Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility

This paper reports the purification and characterization of kinetic parameters of cellulase produced from Trichoderma viride under still culture solid state fermentation technique using cheap and an easily available agricultural waste material, wheat straw as growth supported substrate. Trichoderma viride was cultured in fermentation medium of wheat straw under some previously optimized growth conditions and maximum activity of 398±2.43U/mL obtained after stipulated fermentation time period. Cellulase was purified 2.33 fold with specific activity of 105U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 58kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified enzyme displayed 6.5 and 55oC as an optimum pH and temperature respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 148U/mL with its corresponding KM value of 68μM. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect on purified cellulase whereas, the enzyme activated by Co2+ and Mn2+ at a concentration of 1mM. The purified cellulase was compatible with four local detergent brands with up to 20 days of shelf life at room temperature suggesting its potential as a detergent additive for improved washing therefore, it is concluded that it may be potentially useful for industrial purposes especially for detergent and laundry industry.

Trichoderma viride菌生物量测定及其纤维素酶合成特征

利用HPLC法测定Trichodermaviride菌固态发酵曲中的麦角固醇含量。研究了麦角固醇与菌丝体间的关系。该菌固态曲中麦角固醇分离条件以 1∶2 5 (m/v)的丙酮抽提 1 5h为最佳。当固态发酵培养至 69h时 ,曲中的生物量达到最大值 ,为每克干曲中含有 0 5 75 g菌丝体。此时该菌所产生CMC酶和FP酶活力均达到最大值 ,呈现正相关性 ,说明这 2种酶的合成特征均为同步合成型 ,而C1 酶活力高峰滞后 ,出现在 72h。

Screening for a Novel Trichoderma vride Strain Highly Producing Cellulase via Ultraviolet Mutagenesis

[Objective] The aim of this study was to find out a new Trichoderma vride K strain highly producing cellulase.[Method] Ultraviolet（UV） was used to induce mutagenesis on T.vride K and to select out a new Trichoderma vride strain highly producing cellulase from the first round and further selection.[Result] A new T.vride strain K6 with high yield of cellulase was obtained with the enzyme production amount of 1.39 times over that of starting strain K.This strain showed highest cellulase yield under the culture condition of 28 ℃ for 96 h.[Conclusion] The strain K6 selected out from induced mutation is endowed with better capacity of producing cellulase,which provides a new method for the utilization of straw.

绿色木霉(Trichoderma viride)_(867)产壳聚糖酶的发酵工艺条件的优化

系统研究了碳源、氮源、初始pH、培养温度、培养基装液量、接种量和培养时间等因素对绿色木霉867产壳聚糖酶的影响.结果表明,最佳碳、氮源分别为可溶性壳聚糖和蛋白胨,在初始pH5.0,培养温度28℃,培养基装量75 mL/250 mL,接种量6%和培养时间(180 r/min)40 h时最利于产酶.在此基础上通过均匀设计法优化了发酵培养基配方.优化后的培养基配方为:可溶性壳聚糖0.9%,氨基葡萄糖0.5%,蛋白胨0.9%,K2HPO40.016%,CaCl2.2H2O 0.055%.在该条件下,壳聚糖酶活为0.291 u/mL,比原基础培养条件下酶活提高29.9%.

绿色木霉(Trichoderma viride)基因组文库构建及其CBH I基因阳性克隆筛选

用绿色木霉核DNA部分酶解片段与噬菌体λEMBL3载体左右臂连接,并用高效价包装蛋白对重组分子进行体外包装,侵染宿主菌后,构建了含1.92×10_6个重组噬菌体的绿色木霉基因组文库。以李氏木霉纤维素酶CBH Ⅰ基因的5′、3′两个末端片段为探针,对所建立的基因组文库作轮回噬菌斑原位杂交,筛选到含绿色木霉CBH Ⅰ基因的阳性克隆7个,随机取其中三个克隆进一步用李氏木霉CBH Ⅰ、CBHⅡ基因的5′、3′四个末端片段探针作交叉斑点杂交,证明本实验确实克隆了很可能为全长的绿色木霉CBH Ⅰ基因,并提示CBH Ⅰ与CBHⅡ基因的末端序列之间不存在同源性。

