SCP and crude pectinase production by slurry-state fermentation of lemon pulps

Single cell protein (SCP) and crude pectinolytic enzymes production from citrus pulps is reported. SCP and enzymes were produced by slurry-state flask cultivation of Aspergillus niger and Trichoderma viride on pulps from lemon juice clarification. Production as well as crude pectinase activity was not affected by the high dry matter content of the pulps. Both the protein content in the residue and the enzyme activity in the supernatant were higher in T. viride than in A. niger culture. The crude pectinase of T. viride, whose specific activity was similar to that found for a commercial concentrated preparation, could be utilized in the same citrus processing factory as well as in other factories which use large amounts of pectinolytic crude preparations, for example to enhance depuration plant performance.     

一个抗真菌蛋白在绿色木霉中的分泌表达

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。     

Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride.

Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains.     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Low-molecular-weight xylanase from Trichoderma viride.

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Low-molecular-weight xylanase from Trichoderma viride

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl-β-d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger β-d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

绿色木霉内切几丁质酶cDNA的克隆及其编码蛋白的序列分析

根据已克隆的内切几丁质酶基因序列的同源性比较,设计引物,采用PCR技术从绿色木霉基因组中分离出一个大小为1467bp的特异DNA片段,采用RT．PeR技术从绿色木霉总RNA中分离出大小约1276bp的eDNA片段。序列对比后发现该内切几丁质酶DNA含有三个内含子,大小分别为52bp,69bp,64bp。同源性分析表明其全长eDNA序列和已经报道的内切几丁质酶序列的同源性高达95％以上,预测其编码蛋白的氨基酸序列含424个氨基酸残基,分子量为46kDa,氨基酸序列分析表明该内切几丁质酶164～172位氨基酸是其活性中心,用同源建模法模拟其空间结构模型,为进一步研究其作用机制奠定了良好基础。     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Cloning and characterization of saponin hydrolases from Aspergillus oryzae and Eupenicillium brefeldianum

We purified saponin hydrolases from Aspergillus oryzae PF1224 and Eupenicillium brefeldianum PF1226. It was confirmed that the enzymes from A. oryzae PF1224 (Sda1) and E. brefeldianum PF1226 (Sde1) are glycoproteins with molecular masses of 82 and 90 kDa respectively. The deduced amino acid sequences of each enzyme from the cloned genes (sda1 or sde1) showed approximately 50% homology with that of the saponin hydrolase Sdn1 from Neocosmospora vasinfecta var. vasinfecta PF1225 (DDBJ accession no. AB110615). When sda1 and sde1 were expressed in the host Trichoderma viride under the control of the cellobiohydrolase I gene promoter, recombinant proteins were secreted with molecular masses of 77 and 67 kDa respectively. These recombinant enzymes hydrolyzed soyasaponin I to soyasapogenol B and triose, and its substrate specificities for glycosides were similar to that of Sdn1, but the specific activities of these enzymes were lower than that of Sdn1.     

哈茨木霉与绿色木霉对杉木种子萌发和幼苗生长的影响

以杉木种子为材料,研究不同浓度(0.003、0.03、0.3、3、30、300 mg·L^-1)哈茨木霉和绿色木霉溶液对杉木种子萌发和幼苗生长的影响.结果表明: 各浓度木霉溶液处理对杉木种子萌发和幼苗生长均有一定的促进作用,其促进效果随着处理浓度的增加均呈先升后降的趋势.与对照相比,0.03 mg·L^-1哈茨木霉和绿色木霉处理对提高杉木种子的发芽率、发芽势、发芽指数、活力指数、根长、苗高和鲜质量效果最佳,分别提高了57.6%、125.0%、51.0%、209.2%、114.3%、16.1%、24.6%和42.7%、76.7%、43.9%、185.4%、113.8%、8.6%、22.6%;0.3 mg·L^-1哈茨木霉和绿色木霉显著提高杉木幼苗超氧化物歧化酶和过氧化物酶活性,分别增加了157.6%、179.9%和127.5%、116.2%,而丙二醛含量降低了86.1%和72.4%. 0.03～0.3 mg·L^-1浓度的哈茨木霉和绿色木霉不仅能显著促进杉木种子的萌发和幼苗生长,而且能够提高其抗氧化酶活性,增强杉木幼苗的抗逆性.     

