新报道的由土壤中分离筛选出来的不吸水链霉菌辽宁变种(Streptomyces achygroscopicus var.liaoningensis)产生的,其有效成分是胞嘧啶核苷肽类化合物,已获得农药临时登记,该药剂对烟草花叶病毒(Tobacco mosaic virus,TMV)等多种病毒病害具有很好的防治效果。定量竞争PCR(Quantitative competitive PCR,QCPCR)是一种定量检测细胞因子较为精确的方法,其实质是待测核酸(DNA或RNA)与内参模板一起竞争参与PCR反应,并通过电泳将扩增产物在凝胶上明显的分开,从而进一步进行定性或定量分析。     

Effect of attached carbohydrates on the activity of Trichoderma viride cellulases

Trichoderma viride 1,4-β-d-glucan cellobiohydrolase (exo-cellobiohydrolase, 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) purified from a commercial product to electrophoretic homogeneity by a procedure including affinity and DEAE-Sephadex chromatography, has attached carbohydrates in addition to the glycoprotein constituents. These carbohydrates are lost by consecutive gel filtration steps in Sephadex G-25 columns, whereupon there is a rapid increase in enzymatic activity. A single gel filtration step can eliminate d-glucose or cellobiose added to a solution of this enzyme, but not the carbohydrates attached during incubation with Avicel.After free carbohydrate elimination from crude cellulase complexes by Sephadex G-25 chromatography, liberation of d-glucose following incubation at 50°C and pH 4.8 was observed. This indicates that some carbohydrates remain bound after gel filtration. The elimination of carbohydrate from whole cellulase complex [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was favoured by a yeast treatment, with a simultaneous increase in activity, but the process is not reproducible, as a secondary inactivation process exists.     

防烟叶霉变菌株对烟叶霉变的影响

为有效防止烟叶霉变,采用平板对峙培养的方法,就3个防烟叶霉变菌株对霉变菌的抑制作用进行了研究．结果表明,JMBl42、B112、B329对供试霉变菌皆表现出一定的抑制作用,对不同霉变菌的平均抑制率分别为51．6％、52．9％、53．7％．3个防烟叶霉变菌株分别以每mL 10^7、10^8、10^9 cfu(colony当当forming unit)13个浓度单独处理和每mL10。cfu浓度混合处理烟叶,结果表明,JMBl42菌株每mL10,cfu处理浓度效果最好,与对照差异极显著,其次为B112菌株每mL10^8cfu处理浓度,但它与对照差异不显著,B329菌株处理效果最差,混合施用结果与对照差异不显著．由此确定JMBl42菌株每mL10^9cfu处理浓度为仓储烟叶防霉变最佳处理参数、     

Production of cellulase and xylanase by a wild strain of Trichoderma viride

Summary The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and -gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of -glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of -glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and -glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.     

Influence of Mercury on the Amino Acidic Composition of Tobacco Leaves

In the course of the study on uptake of mercury by plants, tobacco was grown in the presence of mercury vapours and the amino acidic composition of the leaves was examined. The results showed that tobacco can take up more than 5,000 ppm of mercury through the leaves and store it, showing no symptom of poisoning. In relation to the absorption of mercury, an abnormal increase of cystine, glutamic acid and glycine was observed in the proteins of the leaves. A possible role of cystine in the metabolism of mercury is also discussed.     

Effects of Free Radical Scavengers on Stress Ethylene in Soybean Leaves Hypersensitively Reacting to Tobacco Necrosis Virus

The hypersensitive reaction of soybean cuttings to tobacco necrosisvirus is characterized by a large stimulation of stress ethyleneinvolving a marked accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC) and a moderate increase in ethylene-forming enzyme(EFE) activity. The scavengers of hydroxyl radicals (OH^{dot})sodium benzoate, sodium formate, mannitol and dimethylsulphoxide,did not affect stress ethylene biosynthesis. Propyl gallate,an inhibitor of lipoxygenase enzymes, substantially reducedthe release of stress ethylene from hypersensitive leaves. Thisreduction was not attributable to an inhibitory effect on EFEactivity, but to a strong reduction of free ACC accumulationin leaf tissues. The results suggest that OH^{dot} and the lipoxygenasesystem are not involved in stress ethylene produced during thehypersensitive reaction of soybean to this virus. Glycine max Merr, soybean, ethylene, free radicals, hypersensitivity, tobacco necrosis virus     

绿色木霉菌合成金纳米颗粒的可能性及影响因素

【背景】金纳米颗粒(AuNPs)凭借其稳定性、抗氧化性能和生物相容性在许多领域有广泛应用。目前关于微生物合成金纳米颗粒的研究较少。【目的】对微生物合成金纳米颗粒的可能性以及影响因素进行探究,有利于揭示具体的合成机制,发现AuNPs的特性以及合成位置与菌丝和影响因素的关系。【方法】以绿色木霉菌(Trichoderma viride)菌株(GIM3.141)为菌种资源,通过目视检测法、紫外可见分光光度计、X射线衍射和透射电镜等手段分析合成AuNPs的特征。探讨细胞内生物合成金纳米颗粒(AuNPs)的可能性,研究生物量、初始金离子浓度、溶液pH等因素对细胞内合成AuNPs的影响。【结果】X射线衍射分析表明AuNPs以金纳米晶体形态存在。透射电镜分析表明AuNPs主要位于细胞壁膜间隙,一小部分附着在细胞壁上。紫外可见分光光度计分析表明,金纳米颗粒粒径随着生物量添加量和溶液pH的升高而变小,随着初始金离子浓度的升高而变大。【结论】非致病性真菌绿色木霉菌可以在细胞内合成AuNPs,其中包括伪球形、三角形、四边形和六边形等多种形状,粒径范围从几纳米到三百纳米,为大规模、低成本、无污染地生物合成纳米颗粒工艺提供了菌种资源。