黄绿木霉菌(Trichoderma aureoviride)产纤维素酶研究

通过分离获得一株纤维素分解菌——黄绿木霉(Trichoderma aueroviride),在对其发酵条件的研究中发现:以稻草粉与麸皮为发酵固体培养基的条件下,最适稻草与麸皮比例为3:2或2.5:2.5;最适氮源为氯化铵;最适产酶温度为27～30℃,产酶高峰为发酵4d,通气条件变化,产酶能力变化不明显。液体培养中该菌有较高产量的β-葡萄糖苷酶形成,产酶活性高于绿色木霉As3.3711菌株。

Optimization of Some Culture Conditions for Biosynthesis of Cellulase by Trichoderma vivide

[ Objective] To determine the best culture time and inducer for the biosynthesis of cellulase by Trichoderma vivide and thus provide the conditions for its practical application. [ Method] Within the 7 d after the inoculation of Trichoderma vivide ZJ strain, the cultures were collected once every day, and the enzyme yield was respectively determined by 3,5-dinitresalicylic acid assay. The Trichodernm vivide ZJ strain was inoculated into basal medium added by different types of carbon sources or nitrogen sources, and the growth of Trichoderma viride was observed. And the mycalium weight as well as the yield of CMCase enzyme after different culture time was determined. [Result] The optimal culture time for Trichoderma viride ZJ strain was 72-96 h; it grew rapidly in the medium added by monosaccharide or disaccharide as carbon sources, and the production of CMCase enzyme reached a peak after 3 -4 d post inoculation. Cellulose powder was the best carbon inducer. The compound nitrogen source composed of 1 g/L ammonium sulfate and 2 g/L yeast extract was the most suitable for the growth of ZJ strain and produced the highest enzyme activity. [ Condusion] The largest enzyme yield should be obtained after 3-4 d post the inoculation of Trichoderma viride ZJ strain. With cellulose powder as a carbon source and the complex substance composed of ammonium sulfate and yeast extract as a nitrogen source, Trichoderma viride has the highest enzyme activity.

Trichoderma viride Strains against Vegetable Grey Mold in Greenhouse

[Objective] The paper aimed to study the control effects of live spore preparations of Trichoderma viride strains against vegetable grey mold in greenhouse. [Method] Trichoderma viride strains NW-411 live spore preparations were prepared by solid-state fermentation,106-107 spore/g diluent was made to conduct field control experiment,traits change of cucumber and tomato plants inoculated grey mold were investigated,control effect was calculated. [Result] Cucumber and tomato plants without dilution treatment of T. viride spores could be infected with different changes in trait. T. viride spore preparations had a significant preventive effect on greenhouse cucumber and tomato gray mold,the optimal concentration of spores was in the range of 2.3×10^6-2.3×10^7 spore/g. The incidence of cucumber and tomato plants were reduced to 4.2% and 3.1%,the incidence rate decreased 29.8% and 39.1% compared with plants without treatment,biological control effect was over 87%,and the plant growth can be enhanced obviously. [Conclusion] Live spores preparation of T. viride not only had a significant effect on grey mold,but also significantly enhanced the plants growth in greenhouse,which is a safety and environmental protection biological agent,and worthy to be widely spread in large-scale green vegetable production.

Physicochemical properties and release characteristics of calcium alginate microspheres loaded with Trichoderma viride spores