Basic Endochitinases Are Major Proteins in Castanea sativa Cotyledons

Basic endochitinases are abundant proteins in Castanea sativa Mill. cotyledons. Three basic chitinases were purified with molecular masses of 25, 26, and 32 kD (Ch1, Ch2, and Ch3) and with isoelectric points between 8 and 9.5. Antibodies raised against Ch1 cross-reacted with Ch2 and Ch3. However, Ch3 showed differences when compared with the other two enzymes, especially in its higher cysteine content. The size, amino acid composition, and N-terminal sequence of Ch1 indicate that it is a class II endochitinase and, therefore, has no cysteine-rich hevein domain. Ch1 inhibits the growth of the fungus Trichoderma viride. The biological role of these endochitinases is discussed.     

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

绿色木霉产生的葡萄糖苷酶类

由绿色木霉(Trichoderma viride)A10菌株的粗酶制剂中分离纯化得到 6个β-葡萄糖苷酶组分,对它们的物理、化学性质和对20种糖苷类化合物的水解作用进行了测试。其中5个组分都是既能水解β-构型的糖苷化合物,又能水解α-构型的糖苷化合物,表明它们对α-、β-异头专一性的要求并不象文献中报导的那样十分严格,对糖基和糖苷配基专一性的要求也并不十分严格。由此,对葡萄糖苷酶类的命名分类方法提出了讨论,并对在纤维素酶系研究中纤维二糖酶活力的检测方法进行了讨论。     

Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil.

Cultures of Sclerotium rolfsii and Trichoderma viride together in autoclaved soil were assayed at intervals during 8 days of incubation for proteolytic activity (PA) of T. viride. Significant proteolytic activity was detected only in soil containing T. viride (i.e., T. viride alone or S. rolfsii + T. viride); greatest activity occurred between 3 and 4 days after infestation and declined rapidly thereafter. Maximal PA in the mixed-culture soil was accompanied by an increase in soil pH. optimal pH values for PA was 5.5-6.5 with a maximum at 6.0.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

Bio-active composts from rice straw enriched with rock phosphate and their effect on the phosphorous nutrition and microbial community in rhizosphere of cowpea

Composts were produced from rice straw enriched with rock phosphate and inoculated with Aspergillus niger, Trichoderma viride and/or farmyard manure (FYM). The resulting composts were evaluated as organic phosphate fertilizers for cowpea plants in pot experiments. The results showed that the maximum amount of soluble phosphorous (1000 ppm) was produced in composts inoculated with A. niger+T. viride with or without FYM. Any of the produced composts was much better than superphosphate fertilizer in providing the growing cowpea plants with phosphorous. Fertilization of the cowpea plants with the compost inoculated with FYM+A. niger+T. viride resulted in maximum amount of phosphorous uptake (295 ppm). The highest phosphate dissolving fungi numbers in rhizosphere soils of cowpea plants were obtained after fertilization with composts which received A. niger and T. viride treatments, while the highest phosphate dissolving bacterial numbers were found after fertilization with composts which received FYM treatments.     

Production of fungal biomass protein using microfungi from winery wastewater treatment

This study was carried out to investigate the production of fungal biomass protein (FBP) in treatment of winery wastewater using microfungi. Three fungal strains, Trichoderma viride WEBL0702, Aspergillus niger WEBL0901 and Aspergillus oryzae WEBL0401, were selected in terms of microbial capability for FBP production and COD reduction. T. viride appeared to be the best strain for FBP production due to high productivity and less nitrogen requirement. More than 5 g/L of fungal biomass was produced in shake fermentation using T. viride without nitrogen addition, and by A. oryzae and A. niger with addition of 0.5-1.0 g/L (NH4)2SO4. The FBP production process corresponded to 84-90% COD reduction of winery wastewater. Fungal biomass contained approximately 36% protein produced by two Aspergillus strains, while biomass produced by T. viride consisted of 19.8% protein. Kinetic study indicated that maximum fungal cell growth could be achieved in 24h for T. viride and 48 h for A. oryzae and A. niger. Current results indicated that it could be feasible to develop a biotechnological treatment process integrated with FBP production from the winery waste streams.