Novel agroformulations for simultaneous delivery of chemical and biologically active agents to the plants were prepared by encapsulation of Trichoderma viride spores in calcium alginate microspheres.The impact of calcium ions concentration on the viability and sporulation of T.viride spores as well as on the microsphere important physicochemical properties were investigated.Intermolecular interactions in microspheres are complex including mainly hydrogen bonds and electrostatic interactions.T.viride germination inside matrix and germ tubes penetration out of microspheres revealed calcium alginate microspheres provide a supportive environment for T.viride growth.Differences in physicochemical properties and bioactive agents release behaviour from microspheres were ascribed to the changes in microsphere structure.Fitting to Korsmeyer-Peppas empirical model revealed the underlying T.viride release mechanism as anomalous transport kinetics(a combination of two diffusion mechanisms and the Type II transport(polymer swelling and relaxation of the polymeric matrix)).The increasing amount of T.viride spores in the surrounding medium is closely related to the release from microspheres and germination.The rate controlling mechanism of calcium release is Fickian diffusion.A decrease in the release rate with increasing calcium ion concentrations is in accordance with the calcium ions effect on the strength of the alginate network structure.T.viride germination inside microsphere diminished the amount of released calcium ions and slowed release kinetics in comparison with microspheres prepared without T.viride.The results indicated investigated agroformulations have a great potential to be used for plant protection and nutrition.

Construction of biological control strain of Trichoderma viride and study of their ability to induce plant disease resistance

Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type

Using of green fluorescent reporter gene (GFP) to monitor the fate of Fusarium moniliforme mycoparasitized by Trichoderma viride

Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .

Effect of Trichoderma viride on activities of polygalacturonase of Rhizoctonia

The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.

A novel strain of <i>Trichoderma viride</i>shows complete lignocellulolytic activities

In this study we describe a novel dark-green strain of Trichoderma viride exhibiting complete ensemble of cellulase, hemicellulase and ligninase activities on specific plate assays. To assess the cellulase production in detail, basal salt medium (BSM) was fortified with synthetic (carboxymethyl cellulose (CMC), glucose, sucrose, dextrose, lactose or maltose) and natural (flours of banana, banana peel, jack seed, potato or tapioca) carbon as well as nitrogen (yeast extract, beef extract, peptone, NaNO3 or NH4NO3) sources. Temperature and pH optima were 28°C and 4, respectively for the growth of the fungus in CMC-BSM with 137 U/mL cellulase activity, which was enhanced to 173 U/mL at 1.25% CMC concentration. Flours of potato and banana peel supported comparable yields of cellulase to that of CMC, while sodium nitrate was the preferred nitrogen source. The water soluble bluish-green pigment (a probable siderophore) extracted from the spores showed an absorption maximum at 292 nm. To sum up, the complete lignocellulolytic potential of this fungus offers great industrial significance, coupled with the production of a new pigment.

Selection of Trichoderma mutants with enhanced cellulase production and resistant to catabolite repression

Due to high cost and relatively low efficiency of cellulase enzymes used for the saccharification of pretreated lignocelluloses, the improvement of cellulase secreting microorganisms is of vital importance. Trichoderma reesei QM 6a, an excellent source of cellulase was selected in the late 1960’s. at Natick Laboratories by its performance on pure cellulose (Solka Floc, Avicel) . QM 6a is the wild parent strain of best existing hypercellulolytic mutants such as Rut C30, VTT-D-80133, L27, CL-847 and others. Utilization of cheaper carbon sources (e.g., pretreated wood or straw) both in enzyme production and in hydrolysis necessitates to investigate fungal species other than T.reesei. A screening program was initiated to test 150 wild-type Trichoderma strains in shake flask for cellulase production on SO 2-impregnated and steam pretreated spruce and willow, candidate substrates for bioalcohol program in Sweden. Filter paper activity (FPA) method was used to determine the overall cellulase activity. Strain TUB F-1505 was selected as promising candidate for mutagenesis. This wild strain was isolated from a tropical rain forest area near Manaus, Brazil. Isolate F-1505 was subjected to NTG-mutation to select catabolite (glucose, glycerol) resistant mutants. A Petri plate clearing assay using Walseth cellulose, glycerol or glucose and Triton X100 (colony size inhibitor) was applied for pre-screening of the colonies. Over 6000 colonies were evaluated. Best colonies were tested in shake flask fermentation on pretreated spruce and willow as carbon sources. Mutants producing higher levels of cellulase (FPA) were further mutated by either NTG or UV-light. At least 4 mutants were obtained and freeze-dried exhibiting equivalent or higher cellulase production as compared to Trichoderma reesei Rut C30.

Cellulase Production from Species of Fungi and Bacteria from Agricultural Wastes and Its Utilization in Industry: A Review

In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.

Enhanced bio-catalytic and tolerance properties of an indigenous cellulase through xerogel immobilization

Today, demand exists for cost-effective production of industrially important enzymes from entire scientific sectors. By keeping in mind the extensive industrial applications of cellulase, this study was performed to immobilize the indigenous enzyme produced from Trichoderma viride under pre-optimized SSF of an agricultural waste material, wheat straw. To enhance the bio-catalytic and tolerance properties of the present enzyme gel matrix immobilization engineering was applied. Previously, 2.33~fold purified novel cellulase was immobilized in to a xerogel matrix of TMOS and PTMS. FTIR spectroscopy confirmed the successful immobilization of cellulase. The free and immobilized cellulase was characterized and stability profile showed that after 24 h incubation, immobilization enhanced the thermo-stability up to 75% against 80℃ as compare to the free enzyme. Xerogel matrix immobilization enhanced the catalytic efficiency of entrapped enzyme than that of the free cellulase. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect while, gel matrix immobilization enhanced 80% tolerance capacity of the cellulase against inactivating agents.

Selection and Identification of a Cellulase-producing Strain

[ Objective ] This study aimed to identify a cellulase-producing strain selected from tropical rain forestry soils. [ Method ] Morphologic observation and sequence analysis of 18S rDNA were conducted. [Result] A cellulase-preducing strain with high activity was obtained, and morphology of the strain was highly similar to that of Trichoderma reesei. Results of sequence analysis show that the 18S rDNA sequence shares 99% homology with Hypocreajecorina. [ Conclusion] The isolated cellulase-producing strain belongs to Trichoderma reesei.

Evolution and impact of cellulose architecture during enzymatic hydrolysis by fungal cellulases

The enzymatic hydrolysis of cellulose is still considered as a main limiting step of the biological production of biofuels from ligno-cellulosic biomass. Glycoside hydrolases from Trichoderma reesei are currently used to produce fermentable glucose units from degradation of cellulose packed in a complex assembly of cellulose microfibrils. The present work describes the structural evolution of two prototypical samples of cellulose (a micro-crystalline cellulose and a bleached sulfite pulp) over 5 length scale orders of magnitude. The results were obtained through wide angle, small angle and ultra-small angles synchrotron X-ray scattering, completed by Small Angle Neutron Scattering and particle size analyzers. These structural evolutions were followed as a function of enzymatic conversion. The results show that whereas there is no change at the nanometer scale, drastic changes occur at micron. The observed decrease of the size of the cellulose particles is accompanied by a smoothing of the crystalline surfaces that can be explained by a two-step mechanism of the enzymatic hydrolysis.

拟康氏木霉(Trichoderma pseudokoningii)UV III纤维素酶合成的诱导与阻遏

拟康氏木霉 (T .pseudokoningii)TH经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UVIII,纤维素酶产量显著提高。研究表明 ,UVIII对诱导物的敏感性增加了 10 0倍 ,并且对葡萄糖的吸收能力明显下降 ,导致部分解除了葡萄糖阻遏作用 。

拟康氏木霉(Trichoderma pseudokoningii)UV III产纤维素酶液体发酵研究

以拟康氏木霉 (Trichodermapseudokoningii)TH为出发菌株 ,经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UVIII ,其液体发酵最适产酶培养基为 (W /V) :豆皮粉 3% ,硝酸铵 0 .6 % ,磷酸二氢钠 0 .6 5 % ,硫酸镁 0 .2 5 % ,氯化钙 0 .15 % ,pH5 .0 ;最佳发酵条件为 :30℃ ,12 5r/min。发酵 7dCMCase活力可达 10 3.5 5IU/mL ,滤纸酶活可达 5 .5 1IU/mL ,β -葡萄糖苷酶活可达 0 .96IU/mL ,分别比出发菌株TH提高了 1.4 0、2 .34、0 .6 0倍。